JP2014083003A - Sugar producing method via process of removing enzyme inhibitor - Google Patents

Abstract

To provide a method for producing a saccharide capable of sufficiently removing a cellulase activity-inhibiting substance by a simple process and obtaining a saccharide in a high yield.
The present invention relates to a method for producing sugar from cellulosic biomass, in which cellulosic biomass is added to water and boiled, a cooling step of cooling the boiled first mixed liquid, and cooling. A hydrolyzing enzyme is added to the first mixed solution, and the first decomposition step in which hydrolysis is performed by stirring at a temperature of 30 to 50 ° C. and the first mixed solution are separated into a water-soluble fraction and a water-insoluble fraction. A separation step, a contact step of bringing the water-soluble fraction into contact with the adsorption carrier to make a purified water-soluble fraction, and adding the purified water-soluble fraction to the water-insoluble fraction to form a second mixed solution And a second decomposition step of hydrolyzing the second mixed solution by stirring at a temperature of 30 to 50 ° C., and contacting the water-soluble fraction with the adsorption carrier in the contact step, whereby cellulase activity Inhibitors adhere to the adsorption carrier and are excluded from the water-soluble fraction. It is a manufacturing method of that sugar.
[Selection] Figure 1

Description

  The present invention relates to a method for producing sugar. More specifically, the present invention relates to a method for producing sugar through a step of removing an enzyme inhibitor.

About 70% of Japan is surrounded by forests, and Japan has abundant unused cellulosic biomass such as cedar, rice straw and wheat straw.
Such unused cellulosic biomass is the most important on earth not only for the production of biomass ethanol from non-food resources but also for the production of sugar as a raw material for food, chemical industry, and fermentation after the depletion of petroleum resources. It is known that it can be a good carbon source (for example, see Non-Patent Document 1).

  Cellulose biomass existing in nature has a structure in which cellulose, hemicellulose and lignin are intertwined in a complex manner. For this reason, in general, in saccharification, physical pulverization by a cutter mill or the like, structural destruction of lignin and cellulose / hemicellulose by alkali treatment (for example, see Non-Patent Document 2), etc. are performed, and then glycoenzyme treatment Is done.

  However, the saccharified solution obtained by saccharifying cellulosic biomass by performing these steps cannot be said to have a sufficient concentration (yield). For this reason, even if ethanol fermentation etc. were performed using this saccharified liquid (for example, refer to patent documents 1), it has not reached the level which can actually be used in terms of fermentation efficiency (for example, refer to nonpatent documents 3 or 4). .

This is because, in the saccharification reaction by cellulase, the activity of β-glucosidase is not only inhibited by the product cellulose-derived glucose, cellobiose, hemicellulose-derived mannose, galactose, and xylose (for example, see Non-Patent Document 5). ), By-products such as xylan, soluble starch, polysaccharides such as pectin, carbohydrates such as xylose, arabinose, glucuronic acid, furfural, vanillin, syringaldehyde, trans-cinnamic acid, 4-hydroxybenzoic acid, etc. This is probably because the activities of cellulase and β-glucosidase are inhibited (see, for example, Non-Patent Document 6). That is, since these substances are gradually released from the cellulosic biomass during the hydrolysis reaction and inhibit the activity of cellulase, the yield of sugar becomes insufficient.
Moreover, since the activity of cellulase is inhibited, there is a drawback that a large amount of cellulase is required for hydrolysis.

On the other hand, a method for removing a substance that inhibits the activity of cellulase in the production process of sugar from cellulosic biomass has been studied.
For example, in a hydrolyzate that hydrolyzes polysaccharide-based biomass, treats the resulting hydrolyzate with a wood-based carbide, and removes fermentation inhibitors contained in the hydrolyzate or substances that would inhibit fermentation. There are known methods for removing fermentation inhibitors (for example, see Patent Document 2) and methods for obtaining a sugar solution by adsorbing or holding a fermentation inhibitor on a polystyrene resin from a solution containing sugar and a fermentation inhibitor. (For example, refer to Patent Document 3).

  In addition to this, a method for promoting a hydrolysis reaction by separating low molecules such as glucose by ultrafiltration during hydrolysis (for example, Non-Patent Document 7), and a method for removing a fermentation inhibitor by microorganisms (For example, Non-Patent Document 8) is known.

JP 2004-337099 A JP 2005-270056 A JP 2011-78327 A

J. Nowak et al., “Composite structure of woodcells petrified wood.”, “Materials Science and Engineering”, 2005, C, 25, p. 119-130 Y. Yamashita et al., “Effective enzymes accharification and ethanol production from Japanese cedar using various pretreatment methods.”, “Journal of Bioscience and Bioengineering”, 2010, 110, p. 79-86 Kosuke Kimoto et al., “Bioethanol production process from wood-based raw materials”, Mitsui Engineering & Shipbuilding Technical Report, 2010, 201, p. 24-29 Kenji Asano et al., “Bioethanol Production Costs and CO2 Reduction Cost Analysis in Japan” [online], July 2007, AIST / BTRC DISCUSSION PAPER, Internet <URL: https://unit.aist.go.jp/btrc /research_result/documents/DISCUSSIONPAPER.pdf> Z. Xiao et al., “Effect of Sugar Inhibition on Cellulases and β-Glucosidase During Enzymatic Hydrolysis of Softwood Substrates.”, “Applied Biochemistry and Biotechnology”, 2004, 113-116, p. 1115-1126 E. Ximenes et al., “Inhibitor of cellulases by phenols.”, “Enzyme and Microbial Technology”, 2010, 46, p. 170-176 P. Andric ’et al.,“ Effect and Modeling of Glucose Inhibition and In Situ Glucose RemovalDuring Enzymatic Hydrolysis of Pretreated Wheat Straw. ”,“ Applied Biochemistry and Biotechnology ”, 2010, 160, 280-297. N. Nichols et al., “Bioabatement to Remove Inhibitors from Biomass-Derived Sugar Hydrolysates.”, “Applied Biochemistry and Biotechnology”, 2005, 121-124, 379-390.

  However, in the methods described in Patent Documents 2 and 3 described above, it cannot be said that removal of the fermentation inhibitor is sufficient and the yield of sugar is not excellent. In addition, the method described in Non-Patent Document 7 has a disadvantage that a fermentation inhibitor is also collected at the same time as glucose. In the method described in Non-Patent Document 8, a large amount of microorganisms are required, and the microorganisms are removed before fermentation. There is a drawback in that a removal step is required.

  The present invention has been made in view of the above circumstances, and can sufficiently remove a substance that inhibits the activity of cellulase (hereinafter referred to as “cellulase activity inhibitory substance”) with a simple process and has a high yield. It aims at providing the manufacturing method of the saccharide | sugar which can obtain saccharide | sugar by this.

  The present inventors have intensively studied to solve the above-mentioned problems. As a result, the first decomposition step is performed, the water-soluble fraction of the first mixed solution is brought into contact with the adsorption carrier, and then the purified water-soluble fraction is obtained. It was found that the above problem can be solved by returning the minute amount to the second dividing step, and the present invention has been completed.

  The present invention is (1) a method for producing sugar from cellulosic biomass, which comprises adding a cellulosic biomass to water and boiling, a cooling step for cooling the boiled first mixture, 1st decomposition process which hydrolyzes by adding a hydrolase to 1 liquid mixture, and stirs at the temperature of 30-50 degreeC, The isolation | separation which isolate | separates a 1st liquid mixture into a water-soluble fraction and a water-insoluble fraction A step of contacting the water-soluble fraction with an adsorption carrier to form a purified water-soluble fraction, and adding the purified water-soluble fraction to the water-insoluble fraction to form a second mixed solution; A second decomposition step of hydrolyzing the second mixed solution by stirring at a temperature of 30 to 50 ° C., and contacting the water-soluble fraction with the adsorption carrier in the contact step, thereby inhibiting cellulase activity Sugars that are attached to the adsorption carrier and excluded from the water-soluble fraction It resides in the production method.

  The present invention resides in the sugar production method according to the above (1), further comprising (2) an extraction step of separating the saccharified solution from the second mixed solution and extracting the sugar after the second decomposition step.

  This invention exists in the manufacturing method of the saccharide | sugar as described in said (1) or (2) to which (3) 1st decomposition process and 2nd decomposition process are performed continuously.

  In the present invention, (4) any one of the above (1) to (3), wherein the adsorption carrier is at least one selected from the group consisting of celite, diatomaceous earth, bentonite, silica gel, activated carbon, alumina, and calcium carbonate. It exists in the manufacturing method of the described sugar.

  This invention exists in the manufacturing method of the saccharide | sugar as described in any one of said (1)-(3) whose adsorption carrier is ion exchange resin (5).

  This invention exists in the manufacturing method of the saccharide | sugar as described in said (5) whose ion exchange resin is weakly basic ion exchange resin.

  The saccharide according to any one of (1) to (6), wherein (7) the contacting step is performed by filling the column with an adsorption carrier and passing the water-soluble fraction through the column. Exist in the manufacturing method.

  This invention exists in the manufacturing method of the saccharide | sugar as described in any one of said (1)-(7) whose cellulase activity inhibitory substance is a hydroxyl-containing compound and / or a carboxylic acid compound.

  The present invention relates to (9) the sugar according to any one of (1) to (8), wherein the boiling step is performed under an inert gas atmosphere at a temperature of 100 to 150 ° C. and a pressure of 0.1 to 5 MPa. Exist in the manufacturing method.

  The present invention relates to (10) a method for producing ethanol by adding yeast to the sugar obtained by the method for producing sugar according to any one of (1) to (9) above to biodegrade the sugar into ethanol. .

  The present invention is (11) Production of lactic acid by fermenting sugar to lactic acid by adding lactic acid-fermenting bacteria to the sugar obtained by the sugar production method according to any one of (1) to (9) above. Method.

In the sugar production method of the present invention, sugar can be produced by simple steps of a boiling step, a cooling step, a first decomposition step, a separation step, a contact step, and a second decomposition step.
In the sugar production method of the present invention, the cellulase activity-inhibiting substance is attached to the adsorption carrier by subjecting the water-soluble fraction of the first mixed solution to the adsorption carrier by performing the first decomposition step. The activity inhibitory substance can be sufficiently removed. As a result, the activity of cellulase is sustained, so that the amount of cellulase used can be reduced as compared with the conventional case.

  In the method for producing sugar of the present invention, the purified water-soluble fraction from which the cellulase activity inhibitor is excluded is combined with the original water-insoluble fraction and subjected to the second splitting step, so that the sugar can be obtained in high yield. Can be obtained. In addition, when a 1st decomposition process and a 2nd decomposition process are performed continuously, it becomes possible to manufacture sugar more easily.

  In the method for producing sugar of the present invention, a sugar with higher purity can be obtained when an extraction step for separating the saccharified solution from the second mixed solution and extracting the sugar is provided after the second decomposition step.

In the sugar production method of the present invention, when the adsorption carrier is an ion exchange resin, there are advantages in that the production efficiency is good and the affinity with the cellulase activity inhibitor is excellent.
At this time, when the ion exchange resin is a weakly basic ion exchange resin, the sugar can pass through and a wide variety of cellulase activity inhibitors can be attached. Therefore, the cellulase activity inhibitor can be effectively used from the water-soluble fraction. Can be removed.
Moreover, when the cellulase activity inhibitor is a hydroxyl group-containing compound and / or a carboxylic acid compound, the cellulase activity inhibitor can be reliably removed by the adsorption carrier.

  In the sugar production method of the present invention, when the contacting step is performed by filling an adsorption carrier into a column and passing a water-soluble fraction through the column, the contacting step can be performed efficiently and easily. .

  In the sugar production method of the present invention, when the boiling step is performed under an inert gas atmosphere at a temperature of 100 to 150 ° C. and a pressure of 0.1 to 5 MPa, cellulosic biomass can be sufficiently defibrated. At the same time, micropores on the order of several tens of nm are formed. Thereby, the yield of sugar can also be improved.

  In the ethanol production method of the present invention, since the sugar obtained by the above production method is used, the yield is drastically improved.

  In the method for producing lactic acid of the present invention, since the sugar obtained by the above-described production method is used, the yield is drastically improved.

FIG. 1 is a flowchart showing a sugar production method according to the first embodiment. FIG. 2 is a flowchart showing a sugar production method according to the second embodiment. FIG. 3 is a graph showing the relationship between saccharification time and glucose production rate or glucose concentration in Example 1. FIG. 4 is a graph showing the relationship between saccharification time and glucose production rate or glucose concentration in Comparative Example 1. FIG. 5 is a graph showing the relationship between fermentation time (h) and ethanol concentration in Example 2, Comparative Example 2 and the control. FIG. 6 is a graph showing the relationship between fermentation time (h) and lactic acid concentration in Example 3, Comparative Example 3, and control.

In the present invention, cellulosic biomass is used as a raw material.
Here, the cellulosic biomass is obtained by converting a polysaccharide composed of cellulose and hemicellulose into a tree by lignin.
Examples of the cellulosic biomass include agricultural, forestry and fishery resources such as hardwoods and conifers, or wastes of the agricultural, forestry and fishery resources.
More specifically, examples of the cellulosic biomass include bagasse, rice straw, corn stover, oil palm empty fruit bunch, wood fiber, wood chip, veneer scrap, wood flour, pulp, and waste paper. Cellulosic biomass includes sugar resources such as sugar cane and sugar beet, starch resources such as potato, sugar beet, sugar beet, and the like, those having a low tannin and lignin content, and those not containing lignin. Moreover, these raw materials may be used individually by 1 type, or may mix and use 2 or more types.

  Among these, the cellulosic biomass is preferably eucalyptus wood flour (broadleaf tree), cedar wood flour (coniferous tree), bagasse, rice straw, corn stover, and oil palm empty fruit bunch. In these cases, the reason is not clear, but it is easy to defibrate and sugar can be obtained in a relatively high yield.

The cellulose refers to a mixture of a polymer in which glucose is polymerized by a β-1,4 glucoside bond, a derivative obtained by carboxymethylation, aldehyde formation, esterification, or the like. In addition, in this invention, it is preferable that the polymerization degree of glucose in a cellulose is 200 or more from a viewpoint of a saccharification rate.
Cellulose may contain cellooligosaccharides and cellobiose, which are partially decomposed products, and may be crystalline or non-crystalline. In the present invention, the cellulose is preferably crystalline cellulose.
As described above, cellulose is not particularly limited as long as it is derived from biomass, and may be derived from plants, fungi, or bacteria.

[First Embodiment]
FIG. 1 is a flowchart showing a sugar production method according to the first embodiment.
As shown in FIG. 1, in the sugar production method according to the first embodiment, a boiling step S1 in which cellulosic biomass is added to water and boiled, a cooling step S2 in which the boiled first mixed liquid is cooled, and cooling are performed. The hydrolyzing enzyme is added to the first mixed liquid, the first decomposition step S3 for hydrolysis, the separation step S4 for separating the first mixed liquid into a water-soluble fraction and a water-insoluble fraction, and water-soluble Contacting step S5, wherein the fraction is brought into contact with an adsorption carrier to form a purified water-soluble fraction, and the purified water-soluble fraction is added to the water-insoluble fraction to form a second mixed solution, the second mixed solution And a second decomposition step S6 for hydrolyzing, and an extraction step S7 for separating the saccharified solution from the second mixed solution and extracting the sugar.

In the sugar production method according to the first embodiment, the boiling process S1, the cooling process S2, the first decomposition process S3, the separation process S4, the contact process S5, the second decomposition process S6, and the extraction process S7 are relatively simple processes. Thus, sugar can be produced.
In addition, by applying the first decomposition step S3 and bringing the water-soluble fraction of the first mixed solution into contact with the adsorption carrier, the cellulase activity inhibiting substance is attached to the adsorption carrier, and the cellulase activity inhibiting substance is sufficiently removed. can do. As a result, the activity of cellulase is sustained, so that the amount of cellulase used can be reduced as compared with the conventional case.
Hereinafter, each step will be described in more detail.

(Boiling process)
Boiling process S1 is a process of boiling the 1st liquid mixture which added the cellulose biomass to water.
By performing boiling process S1, the entanglement of the lignin of cellulose biomass, cellulose, and hemicellulose is unwound, and it becomes a fibrous form. Thereby, the surface area of a cellulosic biomass increases and it becomes easy to contact an enzyme in the decomposition | disassembly process mentioned later.
At the same time, water-soluble components contained in the cellulosic biomass are eluted.

  In the boiling step S1, the first mixed liquid may contain a water-soluble organic solvent and / or an organic acid. In this case, even under relatively mild conditions, the cellulosic biomass can be fibrillated into a fibrous form.

Here, examples of the water-soluble organic solvent include alcohols such as methanol and ethanol, polyhydric alcohols such as glycerin and ethylene glycol, aprotic polar solvents such as dimethyl sulfoxide, dimethylformamide, and N, N-dimethylacetamide. A water-soluble organic solvent having a specific heat lower than that of water may be mentioned.
Among these, it is preferable that the water-soluble organic solvent is an alcohol. In this case, the entanglement of lignin with cellulose and hemicellulose can be further solved. Moreover, since alcohols are harmless to the human body, they are excellent in safety.

Examples of the organic acid include acetic acid, oxalic acid, formic acid, succinic acid, lactic acid, malic acid, tartaric acid, and citric acid.
Among these, acetic acid, oxalic acid or formic acid is preferable. Since these organic acids have a low boiling point, they can be easily removed by boiling.

  The blending ratio of the cellulosic biomass and water is preferably 1: 1 to 10 by mass ratio. In this case, the entanglement of lignin with cellulose and hemicellulose can be further solved.

When the water-soluble organic solvent and the organic acid are further mixed, the mixing ratio of water, the water-soluble organic solvent, and the organic acid is (water: water-soluble organic solvent: organic acid), 2-30 mass%: 65-97. .8 mass%: It is preferable that it is 0.2-5 mass%.
When the blending ratio of the organic acid exceeds 5% by mass, the hemicellulose component may be excessively decomposed compared to the case where the blending ratio of the organic acid is within the above range, and the blending ratio of water is 30% by mass. When it exceeds%, the hemicellulose component may be excessively decomposed as compared with the case where the mixing ratio of water is within the above range.

The 1st liquid mixture which added the cellulose biomass to water is boiled for 10 to 60 minutes at the temperature of 100-150 degreeC.
At this time, when the temperature is less than 100 ° C., compared to the case where the temperature is within the above range, boiling of the first mixed solution becomes insufficient, and sugar cannot be obtained at a high saccharification rate. On the other hand, when the temperature exceeds 150 ° C., compared to the case where the temperature is within the above range, some sugars are decomposed, and therefore sugar cannot be obtained at a high saccharification rate.

The boiling step is preferably performed in a pressure vessel. The inside of the pressure vessel at this time is preferably saturated with steam, and the pressure is more preferably 1 to 5 times the saturated vapor pressure.
Specifically, the pressure in the pressure vessel is preferably 1 to 5 MPa.
When the pressure is less than 1 MPa, it takes time to boil the first mixed liquid as compared with the case where the pressure is in the above range.

  The inside of the pressure vessel is preferably preliminarily placed in an inert gas atmosphere. Examples of the inert gas include nitrogen, argon, helium and the like. In this case, decomposition of the cellulosic biomass due to oxidation or the like is suppressed.

  In addition, when a boiling process is performed on conditions with a temperature of 100-150 degreeC and a pressure of 0.1-5 MPa in inert gas atmosphere, a cellulose biomass can fully be fibrillated in a 1st liquid mixture. At the same time, micropores on the order of several tens of nm are formed. Thereby, the yield of saccharide | sugar can be improved very much.

(Cooling process)
Cooling process S2 is a process of cooling the 1st liquid mixture boiled in boiling process S1.

  Cooling process S2 is performed by air-cooling a 1st liquid mixture to room temperature. The cooling method is not limited to air cooling, and may be positively cooled with ice, a refrigerator, or the like.

(First decomposition step)
The first decomposition step S3 is a step of adding a hydrolase to the first mixed liquid cooled in the cooling step S2 to perform hydrolysis.
In the first decomposition step S3, the cellulosic biomass is hydrolyzed by the enzymatic saccharification method.
In the first decomposition step S3, part of the cellulosic biomass is saccharified to be eluted in water. At this time, the cellulase activity inhibitor is also released from the cellulosic biomass and eluted into water.

Cellulase is used as the hydrolase.
Such cellulase means an enzyme that decomposes cellulose into glucose or the like.
Examples of such enzymes include, but are not limited to, for example, Acremonium, Trichoderma, Fusarium, Phanerochaete, Tremetes, Peneticium, (Penicillium), Humicola, Aspergillus, Murakia, Murakiella, etc., in addition to these, Clostridium, Pseudomonas bacteria, Cellulomonas bacteria, Ruminococcus bacteria, Bacillus bacteria, etc. Bacteria, Sulfolobus genus (Sulfolobus) bacteria, the genus Streptomyces (Streptomyces) bacteria, the actinomycetes, such as thermo-actinomycin My genus (Thermoactinomyces) bacteria and the like. These enzymes may be artificially modified. Moreover, these enzymes may be used individually by 1 type, or may mix and use 2 or more types.
The cellulase may be a series of enzyme groups.
Such enzyme groups include endoglucanase (EC 3.2.1.74), cellobiohydrolase (EC 3.2.1.91), β-glucosinase (EC 23.2.4.1, EC 3.2). 1.21) and the like.
It is preferable to use a mixture of cellulases derived from different microorganisms. In this case, saccharification of cellulose can be further promoted by their synergistic effect.

  Hydrolysis in the first decomposition step S3 is performed by adjusting the first mixed solution to which the hydrolase is added to pH 3.5 to 5.5 with a buffer and treating the mixture at a temperature of 30 to 50 ° C. for 1 to 50 hours. Done.

The hydrolysis reaction in the first decomposition step S3 may be performed in a batch system (batch system) or in a continuous system using a bioreactor containing an immobilized enzyme.
Moreover, as a reactor used for 1st decomposition process S3, a fixed bed reactor, a fluidized bed reactor, a powder flow phase reactor etc. are mentioned, A fixed bed reactor is used preferably.

  In 1st decomposition process S3, in addition to a hydrolase, you may add an acid to a 1st liquid mixture. In this case, hydrolysis with an acid is also performed at the same time. That is, the enzymatic saccharification method and the acid saccharification method are simultaneously performed on the cellulosic biomass. For this reason, saccharification is promoted more. In addition, it is preferable that addition of this acid does not deviate from the range of pH 3.5-5.5 mentioned above.

(Separation process)
Separation process S4 is a process of isolate | separating a 1st liquid mixture into a water-soluble fraction and a water-insoluble fraction. At this time, the water-soluble fraction contains a sugar and a cellulase activity inhibitor. In the separation step S4, it is not necessary to strictly separate the water-soluble fraction and the water-insoluble fraction. That is, the water-insoluble fraction may partially contain a sugar or a cellulase activity inhibitor.

  In the separation step S4, a method for separating the water-soluble component and the water-insoluble component is not particularly limited, and for example, filtration, suction filtration, decantation, centrifugation, and the like are performed.

(Contact process)
The contact step S5 is a step in which the water-soluble fraction is brought into contact with the adsorption carrier to obtain a purified water-soluble fraction. That is, in the contact step S5, the cellulase activity inhibitor is attached to the adsorption carrier and excluded from the water-soluble fraction by bringing the water-soluble fraction into contact with the adsorption carrier.

  Here, as the adsorption carrier, celite, diatomaceous earth, bentonite, silica gel, activated carbon, alumina, calcium carbonate, etc. inorganic carrier, ceramic powder, polyvinyl alcohol, polypropylene, chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, synthetic adsorption An organic carrier such as a resin can be used.

Among these, the adsorption carrier is preferably at least one selected from the group consisting of celite, diatomaceous earth, bentonite, silica gel, activated carbon, alumina, and calcium carbonate, and more preferably celite.
Moreover, it is preferable that an adsorption carrier is an ion exchange resin from a viewpoint of production efficiency and affinity with a cellulase activity inhibitory substance.

Here, a weakly basic ion exchange resin is preferably used as the ion exchange resin. In this case, sugar can be passed and various cellulase activity inhibitors can be attached. Thereby, the cellulase activity inhibitor is effectively removed from the water-soluble fraction.
Specific examples of weakly basic ion exchange resins include phenol formaldehyde, polystyrene, acrylamide, and divinylbenzene.

The average particle size of the adsorption carrier is preferably less than 300 μm from the viewpoint of improving the reaction rate.
The adsorption carrier is preferably porous. The shape of the adsorption carrier is not particularly limited, and examples thereof include particles, powders, granules, fibers, and sponges.

Examples of the cellulase activity inhibitory substance include a compound containing a hydroxyl group derived from a component of cellulosic biomass (hereinafter referred to as “hydroxyl group-containing compound”), a carboxylic acid compound, and the like.
For example, cellulose, hemicellulose-derived substances containing hydroxyl groups such as melibiose, trehalose, maltose, arabinose, isositol, rhamnose, lactose, inulin, starch, ribitol, xylan, cellobiose, xylose, pullulan, xylitol, fucose, raffinose, mannose, arabitol Examples thereof include carboxylic acid compounds such as compounds, formic acid, glucuronic acid and gluconic acid.
Examples of lignin / tannin-derived substances include hydroxyl group-containing compounds such as vanillin, and carboxylic acid compounds such as vanillic acid, tannic acid, caffeic acid, p-hydroxyphenylacetic acid, trimesic acid, gallic acid, ferulic acid, and glycolic acid.
Examples of coniferous lignin-derived substances include hydroxyl-containing compounds such as methyl guaiacol, ethyl guaiacol, vinyl guaiacol, eugenol, propyl guaiacol, isoeugenol, acetoguaiacon, propioguaiacon, guaiacylacetone, and dihydroconiphenyl alcohol.
As lignin-derived substances of hardwood (eucalyptus wood flour), hydroxyl-containing compounds such as synapyl alcohol, ethyl syringol, vinyl syringol, propyl syringol, allyl syringol, propenyl syringol, acetic acid, tannic acid, gallic acid, etc. A carboxylic acid compound is mentioned.
In addition to the hydroxyl group-containing compound and the carboxylic acid compound, the adsorption carrier can also attach furan compounds such as furfural produced when hydrolyzing lignocellulosic biomass.

  In the contacting step S5, a method for bringing the water-soluble fraction into contact with the adsorption carrier is not particularly limited. For example, the method may be performed by filling the column with the adsorption carrier and passing the water-soluble fraction through the column. . In this case, the contact step S5 can be performed efficiently and easily.

(Second decomposition step)
In the second decomposition step S6, the purified water-soluble fraction obtained in the contact step S5 is added to the water-insoluble fraction separated in the decomposition step S4 to form a second mixed solution, and the second mixed solution is hydrolyzed. It is a process.
In the second decomposition step S6, the cellulosic biomass is hydrolyzed by the enzymatic saccharification method.

Here, since the hydrolase is generally insoluble in water, the water-insoluble fraction separated in the decomposition step S4 includes the hydrolase added in the first decomposition step S3. That is, in the second decomposition step S6, a hydrolase is contained only by combining the purified water-soluble fraction and the water-insoluble fraction, and the hydrolase can be used as it is. From this, in the sugar manufacturing method according to the first embodiment, the amount of hydrolase used can be reduced.
Naturally, a hydrolase may be newly added separately in the second decomposition step S6. Again, cellulase is used as the hydrolase.

  In the second decomposition step S6, undecomposed cellulosic biomass is saccharified to be eluted in water. Note that most of the cellulase activity inhibitor is already liberated from the cellulosic biomass in the first decomposition step S3 and hardly eluted in water in the second decomposition step S6.

  Hydrolysis in the second decomposition step S6 is performed by adjusting the second mixed solution to pH 3.5 to 5.5 with a buffer and treating it at a temperature of 30 to 50 ° C. for 24 hours or more.

The hydrolysis reaction in the second decomposition step S6 may be performed in a batch system (batch system) or in a continuous system using a bioreactor containing an immobilized enzyme.
Moreover, as a reactor used for 2nd decomposition process S6, a fixed bed reactor, a fluidized bed reactor, a powder flow phase reactor etc. are mentioned, A fixed bed reactor is used preferably.

  In the second decomposition step S6, an acid may be added to the second mixed solution. In this case, since the enzymatic saccharification method and the acid saccharification method are performed simultaneously, saccharification is further promoted. In addition, it is preferable that addition of this acid does not deviate from the range of pH 3.5-5.5 mentioned above.

  In the sugar production method according to the first embodiment, the second splitting step S6 is performed on the second mixed solution in which the purified water-soluble fraction from which the cellulase activity inhibitor is excluded is combined with the original water-insoluble fraction. It is possible to obtain a saccharified solution with a high yield.

The obtained saccharified solution can be used as a medium raw material or a carbon source for producing useful materials by biotechnology.
In addition, the saccharides in the saccharified solution can be used by chemically converting them into useful substances such as chemical products, polymer raw materials, and physiologically active substances.
Specifically, it can be suitably used for applications such as ethanol raw materials, lactic acid raw materials, bioethanol raw materials, and biodiesel production.

(Extraction process)
The extraction step S7 is a step of separating the saccharified solution from the second mixed solution obtained in the second decomposition step S6 and extracting the sugar.
By performing extraction process S7, it becomes possible to obtain sugar with higher purity.

The method for extracting the sugar is not particularly limited. For example, the saccharified solution can be obtained as a filtrate by separating impurities by suction filtration.
And the obtained saccharified liquid is dried and sugar is obtained by extracting with water from the dried components.
Incidentally, the sugar thus obtained includes an oligosaccharide composed of a pentasaccharide obtained by hydrolysis of hemicellulose. Oligosaccharides can be used as functional food materials after purification.

(Ethanol production method)
The sugar obtained by the sugar production method according to the first embodiment can be converted to ethanol by a fermentation method.
Specifically, by adding yeast to the obtained sugar and reacting in a thermostatic bath at about 30 ° C., the sugar can be biodegraded to ethanol.
According to the ethanol production method, since the sugar obtained by the above-mentioned sugar production method is used, the yield is drastically improved.

  The ethanol obtained can be used as a raw material for chemical products, a solvent, or a fuel for automobiles by purification. Moreover, the aqueous solution containing ethanol can also be used as an alcoholic beverage.

(Manufacturing method of lactic acid) The saccharide obtained by the saccharide manufacturing method according to the first embodiment may be obtained by adding lactic acid fermentation bacteria and reacting in a thermostatic bath at about 30 ° C. to ferment the sugar into lactic acid. it can.
According to the method for producing lactic acid, since the sugar obtained by the aforementioned method for producing sugar is used, the yield is drastically improved.

[Second Embodiment]
FIG. 2 is a flowchart showing a sugar production method according to the second embodiment.
As shown in FIG. 2, in the method for producing sugar according to the second embodiment, the first decomposition step S3a, the separation step S4a, and the second decomposition step S6a are continuously performed in the same system. This is the same as the sugar manufacturing method according to the first embodiment. That is, the sugar production method according to the second embodiment includes a boiling step S1 for boiling, a cooling step S2 for cooling the boiled first mixed solution, a hydrolase added to the cooled first mixed solution, While the first decomposition step S3a hydrolyzing in the system and the first decomposition step S3a are being performed, the first mixed liquid in the system is separated into a water-soluble fraction and a water-insoluble fraction. The separation step S4a and the water-soluble fraction are brought into contact with an adsorption carrier to make a purified water-soluble fraction, and the purified water-soluble fraction is returned to the original system (water-insoluble fraction). A second decomposition step S6a for hydrolyzing the second mixed solution (returned to minutes), and an extraction step S7 for separating the saccharified solution from the second mixed solution and extracting the sugar.

  In the sugar manufacturing method according to the second embodiment, since the first decomposition step S3a and the second decomposition step S6a are continuously performed, it is possible to manufacture sugar more easily. In addition, what was used in the 1st decomposition | disassembly process S3a is used continuously for the hydrolase in a 2nd decomposition | disassembly process. Naturally, a hydrolase may be newly added separately in the second decomposition step S6a. Again, cellulase is used as the hydrolase.

  In the sugar manufacturing method according to the second embodiment, it is preferable that the separation step S4a and the contact step S5a are continuously performed. By circulating the water-soluble fraction in these steps, the cellulase activity inhibitor can be excluded more and the saccharification reaction of the cellulosic biomass can be performed more efficiently.

  The preferred embodiment of the present invention has been described above, but the present invention is not limited to the above embodiment.

  For example, in the sugar production method according to the present embodiment, the contacting step S5 is performed by filling an adsorption carrier into a column and passing a water-soluble fraction through the column, but includes cellulosic biomass. It may be added to an aqueous solution, and the cellulase activity inhibitor may be attached to the adsorption carrier while stirring the entire solution.

  In the sugar manufacturing method according to the present embodiment, the extraction step S7 is performed, but the extraction step S7 is not necessarily an essential step. That is, you may use the 2nd liquid mixture obtained by 2nd decomposition process S6 as it is.

In the sugar production method according to the present embodiment, the diameter of the cellulosic biomass used in the boiling step S1 is preferably 10 mm or less, more preferably 5 mm or less, and even more preferably 2 mm or less.
Moreover, when the diameter of cellulosic biomass is larger than 10 mm, it is preferable to perform a mechanochemical process before performing boiling process S1.

Here, the mechanochemical process is a process in which the cellulosic biomass is pulverized to have a diameter of 10 mm or less, preferably 5 mm or less, more preferably 2 mm or less.
In the mechanochemical process, the surface area of the cellulosic biomass is increased by pulverizing the cellulosic biomass. If it does so, it will become easy to immerse a cellulose biomass in a chemical | medical solution in boiling process S1.

The mechanochemical process is performed using a grinding machine.
Examples of such a pulverizing machine include a cutter mill, a vibration ball mill, a rotating ball mill, a planetary ball mill, a roll mill, a disk mill, a high-speed rotating blade type mixer, and a homomixer.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example.

Example 1
In the hydrolysis reaction vessel, 800 ml of 20 mM citrate buffer (pH 5.0) was added to 200 g of mecanomi chemical-treated eucalyptus wood flour (cellulose-based biomass) and heated at 121 ° C. for 30 minutes (boiling step).
After cooling to room temperature (cooling step), cellulase (manufactured by Amano Enzyme Inc., T-4) 3FPU / g and β-glucosidase (Genencore, Optimash BG) 6 μl / g were added, and the mixture was heated at 37 ° C. for 24 hours. Stirring (120 rpm) was performed to conduct a hydrolysis reaction (first decomposition reaction).
After the hydrolysis reaction, the reaction solution was recovered and separated into a water-soluble fraction and a water-insoluble fraction by suction filtration (separation step).
The water-soluble fraction was passed through a column packed with an ion exchange resin (Dow Chemical Co., Dowex Marathon WBA, adsorption carrier) to remove the cellulase activity inhibitor (contacting step).
The purified water-soluble fraction that has passed through the ion-exchange resin and the water-insoluble fraction are returned to the hydrolysis reaction vessel again, and cellulase 6FPU / g and β-glucosidase 12 μl / g are added to the hydrolysis reaction to 96. The time was continued (second decomposition step).
After completion of the hydrolysis reaction, the residue was separated again by suction filtration to obtain a saccharified solution (extraction step).

(Comparative Example 1)
A saccharified solution was obtained in the same manner as in Example 1 except that the water-soluble fraction was not treated with an ion exchange resin (no contact step was performed).

(Evaluation)
In Example 1 and Comparative Example 1, a part of the reaction solution is sampled every 24 hours immediately after the start of the reaction, and after boiling for 3 minutes to completely deactivate the enzyme, centrifugation is performed at 15000 rpm for 10 minutes. The water-soluble fraction was recovered.
And about the collect | recovered water-soluble fraction, glucose concentration was measured by the high performance liquid chromatography (HPLC), and glucose production rate was computed.
The quantitative conditions for glucose were as follows: detector: Tosoh RI-8020, column: TSK-gel G2500PWXL, TSK-gel G-3000PWXL, TSK-gel G6000PWXL, mobile phase: ion-exchanged water, flow rate: 0.5 ml / min, Column temperature: 60 ° C.
A graph showing the relationship between saccharification time and glucose production rate or glucose concentration in Example 1 is shown in FIG. 3, and a graph showing the relationship between saccharification time and glucose production rate or glucose concentration in Comparative Example 1 is shown in FIG.
In FIG. 3, after removing the cellulase activity inhibitor under the conditions of Example 1, the glucose production rate when the hydrolysis reaction is carried out is indicated by “◯”, and the glucose concentration is indicated by “Δ”.
In FIG. 4, the glucose production rate when the hydrolysis reaction is performed under the conditions of Comparative Example 1 is indicated by “◯”, and the glucose concentration is indicated by “Δ”.

From the results shown in FIG. 3, the reaction solution passed through the ion exchange resin of Example 1 does not show a decrease in glucose production rate even after 48 hours from the start of the reaction. A 4.5% (w / v) saccharified solution was obtained.
On the other hand, from the results shown in FIG. 4, it was confirmed that the reaction liquid that did not allow the ion exchange resin to pass through in Comparative Example 1 significantly decreased the cellulase activity after 48 hours from the start of the reaction. A saccharified solution having a concentration of about 3.2% (w / v) was obtained.
From these results, it was confirmed that the glucose concentration in the saccharified solution was increased by about 1.4 times compared to the conventional method according to the sugar production method of the present invention.

(Example 2)
The saccharified solution obtained in Example 1 was frozen and concentrated to adjust the glucose concentration to 20% (w / v). Then, yeast extract and malt extract are respectively added to this saccharified solution to 5% (w / v), and 1 platinum ear (10 μl) of Saccharomyces cerevisiae is added to this solution, followed by stirring (120 rpm) at 27 ° C. By culturing, ethanol was obtained.

(Comparative Example 2)
Instead of using the saccharified solution obtained in Example 1, ethanol was obtained in the same manner as in Example 2 except that the saccharified solution obtained in Comparative Example 1 was used.

(Control)
Instead of using the saccharified solution obtained in Example 1, ethanol was obtained in the same manner as in Example 2 except that 20% (w / v) commercially available glucose was used.

(Evaluation)
In Example 2, Comparative Example 2 and the control, the culture solution collected every 24 hours after the start of culture was centrifuged at 15000 rpm for 1 minute, and the supernatant was collected. The collected supernatant was passed through a 0.2 μm filter to completely remove the cells, and the ethanol concentration was measured using high performance liquid chromatography (HPLC).
The ethanol quantification conditions were as follows: detector: Tosoh RI-8020, column: TSK-gel G2500PWXL, TSK-gel G-3000PWXL, TSK-gel G6000PWXL, mobile phase: ion-exchanged water, flow rate 0.5 ml / min, column temperature: 60 C.
FIG. 5 shows a graph of the relationship between fermentation time (h) and ethanol concentration in Example 2, Comparative Example 2 and the control.
In FIG. 5, the ethanol concentration obtained in Example 2 is indicated by “Δ”, the ethanol concentration obtained in Comparative Example 2 is indicated by “□”, and the ethanol concentration obtained by the control is indicated by “●”.

From the results shown in FIG. 5, ethanol fermentation using the saccharified solution of Example 2 was maximized 48 hours after the start of the culture.
Moreover, as a result of measuring the ethanol concentration at 48 hours of culturing, ethanol fermentation using the saccharified solution of Comparative Example 2 showed a fermentation efficiency of about 37.3% compared to the control, whereas the saccharified solution of Example 2 In the ethanol fermentation using No. 1, the fermentation efficiency was about 84.4% compared with the control.
Furthermore, when the ethanol production amount in ethanol fermentation of Example 2 and Comparative Example 2 is compared, ethanol fermentation using the saccharified solution of Example 2 is about 2 compared to ethanol fermentation using the saccharified solution of Comparative Example 2. A 26-fold amount of ethanol was obtained.

(Example 3)
The saccharified solution obtained in Example 1 was freeze-concentrated and adjusted so that the glucose concentration was 5% (w / v). And yeast extract was added to this saccharified liquid so that it might become 2.5% (w / v), it put into the jar fan meter, and it was set as temperature 30 degreeC and pH 9.0 under argon gas atmosphere. Lactic acid was obtained by adding the pre-cultured Enterococcus caseiflavus L-120 strain to this solution and culturing with stirring (180 rpm) at pH 9.0 and 30 ° C. for 72 hours.

(Comparative Example 3)
Lactic acid was obtained in the same manner as in Example 3 except that the saccharified solution obtained in Comparative Example 1 was used instead of the saccharified solution obtained in Example 1.

(Control)
Lactic acid was obtained in the same manner as in Example 3, except that 5% (w / v) of commercially available glucose was used instead of using the saccharified solution obtained in Example 1.

(Evaluation)
In Example 2, Comparative Example 2 and the control, the culture solution collected every 24 hours after the start of culture was centrifuged at 15000 rpm for 1 minute, and the supernatant was collected. The collected supernatant was passed through a 0.2 μm filter to completely remove the cells, and the lactic acid concentration was measured using high performance liquid chromatography (HPLC).
The lactic acid quantification conditions were as follows: Detector: 2420 UV-VIS Detector (440 nm) manufactured by Hitachi High-Tech, column: GL-A180-S + guard column, eluent: ₃ mM HClO ₃ , flow rate: 0.5 ml / min, reaction solution: BTB solution The flow rate was 0.6 ml / min, and the column temperature was 40 ° C.
FIG. 6 shows a graph of the relationship between the fermentation time (h) and the lactic acid concentration in Example 3, Comparative Example 3 and the control.
In FIG. 6, the lactic acid concentration obtained in Example 3 is indicated by “Δ”, the lactic acid concentration obtained in Comparative Example 3 is indicated by “◇”, and the lactic acid concentration obtained by the control is indicated by “●”.

From the results shown in FIG. 6, lactic acid fermentation using the saccharified solution of Example 3 was maximized 48 hours after the start of the culture.
In addition, as a result of measurement of the lactic acid concentration at 48 hours after culturing, the lactic acid fermentation using the saccharified solution of Comparative Example 3 showed a fermentation efficiency of about 73.3% compared to the control, whereas the saccharified solution of Example 3 In the lactic acid fermentation using No. 1, the fermentation efficiency was about 96.1% compared with the control.
Furthermore, when the amount of lactic acid produced in lactic acid fermentation of Example 3 and Comparative Example 3 is compared, lactic acid fermentation using the saccharified solution of Example 3 is about 1 compared to lactic acid fermentation using the saccharified solution of Comparative Example 3. A 31-fold amount of lactic acid was obtained.

  As described above, according to the present invention, sugar can be produced at a sufficiently high saccharification rate even under relatively mild conditions using cellulosic biomass as a raw material, and by using this, ethanol and lactic acid can be produced in a sufficiently high yield. It was confirmed that can be manufactured.

(Reference experiment)
1. Confirmation of Cellulase Activity Inhibition The effect of cellulose activity inhibitors shown in Table 1 on cellulase activity was investigated.
The enzyme shown in Table 1 is added to 100 mM citrate buffer (pH 5.0) containing 20 mM p-nitrophenylglucopyranoside, and the concentration of the cellulose activity inhibitor shown in Table 1 is 6% (w / v). The enzyme reaction was performed at 30 ° C. for 10 minutes. In addition, 6% (w / v) sterilized distilled water was used as a control group.
And relative activity was computed as shown below.
Relative activity (%) = p-nitrophenol production in compound (μmol / ml) × 100 / p-nitrophenol production in sterile distilled water (μmol / ml)
In Table 1, “T4 (Amano)” is cellulase (T-4 manufactured by Amano Enzyme Inc.), and “Ctec (Novozyme)” is cellulase (manufactured by Novozymes A / S, Cellic Ctec). “A3 (Amano)” is cellulase (A-3, manufactured by Amano Enzyme Co., Ltd.).
The obtained results are shown in Table 1.

(Table 1)

  As shown in Table 1, it was found that the enzyme activity of each cellulase was strongly inhibited by carboxylic acid compounds such as tannic acid, trimesic acid, gallic acid, gluconic acid, glycolic acid, and acetic acid.

2. Cellulase activity inhibitor produced in Example 1 and Comparative Example 1 In the hydrolysis reaction of Example 1 and Comparative Example 1, tannic acid, trimesic acid, and gallic acid contained in the reaction solution were quantified over time. . In Example 1, the treatment (contact process) is performed with an ion exchange resin after 24 hours.
The quantification of the aromatic carboxylic acid was carried out using a detector: 2420 UV-VIS Detector (254 nm) manufactured by Hitachi High-Tech, column: Wakosil-II 5C8 RS + guard column, mobile phase: acetonitrile-50 mM potassium phosphate buffer (pH 4.0). (10:90), flow rate: 1.0 ml / min, column temperature: 40 ° C.
Table 2 shows the quantification results of the cellulase activity inhibitor contained in the saccharified solution of Example 1, and Table 3 shows the quantification results of the cellulase activity inhibitor contained in the saccharified solution of Comparative Example 1.

(Table 2)

(Table 3)

As shown in Table 3, tannic acid, trimesic acid, and gallic acid, which are cellulase activity inhibitors, were found to elute continuously as the hydrolysis reaction progressed.
On the other hand, as shown in Table 2, it was confirmed that after treatment with the ion exchange resin of Example 1 (after 48 hours), the amount of the cellulase activity inhibitor was greatly reduced. That is, it was found that the content of the three compounds in the saccharified solution can be reduced by about 60 to 90% by the treatment of the adsorption carrier.

The method for producing saccharides of the present invention can be used for the production of saccharides that can be used as raw materials for media or carbon sources for the production of useful materials by biotechnology.
According to the above sugar production method, the cellulase activity-inhibiting substance can be sufficiently removed by a simple process, and the sugar can be obtained in a high yield.

S1 ... boiling step S2 ... cooling step S3, S3a ... first decomposition step S4, S4a ... separation step S5, S5a ... contact step S6, S6a ... second decomposition step S7 ..Extraction process

Claims (11)

A method for producing sugar from cellulosic biomass,
A boiling step of adding cellulosic biomass to water and boiling;
A cooling step for cooling the boiled first mixture;
Adding a hydrolase to the cooled first mixed liquid and hydrolyzing the mixture by stirring at a temperature of 30 to 50 ° C .;
A separation step of separating the first mixture into a water-soluble fraction and a water-insoluble fraction;
Contacting the water-soluble fraction with an adsorption carrier to obtain a purified water-soluble fraction;
A second decomposition step in which the purified water-soluble fraction is added to the water-insoluble fraction to form a second mixed solution, and the second mixed solution is hydrolyzed by stirring at a temperature of 30 to 50 ° C .;
With
A method for producing a saccharide, wherein in the contacting step, the cellulase activity inhibitor is attached to the adsorption carrier and excluded from the water-soluble fraction by bringing the water-soluble fraction into contact with the adsorption carrier.   The method for producing sugar according to claim 1, further comprising an extraction step of separating the saccharified solution from the second mixed solution and extracting the sugar after the second decomposition step.   The method for producing sugar according to claim 1 or 2, wherein the first decomposition step and the second decomposition step are successively performed.   The method for producing sugar according to any one of claims 1 to 3, wherein the adsorption carrier is at least one selected from the group consisting of celite, diatomaceous earth, bentonite, silica gel, activated carbon, alumina, and calcium carbonate.   The method for producing sugar according to any one of claims 1 to 3, wherein the adsorption carrier is an ion exchange resin.   The method for producing sugar according to claim 5, wherein the ion exchange resin is a weakly basic ion exchange resin.   The sugar production method according to any one of claims 1 to 6, wherein the contacting step is performed by filling the adsorption carrier into a column and passing the water-soluble fraction through the column.   The method for producing sugar according to any one of claims 1 to 7, wherein the cellulase activity inhibitor is a hydroxyl group-containing compound and / or a carboxylic acid compound.   The method for producing sugar according to any one of claims 1 to 8, wherein the boiling step is performed under conditions of a temperature of 100 to 150 ° C and a pressure of 0.1 to 5 MPa in an inert gas atmosphere.   The manufacturing method of ethanol which biodegrades the said saccharide | sugar by adding yeast to the saccharide | sugar obtained by the saccharide | sugar manufacturing method of any one of Claims 1-9. A method for producing lactic acid, wherein a lactic acid-fermenting bacterium is added to the sugar obtained by the method for producing a sugar according to any one of claims 1 to 9, thereby fermenting the sugar to produce lactic acid.

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