WO2021166273A1 - Saccharified liquid - Google Patents

Saccharified liquid Download PDF

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Publication number
WO2021166273A1
WO2021166273A1 PCT/JP2020/013246 JP2020013246W WO2021166273A1 WO 2021166273 A1 WO2021166273 A1 WO 2021166273A1 JP 2020013246 W JP2020013246 W JP 2020013246W WO 2021166273 A1 WO2021166273 A1 WO 2021166273A1
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WIPO (PCT)
Prior art keywords
saccharified solution
content
cellulose
saccharified
less
Prior art date
Application number
PCT/JP2020/013246
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French (fr)
Japanese (ja)
Inventor
敦 古城
Original Assignee
王子ホールディングス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from PCT/JP2020/006443 external-priority patent/WO2021166102A1/en
Application filed by 王子ホールディングス株式会社 filed Critical 王子ホールディングス株式会社
Priority to TW110105795A priority Critical patent/TW202144581A/en
Priority to PCT/JP2021/006346 priority patent/WO2021167064A1/en
Publication of WO2021166273A1 publication Critical patent/WO2021166273A1/en

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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
    • 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
    • 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

Definitions

  • the present invention relates to a saccharified solution. Specifically, the present invention relates to a saccharified solution using cellulose-containing biomass as a raw material.
  • Sugar may be used as a fermentation raw material.
  • a saccharified solution containing monosaccharides such as six-carbon sugar and five-carbon sugar is assimilated by yeast and converted into ethanol.
  • the ethanol thus obtained is effectively used as, for example, ethanol for fuel or ethanol for beverages.
  • a sugar raw material used as a fermentation raw material starch derived from edible raw materials such as sugar beet, potatoes, and corn is widely used industrially.
  • the process of obtaining saccharified liquid from non-edible raw materials such as trees and grass and further producing chemical products such as ethanol by fermentation reaction. Is required to be built.
  • cellulose-containing biomass is hydrolyzed using an enzyme or a microorganism that produces the enzyme to decompose cellulose and hemicellulose contained in the biomass to produce sugar.
  • Patent Document 1 describes a step of hydrolyzing cellulose-containing biomass with cellulase derived from filamentous fungi, filtering the hydrolyzate with an ultrafiltration membrane, recovering cellulase as a non-permeate, and using a saccharified solution as a permeate.
  • a method for producing a saccharified solution containing glucose, a xylooligo, or the like, which comprises a step of recovering and a step of allowing the recovered cellulase to act on a xylan-containing raw material, is disclosed.
  • Patent Document 2 describes a step of reacting a mannanase with a liquid component obtained by hydrotreating woody biomass to obtain a saccharified solution, and a microfiltration membrane and / or an ultrafiltration membrane of the saccharified solution.
  • a method for producing a sugar solution including a step of filtering and recovering the sugar solution from the permeate side is disclosed.
  • Patent Document 3 describes a step of hydrolyzing a cellulose-containing biomass to produce a sugar aqueous solution, and filtering the obtained sugar aqueous solution through a nanofiltration membrane and / or a reverse osmosis membrane from the non-permeable side.
  • a method for producing a sugar solution including a step of recovering a purified sugar solution and removing a fermentation inhibitor such as furfural from the permeation side is disclosed.
  • a saccharified solution is obtained from starch derived from edible raw materials such as tensai, potatoes, and corn
  • the starch degrading enzyme (amylase) cannot cut the branched structural part of the starch, so oligosaccharides are contained in the obtained saccharified solution. Will be included.
  • the saccharified solution there is a desire to obtain a high-concentration glucose solution, but if the saccharified solution contains oligosaccharides, the purity of glucose will decrease, and the efficiency of the subsequent fermentation process will be reduced. There is a problem that it decreases.
  • the conventional method may not sufficiently remove fermentation inhibitors such as furfural, or enzymes used in the saccharification reaction may be mixed in the saccharified solution. there were.
  • the presence of fermentation inhibitors such as furfural is problematic as it reduces the efficiency of subsequent fermentation steps.
  • the enzyme used for the saccharification reaction is mixed in the saccharified solution, the purity of glucose is lowered and the reuse efficiency of the enzyme is lowered, which is a problem.
  • the present inventors have prepared a saccharified solution having a small residual amount of enzyme and capable of increasing fermentation efficiency when subjected to a fermentation step. We proceeded with the study for the purpose of providing it.
  • the present inventors used cellulose-containing biomass as a raw material, and in a saccharified solution having an electrical conductivity of 500 ⁇ S / cm or more, the oligosaccharide content and the content of furfural were found. It has been found that by setting the content and the content of soluble lignin as predetermined conditions, a saccharified solution having a small residual amount of enzyme and capable of increasing fermentation efficiency when subjected to a fermentation step can be obtained.
  • the present invention has the following configuration.
  • the electrical conductivity of the saccharified solution is 500 ⁇ S / cm or more
  • a saccharified solution in which the oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 100 ppm or more.
  • the saccharified solution according to [1] wherein the content of lignin in the cellulose-containing biomass is 1% by mass or more and 3% by mass or less.
  • the present invention may have the following configurations.
  • [6] The purified saccharified solution according to [5], which has an electrical conductivity of 10 ⁇ S / cm or less.
  • [7] The purified saccharified solution according to [5] or [6], wherein the content of soluble lignin is 10 ppm or less.
  • [8] A fermentation raw material containing the saccharified solution according to any one of [1] to [4] or the purified saccharified solution according to any one of [5] to [7].
  • [9] A fermented liquid obtained by fermenting the saccharified liquid according to any one of [1] to [4] or the purified saccharified liquid according to any one of [5] to [7].
  • [10] Ethanol obtained by fermenting the saccharified solution according to any one of [1] to [4] or the purified saccharified solution according to any one of [5] to [7].
  • the present invention may have the following configurations.
  • the electrical conductivity of the saccharified solution is 500 ⁇ S / cm or more
  • the oligosaccharide content in the saccharified solution is 10 ppm or less
  • the furfural content is 1 ppm or less
  • the soluble lignin content is 120 ppm or more.
  • a method for producing a saccharified liquid which comprises a step of membrane-treating the treatment liquid obtained in the saccharification treatment step with an ultrafiltration membrane.
  • the electrical conductivity of the saccharified solution is 500 ⁇ S / cm or more
  • the oligosaccharide content in the saccharified solution is 10 ppm or less
  • the furfural content is 1 ppm or less
  • the soluble lignin content is 120 ppm or more.
  • a method for producing a saccharified solution, wherein the enzymatic activity of the saccharified solution is 10 U / ml or less.
  • a method for producing a purified saccharified solution which comprises a step of treating the saccharified solution with an ion exchange resin, wherein the electric conductivity of the saccharified solution is 500 ⁇ S / cm or more.
  • the oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 120 ppm or more.
  • a method for producing a purified saccharified solution wherein the enzymatic activity of the saccharified solution is 10 U / ml or less.
  • the method for producing a purified saccharified solution according to [17] wherein the electrical conductivity of the purified saccharified solution is 10 ⁇ S / cm or less.
  • the present invention relates to a saccharified solution using cellulose-containing biomass as a raw material.
  • the electrical conductivity of the saccharified solution is 500 ⁇ S / cm or more.
  • the oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 100 ppm or more.
  • the electric conductivity of the saccharified solution is 500 ⁇ S / cm or more, which means that the saccharified solution is a crude saccharified solution.
  • the present invention relates to a crude saccharified solution having an electrical conductivity of 500 ⁇ S / cm or more, in which the contents of oligosaccharide, furfural and soluble lignin are within a predetermined range.
  • the electrical conductivity of the saccharified solution of the present invention is preferably 600 ⁇ S / cm or more, more preferably 700 ⁇ S / cm or more, and even more preferably 750 ⁇ S / cm or more.
  • the electrical conductivity of the saccharified solution of the present invention is preferably 2500 ⁇ S / cm or less, and more preferably 2000 ⁇ S / cm or less. The lower the value of electrical conductivity, the more refined it is.
  • the electric conductivity of the saccharified liquid is a value measured by an electric conductivity meter.
  • the electric conductivity meter for example, LAQUAtwin EC-33B manufactured by HORIBA, Ltd. can be used.
  • the saccharified solution of the present invention has the above-mentioned structure, the residual amount of the enzyme in the saccharified solution is kept low. Specifically, by setting the content of soluble lignin in the saccharified solution of the present invention to 100 ppm or more, the residual amount of the enzyme in the saccharified solution can be suppressed to a low level.
  • cellulose-containing biomass contains lignin
  • lignin contains soluble lignin and insoluble lignin in a certain ratio. And most of the enzymes used in the saccharification step are recovered together with insoluble lignin.
  • the fact that the content of soluble lignin in the saccharified solution is equal to or higher than a predetermined value means that a certain amount of insoluble lignin was present, and that the enzyme was sufficiently recovered after the saccharification step. It is suggested. Therefore, it is considered that the residual amount of the enzyme in the saccharification solution can be kept low by controlling the starting material and the saccharification step so that the content of soluble lignin in the saccharification solution is 100 ppm or more.
  • the enzyme activity of the saccharified solution is preferably 10 U / ml or less, and more preferably 5 U / ml or less.
  • the enzyme activity of the saccharified solution is particularly preferably 0 U / ml. That is, it is preferable that the saccharified solution of the present invention contains substantially no enzyme.
  • the enzyme activity of the saccharified solution is a value measured by the following method. First, 4 ml of 100 mM acetate buffer (pH 5) and 32 ml of 1.25 mM 4-methylhumberiferyl ⁇ -D glucoside (manufactured by Wako Co., Ltd.) are added to 4 ml of the saccharified solution, and the mixture is reacted at 37 ° C.
  • the reaction is stopped with 200 ml of a 0.5 M glycine-NaOH solution (pH 10.5), and the fluorescence intensity (excitation wavelength 355 nm, measurement wavelength 460 nm) is measured with a fluorometer (Infinite 200 manufactured by Tecan Co., Ltd.).
  • the enzyme residual activity is calculated assuming that the enzyme residual activity when 1 mM 4-methylumbelliferone is released per 1 ml of the saccharified solution is 1 U / ml.
  • the saccharified solution of the present invention has the above-mentioned structure, the fermentation efficiency of the saccharified solution can be increased when the saccharified solution is subjected to a fermentation step.
  • the saccharified solution may be used as a fermentation raw material.
  • a saccharified solution containing a monosaccharide such as hexasaccharide or pentasaccharide is assimilated by yeast and converted into ethanol.
  • the ethanol thus obtained is used as, for example, ethanol for fuel or ethanol for beverages.
  • the fermentation efficiency when fermenting the saccharified solution can be increased, and as a result, the ethanol yield can be increased.
  • the ethanol yield in the present specification is a value calculated as the amount of ethanol produced per unit cellulose-containing biomass amount (kg) by measuring the amount of ethanol in the obtained fermented liquid by fermenting the saccharified liquid by the method described later. be. Specifically, 2 ml of corn steep liquor (manufactured by Oji Cornstarch) and 5 ml of 1M acetate buffer (pH 5) were added to 50 ml of the saccharified solution, and 5 ml of the pre-cultured yeast solution (Saccharomyces cerevisiae 1 ⁇ 10 8 cells / mL) was added. Add and hold at 33 ° C. for 18 hours.
  • the amount of ethanol in the culture solution after the reaction is measured using a high performance liquid chromatography device (HP-2200 manufactured by Azilent Co., Ltd.), and the amount of ethanol produced per unit cellulose-containing biomass amount (kg) is calculated.
  • the ethanol yield calculated in this way is preferably 510 mL / kg or more, more preferably 530 mL / kg or more, and even more preferably 540 mL / kg or more.
  • the upper limit of the ethanol yield is not particularly limited, but can be, for example, 600 mL / kg.
  • the oligosaccharide content in the saccharified solution of the present invention may be 10 ppm or less, preferably 5 ppm or less, and more preferably 1 ppm or less.
  • the oligosaccharide content in the saccharified solution is particularly preferably 0 ppm. That is, it is preferable that the saccharified solution of the present invention does not substantially contain oligosaccharides.
  • the oligosaccharide refers to one in which about 2 to 10 monosaccharides are bound by glycosidic bonds. Examples of monosaccharides include glucose, galactose, mannose, fructose and the like.
  • the oligosaccharide content can be measured by ion chromatography.
  • the oligosaccharide content is detected as a peak observed after the monosaccharide elutes and can be quantified by a calibration curve. Since oligosaccharides are not converted to ethanol, the lower the oligosaccharide content, the higher the ethanol yield.
  • the content of the monosaccharide in the saccharified solution of the present invention is preferably 1.0% by mass or more, more preferably 2.0% by mass or more, and further preferably 3.0% by mass or more. It is more preferably 4.0% by mass or more, further preferably 5.0% by mass or more, and particularly preferably 5.2% by mass or more.
  • the content of the monosaccharide in the saccharified solution is preferably 50% by mass or less, more preferably 30% by mass or less, further preferably 20% by mass or less, and 10% by mass or less. Is particularly preferred.
  • the content of monosaccharides in the saccharified solution can be quantified by detecting the peak of monosaccharides by ion chromatography. The quantification of monosaccharides is calculated by measuring a standard sugar solution having a known concentration and comparing it with the peak area thereof. Different types of monosaccharides can be identified because their peak retention times are different.
  • the saccharified solution of the present invention contains glucose. Further, since the saccharified solution of the present invention is made from cellulose-containing biomass, it contains other sugars in addition to glucose. Other sugars include, for example, xylose, arabinose, mannose, xylose, galactose.
  • the saccharified solution of the present invention is a saccharified solution made from cellulose-containing biomass. Whether or not the obtained saccharified solution is made from cellulose-containing biomass can be determined by, for example, whether or not the saccharified solution contains lignin, and the lignin in the saccharified solution can be determined. With the detection, it can be determined that the raw material is cellulose-containing biomass.
  • the cellulose-containing biomass is preferably a tree-derived raw material
  • the saccharified solution of the present invention is preferably a saccharified solution obtained from a tree-derived raw material. Examples of the tree-derived raw material include eucalyptus, acacia, pine, birch, spruce, sugi, and cypress.
  • the cellulose-containing biomass contains lignin, and the content of lignin in the cellulose-containing biomass is preferably 1% by mass or more, more preferably 1.5% by mass or more, and 1.8% by mass or more. Is more preferable, and 2% by mass or more is particularly preferable.
  • the content of lignin in the cellulose-containing biomass is preferably 3% by mass or less, more preferably 2.4% by mass or less, and further preferably 2.2% by mass or less.
  • the amount of soluble lignin remaining in the saccharified solution may be 100 ppm or more, preferably 120 ppm or more, and more preferably 150 ppm or more.
  • the upper limit of the amount of soluble lignin remaining in the saccharified solution is not particularly limited, but is preferably 2000 ppm or less, for example.
  • the soluble lignin content is a value calculated by the following formula by measuring the absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution.
  • Soluble lignin content (ppm) dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution
  • coefficient A is the extinction coefficient of lignin (110 L / g / cm). be.
  • the solid content in the saccharified solution is a value measured by the weight of the residue remaining after the saccharified solution is evaporated to dryness.
  • the saccharified solution of the present invention is a saccharified solution using cellulose-containing biomass as a raw material, and can be obtained by saccharifying the cellulose-containing biomass.
  • the reaction between the water and the cellulose-containing biomass is carried out in the presence of an enzyme or an enzyme-producing microorganism. Therefore, the content of furfural contained in the obtained saccharified solution is 1 ppm or less. That is, the saccharified solution of the present invention does not substantially contain furfural.
  • the content of furfural contained in the saccharified solution is measured by high performance liquid chromatography.
  • the present invention may relate to a purified saccharified solution obtained by purifying the above-mentioned saccharified solution.
  • the purified saccharified solution can be obtained by treating the above-mentioned saccharified solution with an ion exchange resin.
  • an ion exchange resin 10 g of an ion exchange resin (Amber Jet 1024, IRA96SB, manufactured by Organo Corporation) is added to 100 ml of the above-mentioned saccharified solution, and the mixture is stirred at 1 room temperature for 30 minutes. Then, by performing filtration, the filtrate is separated from the ion exchange resin, and the obtained filtrate becomes a purified saccharified solution.
  • the electrical conductivity of the purified saccharified solution is preferably 10 ⁇ S / cm or less, more preferably 8 ⁇ S / cm or less, and even more preferably 7 ⁇ S / cm or less.
  • the electrical conductivity of the purified saccharified solution is preferably 5 ⁇ S / cm or more.
  • the electric conductivity of the purified saccharified liquid is a value measured by an electric conductivity meter.
  • the electric conductivity meter for example, LAQUAtwin EC-33B manufactured by HORIBA, Ltd. can be used.
  • the content of soluble lignin in the purified saccharified solution is preferably 10 ppm or less, more preferably 9 ppm or less, and even more preferably 8 ppm or less.
  • the content of soluble lignin in the purified saccharified solution is preferably 4 ppm or more.
  • the soluble lignin content is a value calculated by the following formula by measuring the absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution.
  • Soluble lignin content (ppm) dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution
  • coefficient A is the extinction coefficient of lignin (110 L / g / cm). be.
  • the solid content in the saccharified solution is a value measured by the weight of the residue remaining after the saccharified solution is evaporated to dryness.
  • the monosaccharide content, oligosaccharide content, and furfural content in the purified saccharified solution are almost the same as those in the above-mentioned saccharified solution because they hardly change before and after the treatment with the ion exchange resin.
  • the content of monosaccharide in the purified saccharified solution is preferably 1.0% by mass or more, more preferably 2.0% by mass or more, and more preferably 3.0% by mass or more. Is more preferably 4.0% by mass or more, further preferably 5.0% by mass or more, and particularly preferably 5.2% by mass or more.
  • the content of monosaccharide in the purified saccharified solution is preferably 50% by mass or less, more preferably 30% by mass or less, further preferably 20% by mass or less, and 10% by mass or less. It is particularly preferable to have.
  • the content of monosaccharides in the purified saccharified solution can be quantified by detecting the peak of monosaccharides by ion chromatography.
  • the content of oligosaccharide in the purified saccharified solution may be 10 ppm or less, preferably 5 ppm or less, and more preferably 1 ppm or less.
  • the oligosaccharide content in the purified saccharified solution is particularly preferably 0 ppm. That is, it is preferable that the purified saccharified solution of the present invention does not substantially contain oligosaccharides.
  • the oligosaccharide content can be measured by ion chromatography. In ion chromatography, the oligosaccharide content is detected as a peak observed after the monosaccharide elutes and can be quantified by a calibration curve.
  • the content of furfural in the purified saccharified solution is preferably 1 ppm or less. That is, the saccharified solution of the present invention does not substantially contain furfural.
  • the content of furfural contained in the purified saccharified solution is measured by high performance liquid chromatography.
  • the method for producing a saccharified solution of the present invention includes a step of saccharifying cellulose-containing biomass.
  • Biomass is a biological resource excluding fossil fuels, and examples of the cellulose-containing biomass include biological resources containing a cellulose component.
  • biological resources containing cellulose components include papermaking trees, forest residue, chips or bark of thinned lumber, sawdust or sawdust generated from sawmills, pruned branches and leaves of roadside trees, construction waste, and the like. Be done.
  • herbaceous resources include agricultural wastes such as kenaf, rice straw, straw, and bagasse, and herbaceous energy crops such as Elianthus, Miscanthus, and Napiergrass.
  • the cellulose-containing biomass wood-derived paper, used paper, pulp, pulp sludge and the like can also be used.
  • the cellulose-containing biomass used in the present invention is preferably a tree-derived raw material.
  • the chips are formed into chips having a size of preferably 10 to 200 mm, more preferably 50 to 100 mm. That is, the method for producing a saccharified solution of the present invention preferably includes a step of saccharifying the chipped cellulose-containing biomass.
  • Cellulose is a chain polymer compound formed by the ⁇ -1,4 glycoside bond between a large number of glucose molecules.
  • cellulose a large number of glucoses are bound by dehydration condensation of the 1-position hydroxyl group of one glucose and the 4-position hydroxyl group of another glucose.
  • 1,4-glycosidic bonds are cleaved, and generally, monosaccharides and oligosaccharides in which about 2 to 10 monosaccharides are bound are produced.
  • the cellulose-containing biomass also includes hemicellulose, which is a polysaccharide existing between cellulose microfibrils. Therefore, when cellulose-containing biomass is saccharified, the hydrolyzate includes glucose, which is a cellulose-derived saccharide, and xylose, arabinose, mannose, and the like, which are hemicellulose-derived saccharides.
  • the method for producing a saccharified solution of the present invention includes a step of saccharifying a broadleaf kraft pulp or a softwood kraft pulp having a lignin content of 1% by mass or more and 3% by mass or less by mixing water and cellulase, and saccharification. It is preferable to include a step of membrane-treating the treatment liquid obtained in the step of treatment with an ultrafiltration membrane.
  • the electrical conductivity of the saccharified solution is 500 ⁇ S / cm or more
  • the oligosaccharide content in the saccharified solution is 10 ppm or less
  • the furfural content is 1 ppm or less
  • the soluble lignin content is 120 ppm or more.
  • the enzymatic activity of the saccharified solution is 10 U / ml or less.
  • the saccharification treatment step is a step of hydrolyzing the polysaccharide contained in the cellulose-containing biomass to obtain a monosaccharide.
  • the reaction with the cellulose-containing biomass is carried out in the presence of water and an enzyme and / or a microorganism producing the enzyme, and stirring is preferably carried out. That is, the saccharification treatment step preferably includes an enzyme treatment step.
  • the solid content concentration in the stirring solution is preferably 1 to 30% by mass, more preferably 3 to 20% by mass, although it depends on the type of enzyme. preferable.
  • the temperature of the stirring liquid is preferably 30 to 75 ° C, more preferably 40 to 70 ° C.
  • the pH of the stirring liquid is preferably 3.0 to 7.0, more preferably 4.0 to 6.5.
  • the saccharification treatment time is preferably 2 to 200 hours, more preferably 5 to 100 hours.
  • the enzyme used in the saccharification treatment step is preferably a cellulolytic enzyme.
  • Cellulose-degrading enzyme is an enzyme having at least one selected from cellobiohydrolase activity, endoglucanase activity and beta-glucosidase activity, and is an enzyme generically called cellulase.
  • the cellulase may have hemicellulase activity.
  • Cellulase a commercially available cellulase preparation can be used.
  • Cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Fanerochaete, the genus Trametes, the genus Trametes, the genus Humicola, and the genus Humicola. Examples of cellulase preparations to be used.
  • cellulase T2 manufactured by HPI
  • Meiserase manufactured by Meiji Seika
  • Novozymes 188 manufactured by Novozymes
  • Multifect CX10L manufactured by Genecore
  • GC220 manufactured by Genecore
  • the amount of the cellulase preparation used with respect to 100 parts by mass of the cellulose-containing biomass is preferably 0.5 to 100 parts by mass, and more preferably 1 to 50 parts by mass.
  • the method for producing a saccharified liquid of the present invention preferably includes a step of membrane-treating the treatment liquid obtained in the saccharification treatment step.
  • UF membranes Materials for ultrafiltration (UF) membranes include polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, sulfone.
  • PES polyethersulfone
  • PS polysulfone
  • PAN polyacrylonitrile
  • PVDF polyvinylidene fluoride
  • regenerated cellulose cellulose, cellulose ester
  • modified polyether sulfone polyolefin
  • polyvinyl alcohol polymethylmethacrylate
  • polytetrafluoroethylene polytetrafluoroethylene
  • a ceramic filter or MF film may be used. Further, in the process of membrane treatment, a filtration method using a filter press or a centrifugation treatment method can also be adopted.
  • the membrane treatment method adopted in the membrane treatment step is not particularly limited, but for example, a simple filtration method (total filtration method) in which treated water is supplied in a direction perpendicular to the membrane surface and a membrane surface.
  • a simple filtration method total filtration method
  • cross-flow filtration in which treated water is supplied in a parallel direction can be mentioned.
  • the membrane permeate obtained in the process of membrane treatment contains a saccharified solution
  • the non-permeate side contains enzymes, raw material residues and the like.
  • the enzyme contained on the non-permeable side can be reused in the above-mentioned saccharification treatment step. By increasing the recycling efficiency of the enzyme, it is possible to suppress the production cost of the saccharified solution.
  • a step of pretreating the cellulose-containing biomass Before the saccharification treatment step, a step of pretreating the cellulose-containing biomass may be provided. Since the cellulose-containing biomass contains lignin, protein, and the like, pretreatment may be performed in order to improve the hydrolysis efficiency by an enzyme such as cellulase. Examples of the pretreatment method include alkali treatment with caustic soda, ammonia and the like, steaming treatment, chemical pulping treatment (sulfite cooking, kraft cooking, etc.) and the like. As a pretreatment step, when cellulose-containing biomass is hydroheat-treated (for example, at a temperature of 100 ° C. or higher), acid-treated or subcritical water-treated, furfural is produced, and furfural in the finally obtained saccharified solution is produced.
  • a pretreatment step when cellulose-containing biomass is hydroheat-treated (for example, at a temperature of 100 ° C. or higher), acid-treated or subcritical water-treated, furfural is produced, and furfural
  • the content may greatly exceed 1 ppm. Therefore, as the pretreatment step, it is preferable to adopt a step in which the formation of furfural can be suppressed.
  • a step in which the formation of furfural can be suppressed For example, alkali treatment with caustic soda, ammonia or the like, steaming treatment and chemical pulping treatment (sulfite cooking, craft cooking, etc.) It is preferable to adopt at least one selected from.
  • cellulose-containing biomass can be mechanically treated to increase the hydrolysis efficiency by cellulase.
  • the mechanical treatment include mechanical means such as cutting, cutting, crushing, and grinding.
  • the mechanical device to be used is not particularly limited, and for example, a cutting device, a uniaxial crusher, a biaxial crusher, a hammer crusher, a refiner, a kneader, a ball mill and the like can be used.
  • a foreign matter removing step may be provided to remove foreign matter (foreign matter such as stone, dust, metal, plastic, etc.).
  • the foreign matter removing step include a cleaning step.
  • a method for cleaning the cellulose-containing biomass for example, a method of spraying water on the cellulose-containing biomass to remove foreign substances mixed in the cellulose-containing biomass, or a method of immersing the cellulose-containing biomass in water to settle and remove the foreign substances. The method and the like can be mentioned.
  • Another method is to separate foreign matter from cellulose-containing biomass by using a device such as a metal trap or a washing drainer.
  • a sterilization treatment step may be provided as a pretreatment step. If germs are mixed in the cellulose-containing biomass, the germs may consume sugar in the saccharification treatment step and the yield of the product may decrease.
  • the sterilization treatment step may be a method of exposing the raw material to a pH at which bacteria are difficult to grow, such as acid or alkali, but a method of treating at a high temperature may be adopted.
  • a step of concentrating the membrane permeate may be provided.
  • the concentration of the saccharified solution contained in the membrane permeate is increased.
  • a concentrated saccharified solution can be obtained by heating the membrane permeate to evaporate the water content.
  • the present invention may relate to a method for producing a purified saccharified solution, which comprises a step of further treating the saccharified solution obtained in the above-mentioned step with an ion exchange resin.
  • the electrical conductivity of the saccharified solution is 500 ⁇ S / cm or more
  • the oligosaccharide content in the saccharified solution is 10 ppm or less
  • the furfural content is 1 ppm or less
  • the soluble lignin content is 120 ppm.
  • the enzymatic activity of the saccharified solution is 10 U / ml or less.
  • the electrical conductivity of the purified saccharified solution obtained by the method for producing the purified saccharified solution is preferably 10 ⁇ S / cm or less, more preferably 8 ⁇ S / cm or less, and further preferably 7 ⁇ S / cm or less.
  • the electrical conductivity of the purified saccharified solution obtained by the method for producing the purified saccharified solution is preferably 5 ⁇ S / cm or more.
  • the electric conductivity of the purified saccharified liquid is a value measured by an electric conductivity meter.
  • the electric conductivity meter for example, LAQUAtwin EC-33B manufactured by HORIBA, Ltd. can be used.
  • the content of soluble lignin in the purified saccharified solution obtained by the method for producing the purified saccharified solution is preferably 10 ppm or less, more preferably 9 ppm or less, and further preferably 8 ppm or less.
  • the content of soluble lignin obtained by the method for producing a purified saccharified solution is preferably 4 ppm or more.
  • the soluble lignin content is a value calculated by the following formula by measuring the absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution.
  • Soluble lignin content (ppm) dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution
  • coefficient A is the extinction coefficient of lignin (110 L / g / cm). be.
  • the solid content in the saccharified solution is a value measured by the weight of the residue remaining after the saccharified solution is evaporated to dryness.
  • the saccharified solution of the present invention is preferably used as a fermentation raw material.
  • the saccharified solution of the present invention is subjected to a fermentation step and becomes a fermentation broth.
  • the fermentation broth is particularly preferably ethanol. Since the content of oligosaccharide in the saccharified solution of the present invention is 10 ppm or less and the content of furfural is 1 ppm or less, fermentation efficiency is good when fermenting the saccharified solution. Therefore, when a fermented liquid (ethanol) is produced using the saccharified liquid of the present invention, a high ethanol yield is achieved.
  • the saccharified solution of the present invention has the above-mentioned structure, the ethanol purity of the fermentation broth (ethanol) is high and the content of impurities can be reduced. Therefore, for example, when ethanol is used for beverages. Can suppress miscellaneous taste and the like.
  • the saccharified solution of the present invention is preferably a saccharified solution for fermentation raw materials, more preferably a saccharified solution for ethanol production, and further preferably a saccharified solution for producing ethanol for beverages. .. Further, the present invention may be related to a fermentation raw material containing the saccharified solution, may be related to a fermented solution obtained by fermenting the saccharified solution, or may be related to ethanol obtained by fermenting the saccharified solution. It may be related to ethanol for drinking obtained by fermenting the saccharified solution.
  • Example 1 ⁇ Saccharification treatment> To 80 mL of water, add 5 g of oxygen-bleached hardwood kraft pulp having a lignin content of 2% by mass (dry weight, oxygen-bleached hardwood kraft pulp is chipped to a size of 50 to 100 mm), and stir to make a pulp slurry. Prepared. Next, 1N sulfuric acid was added to this pulp slurry to adjust the pH to 5.0, and 2 mL of a cellulase solution (Multifect CX10L manufactured by Genencore) and water were added to prepare the final volume to 100 mL. .. The solution was allowed to stand at 50 ° C. for 48 hours, and then the supernatant was filtered through an ultrafiltration membrane (Centricon Plus 70 centrifugal filter unit manufactured by Millipore) to obtain a saccharified solution.
  • an ultrafiltration membrane (Centricon Plus 70 centrifugal filter unit manufactured by Millipore)
  • Example 2 A saccharified solution was obtained in the same manner as in Example 1 except that 5 g (dry weight) of oxygen-bleached softwood kraft pulp having a lignin content of 2% by mass was used as a raw material used for the saccharification treatment.
  • Example 1 A saccharified solution was obtained in the same manner as in Example 1 except that 5 g (dry weight) of bleached hardwood kraft pulp having a lignin content of 0.3% by mass was used as a raw material used for the saccharification treatment.
  • Example 2 A saccharified solution was obtained in the same manner as in Example 1 except that 5 g (dry weight) of bleached softwood kraft pulp having a lignin content of 0.3% by mass was used as a raw material used for the saccharification treatment.
  • ⁇ Furfural content> The content of furfural contained in the saccharified solution was measured using a high performance liquid chromatography device (HP-2200, manufactured by Agilent).
  • Soluble lignin content The absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution was measured, and the soluble lignin content was calculated by the following formula.
  • Soluble lignin content (ppm) dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution
  • coefficient A is the extinction coefficient of lignin (110 L / g / cm).
  • ND (Not Detected) in the above table indicates that it is not more than a measurement limit value. Specifically, the measurement limit value in the measurement of oligosaccharide is 1 ppm, the measurement limit value in the measurement of furfural is 0.1 ppm, the measurement limit value in the measurement of soluble lignin is 1 ppm, and the measurement of electrical conductivity. The measurement limit value in is 0.2 ⁇ S / cm. Further, "-" in Comparative Example 3 in the above table indicates that no enzyme was added, and "-" in Comparative Example 4 indicates that the raw material was starch and did not contain lignin.

Abstract

An objective of the present invention is to provide a saccharified liquid in which the residual enzyme amount is small and with which fermentation efficiency can be improved when subjected to a fermentation process. The present invention pertains to a saccharified liquid that has a cellulose-containing biomass as the source material thereof, wherein the electrical conductivity of the saccharified liquid is at least 500μS/cm, the content of oligosaccharides in the saccharified liquid is at most 10ppm, the content of furfural is at most 1ppm and the content of soluble lignin is at least 100ppm.

Description

糖化液Saccharification liquid
　本発明は、糖化液に関する。具体的には、本発明は、セルロース含有バイオマスを原料とする糖化液に関する。 The present invention relates to a saccharified solution. Specifically, the present invention relates to a saccharified solution using cellulose-containing biomass as a raw material.
　糖は発酵原料として使用されることがある。例えば、六炭糖や五炭糖といった単糖を含む糖化液は酵母により資化されてエタノールに変換される。このようにして得られたエタノールは、例えば燃料用エタノールや飲料用エタノールとして有効利用される。発酵原料となる糖原料としては、テンサイ、芋類、トウモロコシといった可食原料に由来するでん粉が工業的に多く使用されている。しかし、世界人口の増加による食糧需給問題や、食料価格の高騰などの問題から、樹木や草などの非可食原料から糖化液を得て、さらに発酵反応によりエタノール等の化成品を生産するプロセスの構築が求められている。 Sugar may be used as a fermentation raw material. For example, a saccharified solution containing monosaccharides such as six-carbon sugar and five-carbon sugar is assimilated by yeast and converted into ethanol. The ethanol thus obtained is effectively used as, for example, ethanol for fuel or ethanol for beverages. As a sugar raw material used as a fermentation raw material, starch derived from edible raw materials such as sugar beet, potatoes, and corn is widely used industrially. However, due to problems such as food supply and demand due to the increase in the world population and soaring food prices, the process of obtaining saccharified liquid from non-edible raw materials such as trees and grass and further producing chemical products such as ethanol by fermentation reaction. Is required to be built.
　近年、非可食原料としてセルロース含有バイオマスの利用が注目されており、セルロース含有バイオマスを原料とした糖やエタノールの製造プロセスの研究が進められている。糖の製造プロセスにおいては、セルロース含有バイオマスを、酵素やその酵素を生産する微生物を用いて加水分解することにより、バイオマスに含まれるセルロースやヘミセルロースを分解し、糖を生成する。 In recent years, the use of cellulose-containing biomass as a non-edible raw material has attracted attention, and research on sugar and ethanol production processes using cellulose-containing biomass as a raw material is underway. In the sugar production process, cellulose-containing biomass is hydrolyzed using an enzyme or a microorganism that produces the enzyme to decompose cellulose and hemicellulose contained in the biomass to produce sugar.
　例えば、特許文献１には、セルロース含有バイオマスを糸状菌由来セルラーゼにより加水分解する工程と、加水分解物を限外濾過膜によって濾過し、非透過液としてセルラーゼを回収し、透過液として糖化液を回収する工程と、回収セルラーゼをキシラン含有原料に作用させる工程を含む、グルコースを含む糖化液およびキシロオリゴ等の製造方法が開示されている。また、特許文献２には、木質系バイオマスを加水分解処理して得られた液成分にマンナナーゼを反応させて糖化液を得る工程と、糖化液を精密ろ過膜および／または限外ろ過膜してろ過して透過液側から糖液を回収する工程とを含む糖液の製造方法が開示されている。 For example, Patent Document 1 describes a step of hydrolyzing cellulose-containing biomass with cellulase derived from filamentous fungi, filtering the hydrolyzate with an ultrafiltration membrane, recovering cellulase as a non-permeate, and using a saccharified solution as a permeate. A method for producing a saccharified solution containing glucose, a xylooligo, or the like, which comprises a step of recovering and a step of allowing the recovered cellulase to act on a xylan-containing raw material, is disclosed. Further, Patent Document 2 describes a step of reacting a mannanase with a liquid component obtained by hydrotreating woody biomass to obtain a saccharified solution, and a microfiltration membrane and / or an ultrafiltration membrane of the saccharified solution. A method for producing a sugar solution including a step of filtering and recovering the sugar solution from the permeate side is disclosed.
　セルロース含有バイオマスから糖を生成する方法として、濃硫酸等の酸を使用してセルロースやヘミセルロースを加水分解する方法も知られている。例えば、特許文献３には、セルロース含有バイオマスを加水分解し、糖水溶液を製造する工程と、得られた糖水溶液をナノ濾過膜および／または逆浸透膜に通じて濾過して、非透過側から精製糖液を回収し、透過側からフルフラール等の発酵阻害物質を除去する工程を含む糖液の製造方法が開示されている。 As a method for producing sugar from cellulose-containing biomass, a method of hydrolyzing cellulose or hemicellulose using an acid such as concentrated sulfuric acid is also known. For example, Patent Document 3 describes a step of hydrolyzing a cellulose-containing biomass to produce a sugar aqueous solution, and filtering the obtained sugar aqueous solution through a nanofiltration membrane and / or a reverse osmosis membrane from the non-permeable side. A method for producing a sugar solution including a step of recovering a purified sugar solution and removing a fermentation inhibitor such as furfural from the permeation side is disclosed.
国際公開第２０１６／０６８２２３号International Publication No. 2016/06823 国際公開第２０１６／０３５８７５号International Publication No. 2016/035875 国際公開第２０１０／０６７７８５号International Publication No. 2010/06775
　テンサイ、芋類、トウモロコシといった可食原料に由来するでん粉から糖化液を得る場合、でん粉分解酵素（アミラーゼ）はでん粉の枝分かれ構造部分を切断することができないため、得られる糖化液中にはオリゴ糖が含まれることとなる。糖化液の製造工程においては、高濃度グルコース液を得たいという要望があるが、糖化液中にオリゴ糖が含まれる場合、グルコースの純度が低下してしまい、続いて行われる発酵工程の効率が低下するという問題がある。 When a saccharified solution is obtained from starch derived from edible raw materials such as tensai, potatoes, and corn, the starch degrading enzyme (amylase) cannot cut the branched structural part of the starch, so oligosaccharides are contained in the obtained saccharified solution. Will be included. In the process of producing a saccharified solution, there is a desire to obtain a high-concentration glucose solution, but if the saccharified solution contains oligosaccharides, the purity of glucose will decrease, and the efficiency of the subsequent fermentation process will be reduced. There is a problem that it decreases.
　一方、セルロース含有バイオマスから糖化液を得る場合、従来の方法では、フルフラール等の発酵阻害物質の除去が十分ではなかったり、糖化反応に使用される酵素が糖化液中にも混在してしまう場合があった。フルフラール等の発酵阻害物質の存在は、続いて行われる発酵工程の効率を低下させるため問題となる。また、糖化反応に使用される酵素が糖化液に混在する場合、グルコースの純度が低下することに加え、酵素の再利用効率が低下するため問題となる。 On the other hand, when a saccharified solution is obtained from cellulose-containing biomass, the conventional method may not sufficiently remove fermentation inhibitors such as furfural, or enzymes used in the saccharification reaction may be mixed in the saccharified solution. there were. The presence of fermentation inhibitors such as furfural is problematic as it reduces the efficiency of subsequent fermentation steps. Further, when the enzyme used for the saccharification reaction is mixed in the saccharified solution, the purity of glucose is lowered and the reuse efficiency of the enzyme is lowered, which is a problem.
　そこで本発明者らは、このような従来技術の課題を解決するために、酵素の残存量が少ない糖化液であって、かつ、発酵工程に供された際に発酵効率を高め得る糖化液を提供することを目的として検討を進めた。 Therefore, in order to solve the problems of the prior art, the present inventors have prepared a saccharified solution having a small residual amount of enzyme and capable of increasing fermentation efficiency when subjected to a fermentation step. We proceeded with the study for the purpose of providing it.
　上記の課題を解決するために鋭意検討を行った結果、本発明者らは、セルロース含有バイオマスを原料とし、電気伝導度が５００μＳ／ｃｍ以上の糖化液において、オリゴ糖の含有量と、フルフラールの含有量と、可溶性リグニンの含有量を所定条件とすることにより、酵素の残存量が少なく、かつ発酵工程に供された際に発酵効率を高め得る糖化液が得られることを見出した。
　具体的に、本発明は、以下の構成を有する。 As a result of diligent studies to solve the above problems, the present inventors used cellulose-containing biomass as a raw material, and in a saccharified solution having an electrical conductivity of 500 μS / cm or more, the oligosaccharide content and the content of furfural were found. It has been found that by setting the content and the content of soluble lignin as predetermined conditions, a saccharified solution having a small residual amount of enzyme and capable of increasing fermentation efficiency when subjected to a fermentation step can be obtained.
Specifically, the present invention has the following configuration.
［１］　セルロース含有バイオマスを原料とする糖化液であって、
　糖化液の電気伝導度が５００μＳ／ｃｍ以上であり、
　糖化液におけるオリゴ糖の含有量が１０ｐｐｍ以下であり、フルフラールの含有量が１ｐｐｍ以下であり、可溶性リグニンの含有量が１００ｐｐｍ以上である糖化液。
［２］　セルロース含有バイオマスにおけるリグニンの含有量が１質量％以上３質量％以下である［１］に記載の糖化液。
［３］　セルロース含有バイオマスが樹木由来原料である［１］又は［２］のいずれかに記載の糖化液。
［４］　糖化液の酵素活性が１０Ｕ／ｍｌ以下である［１］～［３］のいずれかに記載の糖化液。
［５］　［１］～［４］のいずれかに記載の糖化液をイオン交換樹脂で処理して得られる精製糖化液。 [1] A saccharified solution made from cellulose-containing biomass as a raw material.
The electrical conductivity of the saccharified solution is 500 μS / cm or more,
A saccharified solution in which the oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 100 ppm or more.
[2] The saccharified solution according to [1], wherein the content of lignin in the cellulose-containing biomass is 1% by mass or more and 3% by mass or less.
[3] The saccharified solution according to any one of [1] and [2], wherein the cellulose-containing biomass is a tree-derived raw material.
[4] The saccharified solution according to any one of [1] to [3], wherein the enzymatic activity of the saccharified solution is 10 U / ml or less.
[5] A purified saccharified solution obtained by treating the saccharified solution according to any one of [1] to [4] with an ion exchange resin.
　また、本発明は、以下の構成を有するものであってもよい。
［６］　電気伝導度が１０μＳ／ｃｍ以下である［５］に記載の精製糖化液。
［７］　可溶性リグニンの含有量が１０ｐｐｍ以下である［５］又は［６］に記載の精製糖化液。
［８］　［１］～［４］のいずれかに記載の糖化液、もしくは、［５］～［７］のいずれかに記載の精製糖化液を含む発酵原料。
［９］　［１］～［４］のいずれかに記載の糖化液、もしくは、［５］～［７］のいずれかに記載の精製糖化液を発酵させてなる発酵液。
［１０］［１］～［４］のいずれかに記載の糖化液、もしくは、［５］～［７］のいずれかに記載の精製糖化液を発酵させてなるエタノール。 In addition, the present invention may have the following configurations.
[6] The purified saccharified solution according to [5], which has an electrical conductivity of 10 μS / cm or less.
[7] The purified saccharified solution according to [5] or [6], wherein the content of soluble lignin is 10 ppm or less.
[8] A fermentation raw material containing the saccharified solution according to any one of [1] to [4] or the purified saccharified solution according to any one of [5] to [7].
[9] A fermented liquid obtained by fermenting the saccharified liquid according to any one of [1] to [4] or the purified saccharified liquid according to any one of [5] to [7].
[10] Ethanol obtained by fermenting the saccharified solution according to any one of [1] to [4] or the purified saccharified solution according to any one of [5] to [7].
　さらに、本発明は、以下の構成を有するものであってもよい。
［１１］　リグニン含有量が１質量％以上３質量％以下である広葉樹クラフトパルプまたは針葉樹クラフトパルプを原料として、セルラーゼを添加して加水分解した後に上清を限外濾過膜で濾過して得られた糖化液であって、
　糖化液の電気伝導度が５００μＳ／ｃｍ以上であり、
　糖化液におけるオリゴ糖の含有量が１０ｐｐｍ以下であり、フルフラールの含有量が１ｐｐｍ以下であり、可溶性リグニンの含有量が１２０ｐｐｍ以上であり、
　糖化液の酵素活性が１０Ｕ／ｍｌ以下である、糖化液。
［１２］　セルロース含有バイオマスが樹木由来原料である［１１］に記載の糖化液。
［１３］　［１１］又は［１２］に記載の糖化液をイオン交換樹脂で処理して得られる精製糖化液。
［１４］　電気伝導度が１０μＳ／ｃｍ以下である［１３］に記載の精製糖化液。
［１５］　可溶性リグニンの含有量が１０ｐｐｍ以下である［１３］又は［１４］に記載の精製糖化液。
［１６］　リグニン含有量が１質量％以上３質量％以下である広葉樹クラフトパルプまたは針葉樹クラフトパルプと、水分と、セルラーゼを混合して糖化処理する工程と、
　糖化処理する工程で得られた処理液を限外濾過膜で膜処理する工程とを含む糖化液の製造方法であって、
　糖化液の電気伝導度が５００μＳ／ｃｍ以上であり、
　糖化液におけるオリゴ糖の含有量が１０ｐｐｍ以下であり、フルフラールの含有量が１ｐｐｍ以下であり、可溶性リグニンの含有量が１２０ｐｐｍ以上であり、
　糖化液の酵素活性が１０Ｕ／ｍｌ以下である、糖化液の製造方法。
［１７］　リグニン含有量が１質量％以上３質量％以下である広葉樹クラフトパルプまたは針葉樹クラフトパルプと、水分と、セルラーゼを混合して糖化処理する工程と、
　糖化処理する工程で得られた処理液を限外濾過膜で膜処理し糖化液を得る工程と、
　糖化液をイオン交換樹脂で処理する工程と、を含む精製糖化液の製造方法であって
　糖化液の電気伝導度が５００μＳ／ｃｍ以上であり、
　糖化液におけるオリゴ糖の含有量が１０ｐｐｍ以下であり、フルフラールの含有量が１ｐｐｍ以下であり、可溶性リグニンの含有量が１２０ｐｐｍ以上であり、
　糖化液の酵素活性が１０Ｕ／ｍｌ以下である、精製糖化液の製造方法。
［１８］　精製糖化液の電気伝導度が１０μＳ／ｃｍ以下である［１７］に記載の精製糖化液の製造方法。
［１９］　精製糖化液の可溶性リグニンの含有量が１０ｐｐｍ以下である［１７］又は［１８］に記載の精製糖化液の製造方法。 Furthermore, the present invention may have the following configurations.
[11] Obtained by using hardwood kraft pulp or softwood kraft pulp having a lignin content of 1% by mass or more and 3% by mass or less as a raw material, adding cellulase to hydrolyze the pulp, and then filtering the supernatant with an ultrafiltration membrane. It is a saccharified solution
The electrical conductivity of the saccharified solution is 500 μS / cm or more,
The oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 120 ppm or more.
A saccharified solution having an enzymatic activity of 10 U / ml or less.
[12] The saccharified solution according to [11], wherein the cellulose-containing biomass is a tree-derived raw material.
[13] A purified saccharified solution obtained by treating the saccharified solution according to [11] or [12] with an ion exchange resin.
[14] The purified saccharified solution according to [13], which has an electrical conductivity of 10 μS / cm or less.
[15] The purified saccharified solution according to [13] or [14], wherein the content of soluble lignin is 10 ppm or less.
[16] A step of mixing and saccharifying hardwood kraft pulp or softwood kraft pulp having a lignin content of 1% by mass or more and 3% by mass or less, water, and cellulase.
A method for producing a saccharified liquid, which comprises a step of membrane-treating the treatment liquid obtained in the saccharification treatment step with an ultrafiltration membrane.
The electrical conductivity of the saccharified solution is 500 μS / cm or more,
The oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 120 ppm or more.
A method for producing a saccharified solution, wherein the enzymatic activity of the saccharified solution is 10 U / ml or less.
[17] A step of mixing and saccharifying hardwood kraft pulp or softwood kraft pulp having a lignin content of 1% by mass or more and 3% by mass or less, water, and cellulase.
A step of obtaining a saccharified solution by membrane-treating the treatment solution obtained in the step of saccharification treatment with an ultrafiltration membrane, and a step of obtaining a saccharified solution.
A method for producing a purified saccharified solution, which comprises a step of treating the saccharified solution with an ion exchange resin, wherein the electric conductivity of the saccharified solution is 500 μS / cm or more.
The oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 120 ppm or more.
A method for producing a purified saccharified solution, wherein the enzymatic activity of the saccharified solution is 10 U / ml or less.
[18] The method for producing a purified saccharified solution according to [17], wherein the electrical conductivity of the purified saccharified solution is 10 μS / cm or less.
[19] The method for producing a purified saccharified solution according to [17] or [18], wherein the content of soluble lignin in the purified saccharified solution is 10 ppm or less.
　本発明によれば、酵素の残存量が少なく、かつ発酵工程に供された際に発酵効率を高め得る糖化液を得ることができる。 According to the present invention, it is possible to obtain a saccharified solution having a small residual amount of enzyme and capable of increasing fermentation efficiency when subjected to a fermentation step.
　以下において、本発明について詳細に説明する。以下に記載する構成要件の説明は、代表的な実施形態や具体例に基づいてなされることがあるが、本発明はそのような実施形態に限定されるものではない。なお、本明細書において「～」を用いて表される数値範囲は「～」前後に記載される数値を下限値及び上限値として含む範囲を意味する。 Hereinafter, the present invention will be described in detail. The description of the constituent elements described below may be based on typical embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, the numerical range represented by using "-" means a range including the numerical values before and after "-" as the lower limit value and the upper limit value.
（糖化液）
　本発明は、セルロース含有バイオマスを原料とする糖化液に関する。ここで、糖化液の電気伝導度は５００μＳ／ｃｍ以上である。また、糖化液におけるオリゴ糖の含有量は１０ｐｐｍ以下であり、フルフラールの含有量は１ｐｐｍ以下であり、可溶性リグニンの含有量は１００ｐｐｍ以上である。
　本明細書において、糖化液の電気伝導度が５００μＳ／ｃｍ以上であることは、糖化液が粗精製糖化液であることを意味する。すなわち、本発明は、電気伝導度が５００μＳ／ｃｍ以上の粗精製糖化液において、オリゴ糖、フルフラール及び可溶性リグニンの各含有量を所定範囲とした糖化液に関するものである。本発明の糖化液の電気伝導度は、６００μＳ／ｃｍ以上であることが好ましく、７００μＳ／ｃｍ以上であることがより好ましく、７５０μＳ／ｃｍ以上であることがさらに好ましい。また、本発明の糖化液の電気伝導度は、２５００μＳ／ｃｍ以下であることが好ましく、２０００μＳ／ｃｍ以下であることがより好ましい。なお、電気伝導度の値は低いほど、精製されていることを示している。ここで、糖化液の電気伝導度は、電気伝導率計により測定される値である。電気伝導率計としては、例えば、堀場製作所製のＬＡＱＵＡｔｗｉｎ　ＥＣ－３３Ｂを用いることができる。 (Saccharification solution)
The present invention relates to a saccharified solution using cellulose-containing biomass as a raw material. Here, the electrical conductivity of the saccharified solution is 500 μS / cm or more. The oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 100 ppm or more.
In the present specification, the electric conductivity of the saccharified solution is 500 μS / cm or more, which means that the saccharified solution is a crude saccharified solution. That is, the present invention relates to a crude saccharified solution having an electrical conductivity of 500 μS / cm or more, in which the contents of oligosaccharide, furfural and soluble lignin are within a predetermined range. The electrical conductivity of the saccharified solution of the present invention is preferably 600 μS / cm or more, more preferably 700 μS / cm or more, and even more preferably 750 μS / cm or more. The electrical conductivity of the saccharified solution of the present invention is preferably 2500 μS / cm or less, and more preferably 2000 μS / cm or less. The lower the value of electrical conductivity, the more refined it is. Here, the electric conductivity of the saccharified liquid is a value measured by an electric conductivity meter. As the electric conductivity meter, for example, LAQUAtwin EC-33B manufactured by HORIBA, Ltd. can be used.
　本発明の糖化液は上記構成を有するものであるため、糖化液中における酵素の残存量が低く抑えられている。具体的には、本発明の糖化液において、可溶性リグニンの含有量を１００ｐｐｍ以上とすることにより、糖化液中における酵素の残存量を低く抑えることができる。通常、セルロース含有バイオマスにはリグニンが含まれており、リグニンには、可溶性リグニンと不溶性リグニンが一定の割合で含まれている。そして、糖化工程で用いられる酵素の大部分は不溶性リグニンと共に回収される。このため、糖化液中における可溶性リグニンの含有量が所定値以上であることは、不溶性リグニンがある程度の量存在していたことを意味し、糖化工程後に酵素の回収が十分に行われたことが示唆される。このため、糖化液中の可溶性リグニンの含有量が１００ｐｐｍ以上となるように、出発原料や糖化工程をコントロールすることにより、糖化液中における酵素の残存量が低く抑えられるものと考えられる。 Since the saccharified solution of the present invention has the above-mentioned structure, the residual amount of the enzyme in the saccharified solution is kept low. Specifically, by setting the content of soluble lignin in the saccharified solution of the present invention to 100 ppm or more, the residual amount of the enzyme in the saccharified solution can be suppressed to a low level. Normally, cellulose-containing biomass contains lignin, and lignin contains soluble lignin and insoluble lignin in a certain ratio. And most of the enzymes used in the saccharification step are recovered together with insoluble lignin. Therefore, the fact that the content of soluble lignin in the saccharified solution is equal to or higher than a predetermined value means that a certain amount of insoluble lignin was present, and that the enzyme was sufficiently recovered after the saccharification step. It is suggested. Therefore, it is considered that the residual amount of the enzyme in the saccharification solution can be kept low by controlling the starting material and the saccharification step so that the content of soluble lignin in the saccharification solution is 100 ppm or more.
　糖化液の酵素活性は１０Ｕ／ｍｌ以下であることが好ましく、５Ｕ／ｍｌ以下であることがより好ましい。なお、糖化液の酵素活性は０Ｕ／ｍｌであることが特に好ましい。すなわち、本発明の糖化液には酵素が実質的に含まれないことが好ましい。ここで、糖化液の酵素活性は、以下の方法で測定された値である。まず、糖化液４ｍｌに、１００ｍＭ酢酸緩衝液（ｐＨ５）４ｍｌと１．２５ｍＭ　４－メチルフンベリフェリルβ－Ｄグルコシド（和光社製）３２ｍｌを添加し、３７℃で３０分間反応させる。０．５Ｍグリシン－ＮａＯＨ溶液（ｐＨ１０．５）２００ｍｌで反応を停止させ、蛍光光度計（テカン社製　インフィニット２００）で蛍光強度（励起波長３５５ｎｍ、測定波長４６０ｎｍ）を測定する。糖化液１ｍｌ当たり、１分間に１ｍＭの４－メチルウンベリフェロンが遊離した場合の酵素残存活性を１Ｕ／ｍｌとして、酵素残存活性を算出する。 The enzyme activity of the saccharified solution is preferably 10 U / ml or less, and more preferably 5 U / ml or less. The enzyme activity of the saccharified solution is particularly preferably 0 U / ml. That is, it is preferable that the saccharified solution of the present invention contains substantially no enzyme. Here, the enzyme activity of the saccharified solution is a value measured by the following method. First, 4 ml of 100 mM acetate buffer (pH 5) and 32 ml of 1.25 mM 4-methylhumberiferyl β-D glucoside (manufactured by Wako Co., Ltd.) are added to 4 ml of the saccharified solution, and the mixture is reacted at 37 ° C. for 30 minutes. The reaction is stopped with 200 ml of a 0.5 M glycine-NaOH solution (pH 10.5), and the fluorescence intensity (excitation wavelength 355 nm, measurement wavelength 460 nm) is measured with a fluorometer (Infinite 200 manufactured by Tecan Co., Ltd.). The enzyme residual activity is calculated assuming that the enzyme residual activity when 1 mM 4-methylumbelliferone is released per 1 ml of the saccharified solution is 1 U / ml.
　また、本発明の糖化液は上記構成を有するものであるため、糖化液が発酵工程に供された際に、その発酵効率を高めることができる。糖化液は発酵原料として使用されることがあり、例えば、六炭糖や五炭糖といった単糖を含む糖化液は酵母により資化されてエタノールに変換される。このようにして得られたエタノールは、例えば燃料用エタノールや飲料用エタノールとして利用される。本発明では、糖化液において、オリゴ糖とフルフラールの含有量をそれぞれ所定値以下とすることにより、糖化液を発酵する際の発酵効率を高めることができ、結果としてエタノール収量を高めることができる。 Further, since the saccharified solution of the present invention has the above-mentioned structure, the fermentation efficiency of the saccharified solution can be increased when the saccharified solution is subjected to a fermentation step. The saccharified solution may be used as a fermentation raw material. For example, a saccharified solution containing a monosaccharide such as hexasaccharide or pentasaccharide is assimilated by yeast and converted into ethanol. The ethanol thus obtained is used as, for example, ethanol for fuel or ethanol for beverages. In the present invention, by setting the contents of oligosaccharide and furfural in the saccharified solution to predetermined values or less, the fermentation efficiency when fermenting the saccharified solution can be increased, and as a result, the ethanol yield can be increased.
　本明細書におけるエタノール収量は、後述する方法で糖化液を発酵させて、得られた発酵液中のエタノール量を測定し、単位セルロース含有バイオマス量（ｋｇ）あたりのエタノール生産量として算出した値である。具体的には、糖化液５０ｍｌにコーンスティープリカー（王子コーンスターチ社製）２ｍｌ、１Ｍ酢酸バッファー（ｐＨ５）５ｍｌを添加し、前培養した酵母液（サッカロマイセス・セレビシエ　１×１０８ｃｅｌｌｓ／ｍＬ）を５ｍｌ添加し、３３℃で１８時間保持する。反応後の培養液中のエタノール量を、高速液体クロマトグラフィー装置（アジレント社製　ＨＰ－２２００）を用いて測定し、単位セルロース含有バイオマス量（ｋｇ）あたりのエタノール生産量を算出する。このようにして算出されるエタノール収量は、５１０ｍＬ／ｋｇ以上であることが好ましく、５３０ｍＬ／ｋｇ以上であることがより好ましく、５４０ｍＬ／ｋｇ以上であることがさらに好ましい。エタノール収量の上限値は特に限定されるものではないが、例えば、６００ｍＬ／ｋｇとすることができる。 The ethanol yield in the present specification is a value calculated as the amount of ethanol produced per unit cellulose-containing biomass amount (kg) by measuring the amount of ethanol in the obtained fermented liquid by fermenting the saccharified liquid by the method described later. be. Specifically, 2 ml of corn steep liquor (manufactured by Oji Cornstarch) and 5 ml of 1M acetate buffer (pH 5) were added to 50 ml of the saccharified solution, and 5 ml of the pre-cultured yeast solution (Saccharomyces cerevisiae 1 × 10 8 cells / mL) was added. Add and hold at 33 ° C. for 18 hours. The amount of ethanol in the culture solution after the reaction is measured using a high performance liquid chromatography device (HP-2200 manufactured by Azilent Co., Ltd.), and the amount of ethanol produced per unit cellulose-containing biomass amount (kg) is calculated. The ethanol yield calculated in this way is preferably 510 mL / kg or more, more preferably 530 mL / kg or more, and even more preferably 540 mL / kg or more. The upper limit of the ethanol yield is not particularly limited, but can be, for example, 600 mL / kg.
　本発明の糖化液におけるオリゴ糖の含有量は１０ｐｐｍ以下であればよく、５ｐｐｍ以下であることが好ましく、１ｐｐｍ以下であることがより好ましい。なお、糖化液におけるオリゴ糖の含有量は０ｐｐｍであることが特に好ましい。すなわち、本発明の糖化液にはオリゴ糖が実質的に含まれないことが好ましい。本明細書において、オリゴ糖とは、グリコシド結合によって単糖が２～１０個程度結合したものをいう。単糖としては、例えば、グルコース、ガラクトース、マンノース、フルクトース等が挙げられる。なお、オリゴ糖の含有量は、イオンクロマトグラフィーにより測定できる。イオンクロマトグラフィーにおいて、オリゴ糖の含有量は、単糖が溶出した後に観察されるピークとして検出され、検量線により定量できる。オリゴ糖は、エタノールに変換されないので、オリゴ糖の含有量が少なければ少ないほど、エタノール収量は高くなる。 The oligosaccharide content in the saccharified solution of the present invention may be 10 ppm or less, preferably 5 ppm or less, and more preferably 1 ppm or less. The oligosaccharide content in the saccharified solution is particularly preferably 0 ppm. That is, it is preferable that the saccharified solution of the present invention does not substantially contain oligosaccharides. In the present specification, the oligosaccharide refers to one in which about 2 to 10 monosaccharides are bound by glycosidic bonds. Examples of monosaccharides include glucose, galactose, mannose, fructose and the like. The oligosaccharide content can be measured by ion chromatography. In ion chromatography, the oligosaccharide content is detected as a peak observed after the monosaccharide elutes and can be quantified by a calibration curve. Since oligosaccharides are not converted to ethanol, the lower the oligosaccharide content, the higher the ethanol yield.
　本発明の糖化液における単糖の含有量は、１．０質量％以上であることが好ましく、２．０質量％以上であることがより好ましく、３．０質量％以上であることがさらに好ましく、４．０質量％以上であることが一層好ましく、５．０質量％以上であることがより一層好ましく、５．２質量％以上であることが特に好ましい。また、糖化液における単糖の含有量は、５０質量％以下であることが好ましく、３０質量％以下であることがより好ましく、２０質量％以下であることがさらに好ましく、１０質量％以下であることが特に好ましい。糖化液における単糖の含有量はイオンクロマトグラフィーにおいて単糖のピークを検出することで定量できる。単糖の定量は、既知濃度の標準糖液を測定し、そのピーク面積と比較することで算出する。異なる種類の単糖については、ピークのリテンションタイムが異なるため、それぞれ判別できる。 The content of the monosaccharide in the saccharified solution of the present invention is preferably 1.0% by mass or more, more preferably 2.0% by mass or more, and further preferably 3.0% by mass or more. It is more preferably 4.0% by mass or more, further preferably 5.0% by mass or more, and particularly preferably 5.2% by mass or more. The content of the monosaccharide in the saccharified solution is preferably 50% by mass or less, more preferably 30% by mass or less, further preferably 20% by mass or less, and 10% by mass or less. Is particularly preferred. The content of monosaccharides in the saccharified solution can be quantified by detecting the peak of monosaccharides by ion chromatography. The quantification of monosaccharides is calculated by measuring a standard sugar solution having a known concentration and comparing it with the peak area thereof. Different types of monosaccharides can be identified because their peak retention times are different.
　本発明の糖化液にはグルコースが含まれる。また、本発明の糖化液は、セルロース含有バイオマスを原料とするものであるため、グルコースに加えて他の糖が含まれる。他の糖としては、例えば、キシロース、アラビノース、マンノース、キシロース、ガラクトースが挙げられる。 The saccharified solution of the present invention contains glucose. Further, since the saccharified solution of the present invention is made from cellulose-containing biomass, it contains other sugars in addition to glucose. Other sugars include, for example, xylose, arabinose, mannose, xylose, galactose.
　本発明の糖化液は、セルロース含有バイオマスを原料とする糖化液である。得られた糖化液が、セルロース含有バイオマスを原料としたものであるか否かについては、例えば、糖化液中にリグニンが含まれているか否かで判別することができ、糖化液中のリグニンの検出をもってセルロース含有バイオマスを原料としたものであると判別することができる。セルロース含有バイオマスは、樹木由来原料であることが好ましく、本発明の糖化液は、樹木由来原料から得られる糖化液であることが好ましい。樹木由来原料としては、例えば、ユーカリ、アカシア、マツ、バーチ、トウヒ、スギ、ヒノキ等を挙げることができる。 The saccharified solution of the present invention is a saccharified solution made from cellulose-containing biomass. Whether or not the obtained saccharified solution is made from cellulose-containing biomass can be determined by, for example, whether or not the saccharified solution contains lignin, and the lignin in the saccharified solution can be determined. With the detection, it can be determined that the raw material is cellulose-containing biomass. The cellulose-containing biomass is preferably a tree-derived raw material, and the saccharified solution of the present invention is preferably a saccharified solution obtained from a tree-derived raw material. Examples of the tree-derived raw material include eucalyptus, acacia, pine, birch, spruce, sugi, and cypress.
　セルロース含有バイオマスはリグニンを含むものであり、セルロース含有バイオマスにおけるリグニンの含有量は、１質量％以上であることが好ましく、１．５質量％以上であることがより好ましく、１．８質量％以上であることがさらに好ましく、２質量％以上であることが特に好ましい。また、セルロース含有バイオマスにおけるリグニンの含有量は、３質量％以下であることが好ましく、２．４質量％以下であることがより好ましく、２．２質量％以下であることがさらに好ましい。セルロース含有バイオマスにおけるリグニンの含有量を上記範囲内とすることにより、得られる糖化液中に残存する可溶性リグニン量を所定値以上とすることができ、これにより、酵素量をより効果的に低減することができる。なお、セルロース含有バイオマスにおけるリグニンの含有量は、ＪＩＳ　Ｐ　８２１１：２０１１パルプカッパー価試験方法に準拠して、以下の式で算出された値である。
　リグニン含有量（質量％）＝カッパー価×０．１５ The cellulose-containing biomass contains lignin, and the content of lignin in the cellulose-containing biomass is preferably 1% by mass or more, more preferably 1.5% by mass or more, and 1.8% by mass or more. Is more preferable, and 2% by mass or more is particularly preferable. The content of lignin in the cellulose-containing biomass is preferably 3% by mass or less, more preferably 2.4% by mass or less, and further preferably 2.2% by mass or less. By setting the content of lignin in the cellulose-containing biomass within the above range, the amount of soluble lignin remaining in the obtained saccharified solution can be set to a predetermined value or more, thereby more effectively reducing the amount of enzyme. be able to. The lignin content in the cellulose-containing biomass is a value calculated by the following formula in accordance with the JIS P 8211: 2011 pulp copper value test method.
Lignin content (% by mass) = copper value x 0.15
　糖化液中に残存する可溶性リグニン量は、１００ｐｐｍ以上であればよく、１２０ｐｐｍ以上であることが好ましく、１５０ｐｐｍ以上であることがさらに好ましい。糖化液中に残存する可溶性リグニン量の上限値は特に限定されるものではないが、例えば、２０００ｐｐｍ以下であることが好ましい。ここで、可溶性リグニン含有量は、糖化液のＵＶスペクトル（波長２１０ｎｍ）の吸光度を測定し、下記の式で算出される値である。
　可溶性リグニン含有量（ｐｐｍ）＝希釈倍率×糖化液量×試料溶液吸光度／係数Ａ×糖化液中の固形分量
　但し、上記式中、係数Ａは、リグニンの吸光係数（１１０Ｌ／ｇ／ｃｍ）である。また、糖化液中の固形分量は、糖化液を蒸発乾固することで残存する残渣の重量により測定される値である。 The amount of soluble lignin remaining in the saccharified solution may be 100 ppm or more, preferably 120 ppm or more, and more preferably 150 ppm or more. The upper limit of the amount of soluble lignin remaining in the saccharified solution is not particularly limited, but is preferably 2000 ppm or less, for example. Here, the soluble lignin content is a value calculated by the following formula by measuring the absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution.
Soluble lignin content (ppm) = dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution However, in the above formula, coefficient A is the extinction coefficient of lignin (110 L / g / cm). be. The solid content in the saccharified solution is a value measured by the weight of the residue remaining after the saccharified solution is evaporated to dryness.
　本発明の糖化液は、セルロース含有バイオマスを原料とする糖化液であって、セルロース含有バイオマスを糖化処理することで得られる。糖化処理工程では、水分と酵素または酵素を生産する微生物の存在下で、セルロース含有バイオマスとの反応が行われる。このため、得られる糖化液中に含まれるフルフラールの含有量は、１ｐｐｍ以下となる。すなわち、本発明の糖化液にはフルフラールが実質的に含まれない。なお、糖化液中に含まれるフルフラールの含有量は、高速液体クロマトグラフィーにより測定される。 The saccharified solution of the present invention is a saccharified solution using cellulose-containing biomass as a raw material, and can be obtained by saccharifying the cellulose-containing biomass. In the saccharification process, the reaction between the water and the cellulose-containing biomass is carried out in the presence of an enzyme or an enzyme-producing microorganism. Therefore, the content of furfural contained in the obtained saccharified solution is 1 ppm or less. That is, the saccharified solution of the present invention does not substantially contain furfural. The content of furfural contained in the saccharified solution is measured by high performance liquid chromatography.
（精製糖化液）
　本発明は、上述した糖化液を精製した精製糖化液に関するものであってもよい。例えば、精製糖化液は、上述した糖化液をイオン交換樹脂で処理することで得られる。イオン交換樹脂による処理では、上述した糖化液１００ｍｌにイオン交換樹脂（オルガノ社製、アンバージェット１０２４、ＩＲＡ９６ＳＢ）１０ｇを添加し、１室温で３０分撹拌を行う。その後、ろ別を行うことで、イオン交換樹脂をろ液を分離し、得られたろ液が精製糖化液となる。 (Purified saccharified solution)
The present invention may relate to a purified saccharified solution obtained by purifying the above-mentioned saccharified solution. For example, the purified saccharified solution can be obtained by treating the above-mentioned saccharified solution with an ion exchange resin. In the treatment with an ion exchange resin, 10 g of an ion exchange resin (Amber Jet 1024, IRA96SB, manufactured by Organo Corporation) is added to 100 ml of the above-mentioned saccharified solution, and the mixture is stirred at 1 room temperature for 30 minutes. Then, by performing filtration, the filtrate is separated from the ion exchange resin, and the obtained filtrate becomes a purified saccharified solution.
　精製糖化液における電気伝導度は、１０μＳ／ｃｍ以下であることが好ましく、８μＳ／ｃｍ以下であることがより好ましく、７μＳ／ｃｍ以下であることがさらに好ましい。なお、精製糖化液における電気伝導度は、５μＳ／ｃｍ以上であることが好ましい。ここで、精製糖化液の電気伝導度は、電気伝導率計により測定される値である。電気伝導率計としては、例えば、堀場製作所製のＬＡＱＵＡｔｗｉｎ　ＥＣ－３３Ｂを用いることができる。 The electrical conductivity of the purified saccharified solution is preferably 10 μS / cm or less, more preferably 8 μS / cm or less, and even more preferably 7 μS / cm or less. The electrical conductivity of the purified saccharified solution is preferably 5 μS / cm or more. Here, the electric conductivity of the purified saccharified liquid is a value measured by an electric conductivity meter. As the electric conductivity meter, for example, LAQUAtwin EC-33B manufactured by HORIBA, Ltd. can be used.
　精製糖化液における可溶性リグニンの含有量は、１０ｐｐｍ以下であることが好ましく、９ｐｐｍ以下であることがより好ましく、８ｐｐｍ以下であることがさらに好ましい。なお、精製糖化液における可溶性リグニンの含有量は、４ｐｐｍ以上であることが好ましい。ここで、可溶性リグニン含有量は、糖化液のＵＶスペクトル（波長２１０ｎｍ）の吸光度を測定し、下記の式で算出される値である。
　可溶性リグニン含有量（ｐｐｍ）＝希釈倍率×糖化液量×試料溶液吸光度／係数Ａ×糖化液中の固形分量
　但し、上記式中、係数Ａは、リグニンの吸光係数（１１０Ｌ／ｇ／ｃｍ）である。また、糖化液中の固形分量は、糖化液を蒸発乾固することで残存する残渣の重量により測定される値である。 The content of soluble lignin in the purified saccharified solution is preferably 10 ppm or less, more preferably 9 ppm or less, and even more preferably 8 ppm or less. The content of soluble lignin in the purified saccharified solution is preferably 4 ppm or more. Here, the soluble lignin content is a value calculated by the following formula by measuring the absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution.
Soluble lignin content (ppm) = dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution However, in the above formula, coefficient A is the extinction coefficient of lignin (110 L / g / cm). be. The solid content in the saccharified solution is a value measured by the weight of the residue remaining after the saccharified solution is evaporated to dryness.
　なお、精製糖化液における、単糖の含有量、オリゴ糖の含有量、フルフラールの含有量は、イオン交換樹脂により処理の前後でほとんど変化しないため、上述した糖化液における好ましい範囲と同様である。 The monosaccharide content, oligosaccharide content, and furfural content in the purified saccharified solution are almost the same as those in the above-mentioned saccharified solution because they hardly change before and after the treatment with the ion exchange resin.
　具体的には、精製糖化液における単糖の含有量は、１．０質量％以上であることが好ましく、２．０質量％以上であることがより好ましく、３．０質量％以上であることがさらに好ましく、４．０質量％以上であることが一層好ましく、５．０質量％以上であることがより一層好ましく、５．２質量％以上であることが特に好ましい。また、精製糖化液における単糖の含有量は、５０質量％以下であることが好ましく、３０質量％以下であることがより好ましく、２０質量％以下であることがさらに好ましく、１０質量％以下であることが特に好ましい。精製糖化液における単糖の含有量はイオンクロマトグラフィーにおいて単糖のピークを検出することで定量できる。 Specifically, the content of monosaccharide in the purified saccharified solution is preferably 1.0% by mass or more, more preferably 2.0% by mass or more, and more preferably 3.0% by mass or more. Is more preferably 4.0% by mass or more, further preferably 5.0% by mass or more, and particularly preferably 5.2% by mass or more. The content of monosaccharide in the purified saccharified solution is preferably 50% by mass or less, more preferably 30% by mass or less, further preferably 20% by mass or less, and 10% by mass or less. It is particularly preferable to have. The content of monosaccharides in the purified saccharified solution can be quantified by detecting the peak of monosaccharides by ion chromatography.
　精製糖化液におけるオリゴ糖の含有量は、１０ｐｐｍ以下であればよく、５ｐｐｍ以下であることが好ましく、１ｐｐｍ以下であることがより好ましい。なお、精製糖化液におけるオリゴ糖の含有量は０ｐｐｍであることが特に好ましい。すなわち、本発明の精製糖化液にはオリゴ糖が実質的に含まれないことが好ましい。オリゴ糖の含有量は、イオンクロマトグラフィーにより測定できる。イオンクロマトグラフィーにおいて、オリゴ糖の含有量は、単糖が溶出した後に観察されるピークとして検出され、検量線により定量できる。 The content of oligosaccharide in the purified saccharified solution may be 10 ppm or less, preferably 5 ppm or less, and more preferably 1 ppm or less. The oligosaccharide content in the purified saccharified solution is particularly preferably 0 ppm. That is, it is preferable that the purified saccharified solution of the present invention does not substantially contain oligosaccharides. The oligosaccharide content can be measured by ion chromatography. In ion chromatography, the oligosaccharide content is detected as a peak observed after the monosaccharide elutes and can be quantified by a calibration curve.
　精製糖化液におけるフルフラールの含有量は、１ｐｐｍ以下であることが好ましい。すなわち、本発明の糖化液にはフルフラールが実質的に含まれない。なお、精製糖化液中に含まれるフルフラールの含有量は、高速液体クロマトグラフィーにより測定される。 The content of furfural in the purified saccharified solution is preferably 1 ppm or less. That is, the saccharified solution of the present invention does not substantially contain furfural. The content of furfural contained in the purified saccharified solution is measured by high performance liquid chromatography.
（糖化液の製造方法）
　本発明の糖化液の製造方法は、セルロース含有バイオマスを糖化処理する工程を含む。バイオマスは、化石燃料を除いた生物由来の資源であり、セルロース含有バイオマスとしては、セルロース成分を含む生物由来の資源を挙げることができる。セルロース成分を含む生物由来の資源としては、例えば、製紙用樹木、林地残材、間伐材等のチップ又は樹皮、製材工場等から発生する鋸屑又はおがくず、街路樹の剪定枝葉、建築廃材等が挙げられる。また、草本系の資源としては、ケナフ、稲藁、麦わら、バガスなどの農産廃棄物、草本系エネルギー作物のエリアンサス、ミスカンサス、ネピアグラス等が挙げられる。さらに、セルロース含有バイオマスとして、木材由来の紙、古紙、パルプ、パルプスラッジ等も利用可能である。中でも、本発明で用いるセルロース含有バイオマスは樹木由来原料であることが好ましい。樹木由来原料の場合は、好ましくは１０～２００ｍｍ、より好ましくは５０～１００ｍｍのサイズにチップ化されていることが好ましい。すなわち、本発明の糖化液の製造方法は、チップ化されたセルロース含有バイオマスを糖化処理する工程を含むことが好ましい。 (Manufacturing method of saccharified liquid)
The method for producing a saccharified solution of the present invention includes a step of saccharifying cellulose-containing biomass. Biomass is a biological resource excluding fossil fuels, and examples of the cellulose-containing biomass include biological resources containing a cellulose component. Examples of biological resources containing cellulose components include papermaking trees, forest residue, chips or bark of thinned lumber, sawdust or sawdust generated from sawmills, pruned branches and leaves of roadside trees, construction waste, and the like. Be done. In addition, herbaceous resources include agricultural wastes such as kenaf, rice straw, straw, and bagasse, and herbaceous energy crops such as Elianthus, Miscanthus, and Napiergrass. Further, as the cellulose-containing biomass, wood-derived paper, used paper, pulp, pulp sludge and the like can also be used. Above all, the cellulose-containing biomass used in the present invention is preferably a tree-derived raw material. In the case of a tree-derived raw material, it is preferable that the chips are formed into chips having a size of preferably 10 to 200 mm, more preferably 50 to 100 mm. That is, the method for producing a saccharified solution of the present invention preferably includes a step of saccharifying the chipped cellulose-containing biomass.
　セルロースは、多数のグルコースが分子間でβ－１，４グリコシド結合して生じた鎖状高分子化合物である。セルロースにおいては、１つのグルコースの１位の水酸基と別のグルコースの４位の水酸基とが脱水縮合することにより、多数のグルコースが結合している。セルロースが加水分解（糖化）された場合、１，４－グリコシド結合が切断され、一般的には、単糖や、単糖が２～１０個程度結合したオリゴ糖が生成する。 Cellulose is a chain polymer compound formed by the β-1,4 glycoside bond between a large number of glucose molecules. In cellulose, a large number of glucoses are bound by dehydration condensation of the 1-position hydroxyl group of one glucose and the 4-position hydroxyl group of another glucose. When cellulose is hydrolyzed (saccharified), 1,4-glycosidic bonds are cleaved, and generally, monosaccharides and oligosaccharides in which about 2 to 10 monosaccharides are bound are produced.
　なお、セルロース含有バイオマスには、セルロースミクロフィブリル間に存在する多糖類であるヘミセルロースも含まれる。このため、セルロース含有バイオマスを糖化した場合、その加水分解物には、セルロース由来の糖類であるグルコースの他、ヘミセルロース由来の糖類であるキシロース、アラビノース、マンノースなども含まれる。 The cellulose-containing biomass also includes hemicellulose, which is a polysaccharide existing between cellulose microfibrils. Therefore, when cellulose-containing biomass is saccharified, the hydrolyzate includes glucose, which is a cellulose-derived saccharide, and xylose, arabinose, mannose, and the like, which are hemicellulose-derived saccharides.
　また、本発明の糖化液の製造方法は、リグニン含有量が１質量％以上３質量％以下である広葉樹クラフトパルプまたは針葉樹クラフトパルプと、水分と、セルラーゼを混合して糖化処理する工程と、糖化処理する工程で得られた処理液を限外濾過膜で膜処理する工程とを含むことが好ましい。この場合、糖化液電気伝導度が５００μＳ／ｃｍ以上であり、糖化液におけるオリゴ糖の含有量が１０ｐｐｍ以下であり、フルフラールの含有量が１ｐｐｍ以下であり、可溶性リグニンの含有量が１２０ｐｐｍ以上であり、糖化液の酵素活性が１０Ｕ／ｍｌ以下である、ことがより好ましい。 Further, the method for producing a saccharified solution of the present invention includes a step of saccharifying a broadleaf kraft pulp or a softwood kraft pulp having a lignin content of 1% by mass or more and 3% by mass or less by mixing water and cellulase, and saccharification. It is preferable to include a step of membrane-treating the treatment liquid obtained in the step of treatment with an ultrafiltration membrane. In this case, the electrical conductivity of the saccharified solution is 500 μS / cm or more, the oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 120 ppm or more. , It is more preferable that the enzymatic activity of the saccharified solution is 10 U / ml or less.
＜糖化処理＞
　糖化処理工程は、セルロース含有バイオマスに含まれる多糖類を加水分解し、単糖を得る工程である。糖化処理工程では、例えば、水分と、酵素及び／又はその酵素を生産する微生物の存在下で、セルロース含有バイオマスとの反応が行われ、好ましくは撹拌が行われる。すなわち、糖化処理工程は、酵素処理工程を含むことが好ましい。糖化処理工程においてセルロース含有バイオマスの撹拌を行う際は、酵素の種類により異なるが、撹拌液中の固形分濃度は１～３０質量％であることが好ましく、３～２０質量％であることがより好ましい。また、撹拌液の温度は３０～７５℃であることが好ましく、４０～７０℃であることがより好ましい。撹拌液のｐＨは３．０～７．０であることが好ましく、４．０～６．５であることがより好ましい。なお、糖化処理時間は、２～２００時間であることが好ましく、５～１００時間であることがより好ましい。 <Saccharification treatment>
The saccharification treatment step is a step of hydrolyzing the polysaccharide contained in the cellulose-containing biomass to obtain a monosaccharide. In the saccharification treatment step, for example, the reaction with the cellulose-containing biomass is carried out in the presence of water and an enzyme and / or a microorganism producing the enzyme, and stirring is preferably carried out. That is, the saccharification treatment step preferably includes an enzyme treatment step. When stirring the cellulose-containing biomass in the saccharification treatment step, the solid content concentration in the stirring solution is preferably 1 to 30% by mass, more preferably 3 to 20% by mass, although it depends on the type of enzyme. preferable. The temperature of the stirring liquid is preferably 30 to 75 ° C, more preferably 40 to 70 ° C. The pH of the stirring liquid is preferably 3.0 to 7.0, more preferably 4.0 to 6.5. The saccharification treatment time is preferably 2 to 200 hours, more preferably 5 to 100 hours.
　糖化処理工程で用いられる酵素は、セルロース分解酵素であることが好ましい。セルロース分解酵素は、セロビオヒドロラーゼ活性、エンドグルカナーゼ活性及びベータグルコシダーゼ活性から選択される少なくとも１つを有する酵素であり、いわゆるセルラーゼと総称される酵素である。なお、セルラーゼはヘミセルラーゼ活性を有するものであってもよい。なお、糖化処理工程として、セルロース分解酵素を使わず酸加熱分解により糖を得る場合、酸加熱分解により、セルロースと酸が反応することでフルフラールが生成され、最終的に得られる糖化液中のフルフラールの含有量が１ｐｐｍを大きく超えることとなる。 The enzyme used in the saccharification treatment step is preferably a cellulolytic enzyme. Cellulose-degrading enzyme is an enzyme having at least one selected from cellobiohydrolase activity, endoglucanase activity and beta-glucosidase activity, and is an enzyme generically called cellulase. The cellulase may have hemicellulase activity. When sugar is obtained by acid heating decomposition without using a cellulose-degrading enzyme as a saccharification treatment step, fulfural is produced by the reaction of cellulose and acid by acid heating decomposition, and fulfural in the finally obtained saccharification solution. Content will greatly exceed 1 ppm.
　セルラーゼとしては、市販のセルラーゼ製剤を用いることができる。セルラーゼ製剤としては、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属、アクレモニウム（Ａｃｒｅｍｏｎｉｕｍ）属、アスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属、ファネロケエテ（Ｐｈａｎｅｒｏｃｈａｅｔｅ）属、トラメテス（Ｔｒａｍｅｔｅｓ）属、フーミコラ（Ｈｕｍｉｃｏｌａ）属、バチルス（Ｂａｃｉｌｌｕｓ）属などに由来するセルラーゼ製剤が挙げられる。このようなセルラーゼ製剤の市販品としては、例えば、セルロイシンＴ２（エイチピィアイ社製）、メイセラーゼ（明治製菓社製）、ノボザイム１８８（ノボザイム社製）、マルティフェクトＣＸ１０Ｌ（ジェネンコア社製）、ＧＣ２２０（ジェネンコア社製）等が挙げられる。 As the cellulase, a commercially available cellulase preparation can be used. Cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Fanerochaete, the genus Trametes, the genus Trametes, the genus Humicola, and the genus Humicola. Examples of cellulase preparations to be used. Examples of commercially available cellulase preparations include cellulase T2 (manufactured by HPI), Meiserase (manufactured by Meiji Seika), Novozymes 188 (manufactured by Novozymes), Multifect CX10L (manufactured by Genecore), and GC220 (manufactured by Genecore). (Manufactured by the company) and the like.
　セルロース含有バイオマス１００質量部に対するセルラーゼ製剤の使用量は、０．５～１００質量部であることが好ましく、１～５０質量部であることがより好ましい。 The amount of the cellulase preparation used with respect to 100 parts by mass of the cellulose-containing biomass is preferably 0.5 to 100 parts by mass, and more preferably 1 to 50 parts by mass.
＜膜処理をする工程＞
　本発明の糖化液の製造方法は、糖化処理工程で得られた処理液を膜処理する工程を含むことが好ましい。 <Process of film treatment>
The method for producing a saccharified liquid of the present invention preferably includes a step of membrane-treating the treatment liquid obtained in the saccharification treatment step.
　膜処理する工程では、限外濾過（ＵＦ）膜を用いることが好ましい。限外濾過（ＵＦ）膜の素材としては、ポリエーテルスルホン（ＰＥＳ）、ポリスルホン（ＰＳ）、ポリアクリロニトリル（ＰＡＮ）、ポリフッ化ビニルデン（ＰＶＤＦ）、再生セルロース、セルロース、セルロースエステル、スルホン化ポリスルホン、スルホン化ポリエーテルスルホン、ポリオレフィン、ポリビニルアルコール、ポリメチルメタクリレート、ポリ４フッ化エチレンなどを挙げることができる。 In the process of membrane treatment, it is preferable to use an ultrafiltration (UF) membrane. Materials for ultrafiltration (UF) membranes include polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, sulfone. Examples thereof include modified polyether sulfone, polyolefin, polyvinyl alcohol, polymethylmethacrylate, polytetrafluoroethylene and the like.
　膜処理する工程では、セラミックフィルターやＭＦ膜を用いてもよい。また、膜処理する工程では、フィルタープレスによるろ過方法や遠心分離処理方法を採用することもできる。 In the process of film treatment, a ceramic filter or MF film may be used. Further, in the process of membrane treatment, a filtration method using a filter press or a centrifugation treatment method can also be adopted.
　膜処理する工程で採用される膜処理の方式は特に限定されるものではないが、例えば、膜面に対して垂直方向に処理水を供給する単純ろ過方式（全量ろ過方式）と、膜面に対して平行方向に処理水を供給するクロスフローろ過が挙げられる。膜処理する工程で得られる膜透過液には糖化液が含まれ、非透過側には酵素や原料残渣等が含まれる。なお、非透過側に含まれる酵素は、上述した糖化処理工程で再利用され得る。酵素の再利用効率を高めることで、糖化液の製造コストを抑制することが可能となる。 The membrane treatment method adopted in the membrane treatment step is not particularly limited, but for example, a simple filtration method (total filtration method) in which treated water is supplied in a direction perpendicular to the membrane surface and a membrane surface. On the other hand, cross-flow filtration in which treated water is supplied in a parallel direction can be mentioned. The membrane permeate obtained in the process of membrane treatment contains a saccharified solution, and the non-permeate side contains enzymes, raw material residues and the like. The enzyme contained on the non-permeable side can be reused in the above-mentioned saccharification treatment step. By increasing the recycling efficiency of the enzyme, it is possible to suppress the production cost of the saccharified solution.
＜その他の工程＞
　糖化処理工程と、膜処理工程との間には他の工程が設けられてもよいが、本発明においては、糖化処理工程と膜処理工程の間には他の工程を設けず、糖化処理工程の次工程として膜処理工程を設けることが好ましい。これにより、糖化液の製造工程を簡略化することが可能となり、さらに設備をコンパクトにすることも可能となる。 <Other processes>
Although another step may be provided between the saccharification treatment step and the membrane treatment step, in the present invention, no other step is provided between the saccharification treatment step and the membrane treatment step, and the saccharification treatment step It is preferable to provide a film treatment step as the next step. This makes it possible to simplify the manufacturing process of the saccharified liquid and further make the equipment compact.
＜前処理工程＞
　糖化処理工程の前には、セルロース含有バイオマスを前処理する工程を設けてもよい。セルロース含有バイオマスにはリグニンやタンパク質などが含まれるため、セルラーゼといった酵素による加水分解効率を向上させるために前処理を施してもよい。前処理の方法としては、苛性ソーダ、アンモニアなどによるアルカリ処理、蒸煮処理、化学パルプ化処理（サルファイト蒸解やクラフト蒸解等）などが挙げられる。なお、前処理工程として、セルロース含有バイオマスを水熱処理（一例として、温度１００℃以上）、酸処理または亜臨界水処理を行うと、フルフラールが生成され、最終的に得られる糖化液中のフルフラールの含有量が１ｐｐｍを大きく超える場合がある。このため、前処理工程としては、フルフラールが生成が抑制され得る工程を採用することが好ましく、例えば、苛性ソーダ、アンモニアなどによるアルカリ処理、蒸煮処理及び化学パルプ化処理（サルファイト蒸解やクラフト蒸解等）から選択される少なくとも１種を採用することが好ましい。 <Pretreatment process>
Before the saccharification treatment step, a step of pretreating the cellulose-containing biomass may be provided. Since the cellulose-containing biomass contains lignin, protein, and the like, pretreatment may be performed in order to improve the hydrolysis efficiency by an enzyme such as cellulase. Examples of the pretreatment method include alkali treatment with caustic soda, ammonia and the like, steaming treatment, chemical pulping treatment (sulfite cooking, kraft cooking, etc.) and the like. As a pretreatment step, when cellulose-containing biomass is hydroheat-treated (for example, at a temperature of 100 ° C. or higher), acid-treated or subcritical water-treated, furfural is produced, and furfural in the finally obtained saccharified solution is produced. The content may greatly exceed 1 ppm. Therefore, as the pretreatment step, it is preferable to adopt a step in which the formation of furfural can be suppressed. For example, alkali treatment with caustic soda, ammonia or the like, steaming treatment and chemical pulping treatment (sulfite cooking, craft cooking, etc.) It is preferable to adopt at least one selected from.
　前処理工程として、セルロース含有バイオマスに機械的処理を施し、セルラーゼによる加水分解効率を高めることもできる。機械的処理としては、切断、裁断、破砕、磨砕等の機械的手段が挙げられる。使用する機械装置については特に限定されないが、例えば、切出し装置、一軸破砕機、二軸破砕機、ハンマークラッシャー、レファイナー、ニーダー、ボールミル等を用いることができる。 As a pretreatment step, cellulose-containing biomass can be mechanically treated to increase the hydrolysis efficiency by cellulase. Examples of the mechanical treatment include mechanical means such as cutting, cutting, crushing, and grinding. The mechanical device to be used is not particularly limited, and for example, a cutting device, a uniaxial crusher, a biaxial crusher, a hammer crusher, a refiner, a kneader, a ball mill and the like can be used.
　また、前処理工程として、異物（石、ゴミ、金属、プラステック等の異物）を除去するために異物除去工程を設けてもよい。異物除去工程としては、例えば、洗浄工程を挙げることができる。セルロース含有バイオマスを洗浄する方法としては、例えば、セルロース含有バイオマスに水を噴射してセルロース含有バイオマスに混合されている異物を除く方法、あるいは、セルロース含有バイオマスを水中に浸漬し異物を沈降させて取り除く方法等が挙げられる。また、メタルトラップ、洗浄ドレーナー等の装置を用いて、異物をセルロース含有バイオマスから分離する方法も挙げられる。 Further, as a pretreatment step, a foreign matter removing step may be provided to remove foreign matter (foreign matter such as stone, dust, metal, plastic, etc.). Examples of the foreign matter removing step include a cleaning step. As a method for cleaning the cellulose-containing biomass, for example, a method of spraying water on the cellulose-containing biomass to remove foreign substances mixed in the cellulose-containing biomass, or a method of immersing the cellulose-containing biomass in water to settle and remove the foreign substances. The method and the like can be mentioned. Another method is to separate foreign matter from cellulose-containing biomass by using a device such as a metal trap or a washing drainer.
　さらに、前処理工程として、殺菌処理工程を設けてもよい。セルロース含有バイオマスに雑菌が混入していると糖化処理工程で雑菌が糖を消費して生成物の収量が低下する場合がある。殺菌処理工程は酸やアルカリなど、菌の生育困難なｐＨに原料を晒す方法でもよいが、高温下で処理する方法を採用してもよい。 Further, a sterilization treatment step may be provided as a pretreatment step. If germs are mixed in the cellulose-containing biomass, the germs may consume sugar in the saccharification treatment step and the yield of the product may decrease. The sterilization treatment step may be a method of exposing the raw material to a pH at which bacteria are difficult to grow, such as acid or alkali, but a method of treating at a high temperature may be adopted.
＜濃縮工程＞
　膜処理工程の後には、膜透過液を濃縮する工程を設けてもよい。濃縮する工程では、膜透過液に含まれる糖化液の濃度を高める。例えば、膜透過液を加熱して水分を蒸散させることで濃縮された糖化液を得ることができる。 <Concentration process>
After the membrane treatment step, a step of concentrating the membrane permeate may be provided. In the step of concentration, the concentration of the saccharified solution contained in the membrane permeate is increased. For example, a concentrated saccharified solution can be obtained by heating the membrane permeate to evaporate the water content.
（精製糖化液の製造方法）
　本発明は、上述した工程で得られる糖化液を、さらにイオン交換樹脂で処理する工程を含む精製糖化液の製造方法に関するものであってもよい。上述したように、糖化液の電気伝導度は５００μＳ／ｃｍ以上であり、糖化液におけるオリゴ糖の含有量は１０ｐｐｍ以下であり、フルフラールの含有量は１ｐｐｍ以下であり、可溶性リグニンの含有量は１２０ｐｐｍ以上であり、糖化液の酵素活性は１０Ｕ／ｍｌ以下である。 (Manufacturing method of purified saccharified liquid)
The present invention may relate to a method for producing a purified saccharified solution, which comprises a step of further treating the saccharified solution obtained in the above-mentioned step with an ion exchange resin. As described above, the electrical conductivity of the saccharified solution is 500 μS / cm or more, the oligosaccharide content in the saccharified solution is 10 ppm or less, the furfural content is 1 ppm or less, and the soluble lignin content is 120 ppm. As described above, the enzymatic activity of the saccharified solution is 10 U / ml or less.
　精製糖化液の製造方法で得られる精製糖化液の電気伝導度は１０μＳ／ｃｍ以下であることが好ましく、８μＳ／ｃｍ以下であることがより好ましく、７μＳ／ｃｍ以下であることがさらに好ましい。なお、精製糖化液の製造方法で得られる精製糖化液の電気伝導度は、５μＳ／ｃｍ以上であることが好ましい。ここで、精製糖化液の電気伝導度は、電気伝導率計により測定される値である。電気伝導率計としては、例えば、堀場製作所製のＬＡＱＵＡｔｗｉｎ　ＥＣ－３３Ｂを用いることができる。 The electrical conductivity of the purified saccharified solution obtained by the method for producing the purified saccharified solution is preferably 10 μS / cm or less, more preferably 8 μS / cm or less, and further preferably 7 μS / cm or less. The electrical conductivity of the purified saccharified solution obtained by the method for producing the purified saccharified solution is preferably 5 μS / cm or more. Here, the electric conductivity of the purified saccharified liquid is a value measured by an electric conductivity meter. As the electric conductivity meter, for example, LAQUAtwin EC-33B manufactured by HORIBA, Ltd. can be used.
　精製糖化液の製造方法で得られる精製糖化液の可溶性リグニンの含有量は、１０ｐｐｍ以下であることが好ましく、９ｐｐｍ以下であることがより好ましく、８ｐｐｍ以下であることがさらに好ましい。なお、精製糖化液の製造方法で得られる可溶性リグニンの含有量は、４ｐｐｍ以上であることが好ましい。ここで、可溶性リグニン含有量は、糖化液のＵＶスペクトル（波長２１０ｎｍ）の吸光度を測定し、下記の式で算出される値である。
　可溶性リグニン含有量（ｐｐｍ）＝希釈倍率×糖化液量×試料溶液吸光度／係数Ａ×糖化液中の固形分量
　但し、上記式中、係数Ａは、リグニンの吸光係数（１１０Ｌ／ｇ／ｃｍ）である。また、糖化液中の固形分量は、糖化液を蒸発乾固することで残存する残渣の重量により測定される値である。 The content of soluble lignin in the purified saccharified solution obtained by the method for producing the purified saccharified solution is preferably 10 ppm or less, more preferably 9 ppm or less, and further preferably 8 ppm or less. The content of soluble lignin obtained by the method for producing a purified saccharified solution is preferably 4 ppm or more. Here, the soluble lignin content is a value calculated by the following formula by measuring the absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution.
Soluble lignin content (ppm) = dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution However, in the above formula, coefficient A is the extinction coefficient of lignin (110 L / g / cm). be. The solid content in the saccharified solution is a value measured by the weight of the residue remaining after the saccharified solution is evaporated to dryness.
（用途）
　本発明の糖化液は、発酵原料として好ましく用いられる。本発明の糖化液が発酵原料として用いられる場合、糖化液は、発酵工程に供され、発酵液となる。発酵液はエタノールであることが特に好ましい。本発明の糖化液におけるオリゴ糖の含有量は１０ｐｐｍ以下であり、フルフラールの含有量は１ｐｐｍ以下であるため、糖化液を発酵する際に発酵効率がよい。このため、本発明の糖化液を用いて発酵液（エタノール）を生成した際には、高いエタノール収量が達成される。また、本発明の糖化液は上記構成を有するものであるため、発酵液（エタノール）のエタノール純度が高く、不純物の含有量を少なくすることができるため、例えば、エタノールを飲料用として用いる場合には、雑味等を抑えることができる。 (Use)
The saccharified solution of the present invention is preferably used as a fermentation raw material. When the saccharified solution of the present invention is used as a fermentation raw material, the saccharified solution is subjected to a fermentation step and becomes a fermentation broth. The fermentation broth is particularly preferably ethanol. Since the content of oligosaccharide in the saccharified solution of the present invention is 10 ppm or less and the content of furfural is 1 ppm or less, fermentation efficiency is good when fermenting the saccharified solution. Therefore, when a fermented liquid (ethanol) is produced using the saccharified liquid of the present invention, a high ethanol yield is achieved. Further, since the saccharified solution of the present invention has the above-mentioned structure, the ethanol purity of the fermentation broth (ethanol) is high and the content of impurities can be reduced. Therefore, for example, when ethanol is used for beverages. Can suppress miscellaneous taste and the like.
　上記のように、本発明の糖化液は発酵原料用の糖化液であることが好ましく、エタノール生成用の糖化液であることがより好ましく、飲料用エタノール生成用の糖化液であることがさらに好ましい。また、本発明は、上記糖化液を含む発酵原料に関するものであってもよく、上記糖化液を発酵させてなる発酵液に関するものであってもよく、上記糖化液を発酵させてなるエタノールに関するものであってもよく、上記糖化液を発酵させてなる飲料用エタノールに関するものであってもよい。 As described above, the saccharified solution of the present invention is preferably a saccharified solution for fermentation raw materials, more preferably a saccharified solution for ethanol production, and further preferably a saccharified solution for producing ethanol for beverages. .. Further, the present invention may be related to a fermentation raw material containing the saccharified solution, may be related to a fermented solution obtained by fermenting the saccharified solution, or may be related to ethanol obtained by fermenting the saccharified solution. It may be related to ethanol for drinking obtained by fermenting the saccharified solution.
　以下に実施例と比較例を挙げて本発明の特徴をさらに具体的に説明する。以下の実施例に示す材料、使用量、割合、処理内容、処理手順等は、本発明の趣旨を逸脱しない限り適宜変更することができる。したがって、本発明の範囲は以下に示す具体例により限定的に解釈されるべきものではない。 The features of the present invention will be described in more detail below with reference to Examples and Comparative Examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the present invention. Therefore, the scope of the present invention should not be construed as limited by the specific examples shown below.
（実施例１）
＜糖化処理＞
　水８０ｍＬに、リグニンの含有量が２質量％の酸素晒広葉樹クラフトパルプ５ｇ（乾燥重量、酸素晒広葉樹クラフトパルプはサイズ５０～１００ｍｍにチップ化されている）を添加し、撹拌してパルプスラリーを調製した。次いで、このパルプスラリーに１Ｎ硫酸を加えてｐＨを５．０に調整し、セルラーゼ溶液（ジェネンコア社製、マルティフェクトＣＸ１０Ｌ）２ｍＬと、水を添加することで最終容量が１００ｍＬとなるように調製した。該溶液を５０℃で４８時間静置した後、上清を限外濾過膜（ミリポア社製、セントリコンプラス７０遠心フィルターユニット）でろ過することで糖化液を得た。 (Example 1)
<Saccharification treatment>
To 80 mL of water, add 5 g of oxygen-bleached hardwood kraft pulp having a lignin content of 2% by mass (dry weight, oxygen-bleached hardwood kraft pulp is chipped to a size of 50 to 100 mm), and stir to make a pulp slurry. Prepared. Next, 1N sulfuric acid was added to this pulp slurry to adjust the pH to 5.0, and 2 mL of a cellulase solution (Multifect CX10L manufactured by Genencore) and water were added to prepare the final volume to 100 mL. .. The solution was allowed to stand at 50 ° C. for 48 hours, and then the supernatant was filtered through an ultrafiltration membrane (Centricon Plus 70 centrifugal filter unit manufactured by Millipore) to obtain a saccharified solution.
（実施例２）
　糖化処理に使用する原料として、リグニンの含有量が２質量％の酸素晒針葉樹クラフトパルプ５ｇ（乾燥重量）を使用した以外は実施例１と同様に処理を行い、糖化液を得た。 (Example 2)
A saccharified solution was obtained in the same manner as in Example 1 except that 5 g (dry weight) of oxygen-bleached softwood kraft pulp having a lignin content of 2% by mass was used as a raw material used for the saccharification treatment.
（比較例１）
　糖化処理に使用する原料として、リグニンの含有量が０．３質量％の晒広葉樹クラフトパルプ５ｇ（乾燥重量）を使用した以外は実施例１と同様に処理を行い、糖化液を得た。 (Comparative Example 1)
A saccharified solution was obtained in the same manner as in Example 1 except that 5 g (dry weight) of bleached hardwood kraft pulp having a lignin content of 0.3% by mass was used as a raw material used for the saccharification treatment.
（比較例２）
　糖化処理に使用する原料として、リグニンの含有量が０．３質量％の晒針葉樹クラフトパルプ５ｇ（乾燥重量）を使用した以外は実施例１と同様に処理を行い、糖化液を得た。 (Comparative Example 2)
A saccharified solution was obtained in the same manner as in Example 1 except that 5 g (dry weight) of bleached softwood kraft pulp having a lignin content of 0.3% by mass was used as a raw material used for the saccharification treatment.
（比較例３）
＜酸加水分解処理＞
　リグニンの含有量が２質量％の酸素晒広葉樹クラフトパルプ３００ｍｇ（乾燥重量）に７２％の硫酸３ｍｌを添加し、３０℃の水浴中で１時間処理した後、イオン交換水８４ｍｌを添加した。次いで、１２０℃、１時間の条件でオートクレーブ処理し、１Ｇ－３ガラスフィルターでろ過することで糖化液を得た。 (Comparative Example 3)
<Acid hydrolysis treatment>
To 300 mg (dry weight) of oxygen-bleached hardwood kraft pulp having a lignin content of 2% by mass, 3 ml of 72% sulfuric acid was added, treated in a water bath at 30 ° C. for 1 hour, and then 84 ml of ion-exchanged water was added. Then, it was autoclaved at 120 ° C. for 1 hour and filtered through a 1G-3 glass filter to obtain a saccharified solution.
（比較例４）
＜でん粉糖化処理＞
　水８０ｍＬに、王子コーンスターチ社製のトウモロコシ由来でん粉５ｇ（乾燥重量）を添加し、撹拌してでん粉スラリーを調製した。次いで、このでん粉スラリーに１Ｎ硫酸を加えてｐＨを５．０に調整し、アミラーゼ溶液（三菱ケミカルフーズ社製、コクラーゼ）２ｍＬと、水を添加することで最終容量が１００ｍＬとなるように調製した。該溶液を５０℃で４８時間静置した後、上清を限外濾過膜（ミリポア社製、セントリコンプラス７０遠心フィルターユニット）でろ過することで糖化液を得た。 (Comparative Example 4)
<Starch saccharification treatment>
To 80 mL of water, 5 g (dry weight) of corn-derived starch manufactured by Oji Cornstarch was added and stirred to prepare a starch slurry. Next, 1N sulfuric acid was added to this starch slurry to adjust the pH to 5.0, and 2 mL of an amylase solution (Coclase manufactured by Mitsubishi Chemical Foods Co., Ltd.) and water were added to prepare the final volume to 100 mL. .. The solution was allowed to stand at 50 ° C. for 48 hours, and then the supernatant was filtered through an ultrafiltration membrane (Centricon Plus 70 centrifugal filter unit manufactured by Millipore) to obtain a saccharified solution.
（測定及び評価）
＜セルロース含有バイオマス（パルプ）中のリグニン含有量測定＞
　ＪＩＳ　Ｐ　８２１１：２０１１パルプカッパー価試験方法に準拠してセルロース含有バイオマス（パルプ）のカッパー価を測定し、下記の式でリグニン含有量を算出した。
　リグニン含有量（質量％）＝カッパー価ｘ０．１５ (Measurement and evaluation)
<Measurement of lignin content in cellulose-containing biomass (pulp)>
The copper value of cellulose-containing biomass (pulp) was measured according to the JIS P 8211: 2011 pulp copper value test method, and the lignin content was calculated by the following formula.
Lignin content (% by mass) = copper value x 0.15
＜電気伝導度＞
　実施例及び比較例で得られた糖化液の電気伝導度を電気伝導率計（堀場製作所　ＬＡＱＵＡｔｗｉｎ　ＥＣ－３３Ｂ）で測定した。 <Electrical conductivity>
The electric conductivity of the saccharified liquids obtained in Examples and Comparative Examples was measured with an electric conductivity meter (HORIBA, Ltd. LAQUAtwin EC-33B).
＜オリゴ糖及び単糖の含有量＞
　イオンクロマトグラフィー装置（ダイオネクス社製、ＩＣＳ－２０００）を用いて、オリゴ糖分析モードにて糖化液中に含まれるオリゴ糖及び単糖の含有量を測定した。 <Contents of oligosaccharides and monosaccharides>
Using an ion chromatography device (ICS-2000 manufactured by Dionex Co., Ltd.), the contents of oligosaccharides and monosaccharides contained in the saccharified solution were measured in the oligosaccharide analysis mode.
＜フルフラール含有量＞
　高速液体クロマトグラフィー装置（アジレント社製、ＨＰ－２２００）を用いて糖化液中に含まれるフルフラールの含有量を測定した。 <Furfural content>
The content of furfural contained in the saccharified solution was measured using a high performance liquid chromatography device (HP-2200, manufactured by Agilent).
＜可溶性リグニン含有量＞
　糖化液のＵＶスペクトル（波長２１０ｎｍ）の吸光度を測定し、下記の式で可溶性リグニン含有量を算出した。
　可溶性リグニン含有量（ｐｐｍ）＝希釈倍率×糖化液量×試料溶液吸光度／係数Ａ×糖化液中の固形分量
　但し、上記式中、係数Ａは、リグニンの吸光係数（１１０Ｌ／ｇ／ｃｍ）である。 <Soluble lignin content>
The absorbance of the UV spectrum (wavelength 210 nm) of the saccharified solution was measured, and the soluble lignin content was calculated by the following formula.
Soluble lignin content (ppm) = dilution ratio x amount of saccharified solution x sample solution extinction / coefficient A x amount of solids in saccharified solution However, in the above formula, coefficient A is the extinction coefficient of lignin (110 L / g / cm). be.
＜酵素残存活性＞
　実施例及び比較例で得られた糖化液４ｍｌに、１００ｍＭ酢酸緩衝液（ｐＨ５）４ｍｌと１．２５ｍＭ　４－メチルフンベリフェリルβ－Ｄグルコシド（和光社製）３２ｍｌを添加し、３７℃で３０分間反応させた。０．５Ｍグリシン－ＮａＯＨ溶液（ｐＨ１０．５）２００ｍｌで反応を停止させ、蛍光光度計（テカン社製　インフィニット２００）で蛍光強度（励起波長３５５ｎｍ、測定波長４６０ｎｍ）を測定した。糖化液１ｍｌ当たり、１分間に１ｍＭの４－メチルウンベリフェロンが遊離した場合の酵素残存活性を１Ｕ／ｍｌとして、酵素残存活性を算出した。 <Enzyme residual activity>
To 4 ml of the saccharified solution obtained in Examples and Comparative Examples, 4 ml of 100 mM acetate buffer (pH 5) and 32 ml of 1.25 mM 4-methylhumberiferyl β-D glucoside (manufactured by Wako Co., Ltd.) were added, and 30 at 37 ° C. Reacted for minutes. The reaction was stopped with 200 ml of a 0.5 M glycine-NaOH solution (pH 10.5), and the fluorescence intensity (excitation wavelength 355 nm, measurement wavelength 460 nm) was measured with a fluorometer (Infinite 200 manufactured by Tecan). The enzyme residual activity was calculated assuming that the enzyme residual activity when 1 mM 4-methylumbelliferone was released per 1 ml of the saccharified solution was 1 U / ml.
＜エタノール収量＞
　実施例及び比較例で得られた糖化液５０ｍｌにコーンスティープリカー（王子コーンスターチ社製）２ｍｌ、１Ｍ酢酸バッファー（ｐＨ５）５ｍｌを添加した。次いで、前培養した酵母液（サッカロマイセス・セレビシエ　１×１０８ｃｅｌｌｓ／ｍＬ）を５ｍｌ添加し、３３℃で１８時間保持した。反応後の培養液中のエタノール量を、高速液体クロマトグラフィー装置（アジレント社製　ＨＰ－２２００）を用いて測定し、単位セルロース含有バイオマス量（ｋｇ）あたりのエタノール生産量を算出した。 <Ethanol yield>
To 50 ml of the saccharified solution obtained in Examples and Comparative Examples, 2 ml of corn steep liquor (manufactured by Oji Corn Starch) and 5 ml of 1M acetate buffer (pH 5) were added. Then, 5 ml of pre-cultured yeast solution (Saccharomyces cerevisiae 1 × 10 8 cells / mL) was added, and the mixture was kept at 33 ° C. for 18 hours. The amount of ethanol in the culture solution after the reaction was measured using a high performance liquid chromatography device (HP-2200 manufactured by Azilent Co., Ltd.), and the amount of ethanol produced per unit cellulose-containing biomass amount (kg) was calculated.
＜イオン交換樹脂処理＞
　実施例及び比較例で得られた糖化液１００ｍｌにイオン交換樹脂（オルガノ社製　アンバージェット１０２４、ＩＲＡ９６ＳＢ）１０ｇを添加し、１室温で３０分撹拌した後、ろ別した。得られたろ液は、精製糖化液であり、精製糖化液についても糖化液と同様に、上述した方法で各種分析を行った。 <Ion exchange resin treatment>
To 100 ml of the saccharified solution obtained in Examples and Comparative Examples, 10 g of an ion exchange resin (Amber Jet 1024 manufactured by Organo Corporation, IRA96SB) was added, and the mixture was stirred at 1 room temperature for 30 minutes and then filtered. The obtained filtrate was a purified saccharified solution, and the purified saccharified solution was subjected to various analyzes by the above-mentioned method in the same manner as the saccharified solution.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　なお、上記表における「ＮＤ（Ｎｏｔ　Ｄｅｔｅｃｔｅｄ）」とは、測定限界値以下であることを示している。具体的には、オリゴ糖の測定における測定限界値は１ｐｐｍであり、フルフラールの測定における測定限界値は０．１ｐｐｍであり、可溶性リグニンの測定における測定限界値は１ｐｐｍであり、電気伝導度の測定における測定限界値は０．２μＳ／ｃｍである。
　また、上記表における比較例３における「－」は、酵素未添加であることを表し、比較例４における「－」は、原料がデンプンであり、リグニンが含まれていないことを示している。 In addition, "ND (Not Detected)" in the above table indicates that it is not more than a measurement limit value. Specifically, the measurement limit value in the measurement of oligosaccharide is 1 ppm, the measurement limit value in the measurement of furfural is 0.1 ppm, the measurement limit value in the measurement of soluble lignin is 1 ppm, and the measurement of electrical conductivity. The measurement limit value in is 0.2 μS / cm.
Further, "-" in Comparative Example 3 in the above table indicates that no enzyme was added, and "-" in Comparative Example 4 indicates that the raw material was starch and did not contain lignin.
　実施例では、酵素残存量の少ない糖化液が得られた。また、実施例で得られた糖化液を発酵させることでエタノールを製造した場合、高いエタノール収量が得られた。 In the examples, a saccharified solution having a small residual amount of enzyme was obtained. Further, when ethanol was produced by fermenting the saccharified solution obtained in the example, a high ethanol yield was obtained.

Claims (5)

  1. 　セルロース含有バイオマスを原料とする糖化液であって、
    　前記糖化液の電気伝導度が５００μＳ／ｃｍ以上であり、
    　前記糖化液におけるオリゴ糖の含有量が１０ｐｐｍ以下であり、フルフラールの含有量が１ｐｐｍ以下であり、可溶性リグニンの含有量が１００ｐｐｍ以上である糖化液。     A saccharified solution made from cellulose-containing biomass.
    The electrical conductivity of the saccharified solution is 500 μS / cm or more.
    A saccharified solution having an oligosaccharide content of 10 ppm or less, a furfural content of 1 ppm or less, and a soluble lignin content of 100 ppm or more in the saccharified solution.
  2. 　前記セルロース含有バイオマスにおけるリグニンの含有量が１質量％以上３質量％以下である請求項１に記載の糖化液。 The saccharified solution according to claim 1, wherein the content of lignin in the cellulose-containing biomass is 1% by mass or more and 3% by mass or less.
  3. 　前記セルロース含有バイオマスが樹木由来原料である請求項１又は２に記載の糖化液。 The saccharified solution according to claim 1 or 2, wherein the cellulose-containing biomass is a tree-derived raw material.
  4. 　前記糖化液の酵素活性が１０Ｕ／ｍｌ以下である請求項１～３のいずれか１項に記載の糖化液。 The saccharified solution according to any one of claims 1 to 3, wherein the enzyme activity of the saccharified solution is 10 U / ml or less.
  5. 　請求項１～４のいずれか１項に記載の糖化液をイオン交換樹脂で処理して得られる精製糖化液。 A purified saccharified solution obtained by treating the saccharified solution according to any one of claims 1 to 4 with an ion exchange resin.
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