JP5190858B2 - Production method of low molecular weight carbohydrates from materials containing polysaccharides - Google Patents

Description

  The present invention relates to a technique for effectively using a polysaccharide, particularly a material containing a hardly degradable polysaccharide, and specifically to a technique for producing a low molecular weight carbohydrate or the like from a material containing a hardly biodegradable polysaccharide derived from a living body. Furthermore, the present invention relates to advanced utilization technology of carbohydrate biomass targeting ethanol and petroleum substitute products.

As environmental policies and defossil resources progress on a global scale, the mixing of bioethanol into gasoline has become a policy goal in countries around the world, and the development of energy conversion technology for starch and sucrose has been promoted rapidly. ing. In Brazil, 11 million kiloliters of ethanol is made from sugarcane sucrose, and in the United States, over 10 million kiloliters of ethanol is produced using glucose produced by saccharification of corn starch. In Thailand, India, etc., plans have been made to dramatically increase the scale of ethanol production using sugarcane, cassava and the like.
However, most of the plant fiber biomass produced as a by-product from sugarcane, cereals, potatoes, etc. as sucrose squeezed residue and starch extraction residue is burned as a highly septic residue while leaving a small amount of sucrose and starch. Currently, it is disposed of by stagnation.

In general, plant fiber biomass contains many useful carbohydrates such as xylose constituting hemicellulose, galacturonic acid constituting arabinose and pectin in addition to glucose constituting cellulose, and in particular glucose obtained mainly from cellulose. Is extremely useful as a raw material for fermentation and petroleum substitutes.
Under such circumstances, in the United States, a comprehensive technology development project for ethanol conversion of cellulosic resources is being led by the US Department of Energy. In Brazil, ethanol fermentation technology from cellulose is being developed.
However, due to the low decomposition efficiency of cellulose and the like, an effective utilization technique at an economically viable level has not been developed at present.

For example, in the United States, a method of performing enzymatic saccharification after alkali / hydrogen peroxide treatment has been reported (see Non-Patent Document 1).
However, this method is extremely difficult to handle alkali / hydrogen peroxide, and it is necessary to use a large amount of acid for neutralization. The main purpose of the alkali / hydrogen peroxide treatment is delignification / hemicellulose. In the saccharification step after the neutralization step, various salts are generated in the reaction solution.

In addition, conventional biomass saccharification techniques have used heat treatment with concentrated sulfuric acid or dilute sulfuric acid, or steam explosion treatment.
However, these treatment conditions are harsh and the equipment becomes sophisticated, so that there is an increasing need for the development of original technologies centering on enzymatic methods with low environmental impact.

To date, researchers around the world have conducted many studies to optimize the amount of various cellulolytic enzymes produced by living organisms and their catalytic properties to improve the degradation efficiency.
However, cellulose is tightly packed by strong hydrogen bonds and hydrophobic bonds, and it has been considered that it is extremely difficult to significantly improve the decomposition efficiency.

In addition, for the purpose of reducing the molecular weight and swelling of polysaccharides contained in materials containing polysaccharides, acid hydrolysis and fibrillation methods have been used. For example, concentrated hydrochloric acid treatment is performed in the production of colloidal chitin swollen by chitin and chitin oligosaccharides and N-acetylglucosamine which are partial hydrolysates of chitin.
However, under conditions in which a strong acid such as concentrated hydrochloric acid is used in large quantities, it is necessary to pay close attention to the handling of the acid used, and it is also necessary to pay attention to side reactions. Development of effective low molecular weight and swelling technology that suppresses the amount of acid used is required.

One of the forms of biomass energy use in Japan is considered to be community-based, and the Ministry of Agriculture, Forestry and Fisheries uses materials produced from the region as materials or based on the biomass town concept based on the biomass and Japan comprehensive strategy. Comprehensive research is underway to convert to energy. In addition to agriculture, forestry and fishery products themselves, sugar squeezed residue, starch extraction residue, okara, fruit juice residue, agriculture, forestry and fishery products discarded for production adjustment, unused plants, resource crops, unused algae, rice husks, straw, crops Stalks and leaves, corn cob, tea grounds, coffee grounds, pruned branches, sawdust, wood chips, driftwood, thinned wood, construction waste, paper sludge, waste paper, old cloth, fermentation residue, crustacean shell, etc.
In order to saccharify these materials or use them effectively as new materials, the development of basic technologies for promoting local efforts is strongly demanded. It is also known that a considerable amount of starch granules remain in the starch pomace. Since it is difficult to recover by physical treatment and causes rot, development of its utilization technology is desired.

Biotechnol. Prog., 22, pp. 449-453 (2006) Chitin and Chitosan Experiment Manual, Gigodo Publishing, (ISBN 4-7655-0734-7C3043), pp.5-6 and pp.79-81 (1991)

  The present invention leads to advanced utilization of carbohydrate biomass targeting ethanol, petroleum substitute products, and the like by providing a technique for producing low molecular weight carbohydrates and the like from materials containing polysaccharides, in particular, hardly degradable polysaccharides. The purpose is that.

  The present invention aims to produce biomaterials and bioethanol using the individuality of each region in Japan by developing effective utilization technologies for a wide variety of carbohydrate-based materials that are generated directly or indirectly from the agriculture, forestry and fisheries and food industries. Breakthroughs in the development of biomass utilization technologies all over the world by greatly impacting overseas bioethanol production strategies such as the United States, Brazil, China, India, and Thailand. It becomes through.

In view of the above problems, the present inventor has conducted extensive studies and, after treatment with a salt having an activity to modify the crystal structure of a polysaccharide or an aqueous solution thereof, or a substance having substantially the same activity as that, It has been found that the saccharification process can be made more efficient by washing the material containing the polysaccharide as necessary and enzymatically saccharifying it.
In addition, when a polysaccharide-containing material is treated with a high-concentration salt of a strong acid or an aqueous solution thereof, or a substance having substantially the same activity as that, a polysaccharide is swelled or reduced in molecular weight. Found to be promoted.
The present inventor has found that the above problems can be solved by the above-described means, and has completed the present invention based on such knowledge.

That is, the invention provided by the present application is as follows.
The present invention according to claim 1 is to cause calcium thiocyanate, sodium thiocyanate, hydrates thereof, or an aqueous solution thereof to act on a material containing polysaccharide at minus 20 degrees Celsius to 40 degrees Celsius. included in the material containing the polysaccharide, it was modified crystal structure while minimizing the lower molecular weight, solubilization of the polysaccharide was subjected to the enzymatic hydrolysis reaction, carrying out the low molecular weight, solubilization of the polysaccharide The present invention provides a method for producing a low molecular weight carbohydrate.
The present invention according to claim 2 is a material containing a polysaccharide using calcium thiocyanate or a hydrate thereof, or an aqueous solution thereof and having a concentration of 20% (w / v) or more in terms of calcium thiocyanate. The method according to claim 1 is provided.
In the present invention according to claim 3 , at least a part of the polysaccharide contained in the polysaccharide-containing material is cellulose, and at least a part of the enzyme has an activity of hydrolyzing a cellulose hydrolase or a partial hydrolyzate of cellulose. The method according to claim 1 or 2 is provided.
In the present invention according to claim 4 , at least a part of the polysaccharide contained in the polysaccharide-containing material is starch, and at least a part of the enzyme has an activity to hydrolyze starch hydrolase or a partial hydrolyzate of starch. The method according to claim 1 or 2 is provided.
The present invention according to claim 5 provides the method according to any one of claims 1 to 4 , wherein at least a part of the low molecular weight and solubilized carbohydrate is glucose.
The present invention according to claim 6 is a method in which the polysaccharide-containing material is at least a part of an organ of a plant in which starch or sugar is accumulated, or starch extracted from a starch or sugar accumulation site in the plant, or sugar-squeezed. The method according to any one of claims 1 to 5 , which is obtained from soup stock.
The present invention according to claim 7 is the material according to any one of claims 1 to 6 , wherein the material containing polysaccharide is a material containing cellulose by-produced during pulp production, pulp, waste paper, or fiber containing cellulose. A method is provided.
The present invention according to claim 8, calcium thiocyanate, sodium thiocyanate, their hydrates or their aqueous solution, characterized in that it comprises a polar organic solvent, according to any one of claims 1-7 This method is provided.
The present invention according to claim 9 provides the method according to any one of claims 1 to 8 , wherein the material containing the polysaccharide is washed after modifying the crystal structure of the polysaccharide.
In the present invention according to claim 10 , after adding calcium chloride or a hydrate thereof, or a solution in which they are dissolved to a material containing a polysaccharide and subjecting it to a high temperature treatment at a temperature higher than 50 degrees Celsius, The method according to any one of claims 1 to 9 , wherein the method for producing a low molecular weight carbohydrate according to any one of claims 1 to 9 is performed after the step of performing a low-temperature treatment at a temperature of less than or equal to 10 degrees. Is to provide.
The present invention according to claim 11, in the step of modifying the crystalline structure of the polysaccharide, salt or hydrate thereof of a strong acid or a solution which was dissolved, characterized in that the material containing the polysaccharide to bacteriostatic and storage The method according to any one of claims 1 to 10 is provided.
The present invention according to claim 12 is characterized in that the salt of the strong acid is calcium chloride, and the concentration thereof is 10% (w / v) or more in terms of calcium chloride dihydrate in the solution. Item 12. A method according to Item 11 , is provided.
The present invention according to claim 13, performs a step of oxidizing the material comprising the polysaccharide in a solution containing an alkali and hydrogen peroxide, characterized in that a step of subsequently modifying the crystal structure of the polysaccharide, A method according to any one of claims 1 to 12 is provided.
The present invention according to claim 14 provides the method according to claim 13 , characterized in that the alkali is calcium hydroxide.
The present invention according to claim 15, to produce a low molecular saccharides by the method according to any one of claims 1 to 14 to provide a method for producing ethanol, which comprises fermenting the low molecular carbohydrates Is .

According to the present invention, a useful substance having a low molecular weight carbohydrate can be produced from a material containing a polysaccharide, particularly a hardly degradable polysaccharide.
According to the present invention, by providing a technique for producing a low molecular weight carbohydrate from a material containing a polysaccharide, in particular, a hardly degradable polysaccharide, it is possible to lead to advanced utilization of carbohydrate biomass targeting ethanol or petroleum substitute products. it can. That is, according to the present invention, the enzymatic saccharification of insoluble saccharides such as cellulose, starch and chitin and the saccharification by acid are dramatically improved, and thus the management cost of the saccharification process, the enzyme reuse efficiency, etc. are improved. Through this, it becomes possible to develop biomass conversion technology at low cost.
Furthermore, according to the present invention, a new material imparted with solubility and dispersibility can be produced from a material containing an insoluble, hardly degradable polysaccharide, and also from the low molecular weight carbohydrate recovery residue, leading to a new use. It can also be provided as a polysaccharide resource.

  In the present invention, swelling agents such as zinc chloride, calcium thiocyanate, sodium thiocyanate, calcium chloride, magnesium chloride, sodium sulfate, ammonium sulfate, and urea effectively act at normal temperature and normal pressure. Enzymatic saccharification and acid saccharification Speed can be significantly improved. Moreover, since an effect is recognized on cellulose, starch and chitin, it can be a breakthrough of a wide variety of biomass conversion technologies.

  According to the present invention, an effective utilization technique of a material containing a polysaccharide is developed. This greatly accelerates the conversion of biomass such as agriculture, forestry and fishery waste, food manufacturing waste and resource crops to a wide variety of useful materials. Therefore, it is expected to contribute to the mitigation of global energy and environmental problems. The scope of use includes domestic and overseas areas where sugarcane, corn, and moss are used as large-scale plantations as resource crops. It covers urban areas where waste is generated, and areas where agricultural, forestry and fishery waste and community-based food manufacturing waste are generated.

The present invention will be described below.
The present invention according to claim 1 uses a salt having an activity to modify the crystal structure of a polysaccharide, an aqueous solution thereof, or a substance having substantially the same activity as that of the polysaccharide. A method for producing a low-molecular-weight carbohydrate, characterized in that the crystal structure is modified while minimizing the low-molecular-weight and solubilization, and then the enzyme is hydrolyzed to lower the molecular weight and solubilize the polysaccharide. It is.
Further, the present invention according to claim 2 uses an aqueous solution of urea or a substance having substantially the same activity, and minimizes the low molecular weight and solubilization of the polysaccharide contained in the polysaccharide-containing material. This is a method for producing a low molecular weight carbohydrate, characterized in that after the crystal structure is modified while being suppressed, an enzymatic hydrolysis reaction is performed to lower the molecular weight and solubilize the polysaccharide.

  The present invention according to claims 1 to 13 uses a salt (en) having an activity to modify the crystal structure of a polysaccharide, an aqueous solution thereof, or a substance having substantially the same activity as the material containing the polysaccharide. By modifying the crystal structure while minimizing the low molecular weight and solubilization of the polysaccharides contained, and then performing an enzymatic hydrolysis reaction, the enzyme-degrading properties of the polysaccharide-containing material are significantly improved. It is based on a completely new discovery that it can be used for the production of useful substances such as ethanol.

With respect to substances having an activity of modifying the crystal structure of polysaccharides, research has been conducted for a long time to modify the structure of cellulose fibers.
So far, for example, acids such as hydrochloric acid and sulfuric acid, alkalis such as lithium hydroxide and sodium hydroxide, inorganic salts such as zinc chloride, calcium thiocyanate, ammonium thiocyanate / ammonia, hydrazine, metals such as cadoxene and copper ammonia Complexes, organic salts / salt mixtures such as N, N-dimethylacetamide / lithium chloride, and organic solvents such as pyridine / chloral are well known (“Cellulose Dictionary” edited by Cellulose Society, Asakura Shoten Co., Ltd. ISBN 4 -254-47030-4 C 3561). There is also a paper investigating the thermal decomposition behavior when cellulose is dissolved in a solvent (Acta Polym., 35, 339-343 (1984)).
However, all of these have been studied as solvents for fiber processing, and have hardly been studied in order to perform enzymatic reactions and obtain saccharified products.
The only pretreatment method using cadoxen has been reported, but cadoxene is a concentrated alkaline substance and contains cadmium, so adjustment of pH and removal of cadmium remaining inside the substrate are major problems. Is easily conceived. For this reason, a pretreatment technique with low environmental impact and high work safety has been demanded (Science 201, pp. 743-745 (1978)).

As a result of intensive studies, the present inventor has clarified that, even with a hardly degradable polysaccharide, the enzyme structure is improved by modifying (swelling) the crystal structure using an appropriate substance.
An important point of the present invention is that the release of polysaccharide degradation products is minimized in the swelling treatment, and a mixture of a substance (swelling agent) having an activity of modifying the crystal structure of the polysaccharide and a low molecular weight carbohydrate is not generated as much as possible. In this case, the swelling agent is recovered in a high purity state, and the subsequent enzymatic saccharification is performed smoothly.
Therefore, as the swelling agent in the present invention according to claims 1 to 13, a strong acid such as sulfuric acid, hydrochloric acid or phosphoric acid or a strong alkali such as lithium hydroxide or sodium hydroxide is not used as a main component. In the present invention, when a strong acid or strong alkali is used as a swelling agent as a secondary component for optimizing the activity of salt or urea, it is necessary to suppress the solubilization of polysaccharides by hydrolysis as much as possible. Use is desirable.

  Moreover, starch etc. will swell and gelatinize the crystal structure only by heating aqueous solution. The method of gelatinizing a starch suspension by heating and then saccharifying with amylase, glucoamylase or the like is a technique that has been widely used in the saccharification industry. Such a saccharification process using water as the main swelling agent is not included in the present invention.

However, the present inventor applied the result of the present invention, that is, a salt having an activity to modify the crystal structure of a polysaccharide or an aqueous solution thereof, or a substance having substantially the same activity to a material containing starch. I found that there is a case that can be done. Theoretically, the enzymatic degradability is dramatically improved by gelatinization. However, when starch existing in a matrix such as a plant cell wall is gelatinized by heating, a starch paste layer is formed in the matrix. There is a concern that the saccharification efficiency may be reduced by preventing the entry of hydrolase. In addition, the heat treatment may increase the viscosity of the sample and the hot water extract may be eluted, which may complicate the treatment of the saccharification reaction solution that has undergone heat gelatinization. In order to improve the enzymatic decomposition efficiency of the material containing starch, it is desirable to moderately swell the starch material by a reaction near room temperature.
In the present invention, in order to solve such a problem in the enzyme reaction, a salt or urea having an activity for modifying the crystal structure of the polysaccharide is used, and the polysaccharides contained in the polysaccharide-containing material are minimized in molecular weight and solubilized. It is characterized by modifying the crystal structure while limiting to the limit. The idea of swelling starch with salt and performing enzyme treatment is known, and there is an example used in structural analysis studies of starch (Carbohydr. Res., 247, 279-290 (1993)). However, the concept of improving the enzymatic saccharification efficiency while modifying the starch structure near normal temperature and maintaining the insolubility of the starch-containing material is novel. In addition, for materials containing both cellulose and starch, it is expected that the polysaccharide modification / saccharification step in the present invention is effective.

The substance having an activity to modify the crystal structure of the polysaccharide used in the present invention according to claim 1 includes a salt, a molecule forming a complex with the salt, a molecule having water of crystallization or a part thereof, hydrogen ion or hydroxylation. It also includes molecules composed of product ions.
As another substance having an activity of modifying the crystal structure of a non-salt polysaccharide, urea may be mentioned as described in claim 2.

  In addition, an aqueous solution of a salt having an activity to modify the crystal structure of a polysaccharide is an aqueous solution of a salt having an activity to modify the crystal structure of a polysaccharide, a solution in which an acid, an alkali, or another salt is dissolved, or a polar solution to them A solution in which an organic solvent is dissolved is shown. Even if an acid or alkali is added to an aqueous solution in which salt or urea is dissolved, the crystal structure of the polysaccharide is modified in that the action of a substance having an activity to modify the crystal structure of the polysaccharide is used for enzymatic saccharification. It is substantially the same as a salt, urea, or an aqueous solution thereof having an activity to act, and is included in a substance having an activity to modify the crystal structure of the polysaccharide of the present invention.

As a salt having an activity of modifying the crystal structure of a polysaccharide, an aqueous solution thereof, or a substance having substantially the same activity as those, a metal salt is preferable as described in claim 3.
In the present invention, a saccharification technique using the fact that various salts, particularly metal salts, swell polysaccharides such as cellulose and starch is proposed. Examples of such salts include many salts such as sodium thiocyanate, calcium thiocyanate, copper ammonia, zinc chloride, calcium chloride, magnesium chloride, sodium sulfate and the like, but the target salt is not limited thereto. Biomass conversion technology itself, which is characterized by pretreatment and enzymatic saccharification under a mild environment-friendly condition using a salt having high swelling activity or an aqueous solution thereof, is based on a novel viewpoint.
For example, when various chaotropic ions are constituent components of the salt, the structure of water in the aqueous solution is destroyed, and it is expected that the decrease in water entropy is suppressed. Many of the above-mentioned substances have been developed as cellulose swelling agents, solvents, etc. in the textile industry, and their effective concentrations and use conditions have been investigated in detail.
In the present invention, based on these known information, it is known from the viewpoint of not dissolving the polysaccharide molecule or the low molecular weight product thereof existing in the polysaccharide-containing material in the aqueous layer in the swelling treatment stage, and from the viewpoint of cost calculation. The optimum condition can be determined by the condition examination method.

For example, one of these substances, calcium thiocyanate, will be described as an example. The expression “acting calcium thiocyanate” means that calcium thiocyanate, its hydrate or a solution thereof, or a solution having a functionally equivalent activity to them is allowed to act.
The salt other than calcium thiocyanate or an aqueous solution thereof conforms to the contents described for calcium thiocyanate, and the essence of the important point of focus of the present invention is not changed. It is possible to optimize the production method and system in accordance with the characteristics of each substance while referring to the contents of the explanation regarding calcium thiocyanate. For example, for recovery of zinc ions from a zinc chloride aqueous solution, a known zinc ion recovery technique such as a recovery method using an ion exchange resin can be used.

  Various solvents including calcium thiocyanate have long been known as typical reagents for swelling and dissolving cellulose. Calcium thiocyanate dissolves at a concentration of 50% or more and about 100 ° C. or more. However, it is reported that it exhibits a decomposition action at higher temperatures (“Dictionary of Cellulose” edited by Cellulose Society, Asakura Shoten Co., Ltd. ISBN 4-254-47030-4 C 3561). The interaction between cellulose and calcium thiocyanate aqueous solution has been studied vigorously, and it is presumed that it interacts with calcium thiocyanate water complex and glucose residues 05 and 06.

  Regarding modification of cellulose fibers using aqueous calcium thiocyanate solution, a method for producing a blend molded product with polyacrylonitrile (Japanese Patent Laid-Open No. 10-316767), swelling treatment for the purpose of enzyme treatment and processing of fiber products (Tokyo Metropolitan Industrial Co., Ltd.) Technical Research Institute Research Report No.5 pp.113-116 (2002), the same magazine, No.6 pp81-84 (2003)) and research on gelation technology (Polymer, Vol.48, June, p425 (1999)) Many are known. Among them, a technique for increasing the degradability of the enzyme and performing complicated processing on the fiber by swelling the fiber using calcium thiocyanate is included.

  On the other hand, starch has been reported to be gelled with salt at a low temperature to leach out the internal amylase (Starch / Staerke, 31, 195-200 (1979)).

  However, there has been no concept of using calcium thiocyanate as a pretreatment method for reducing the molecular weight of saccharide biomass and improving its utilization. Since calcium thiocyanate is decomposed or insolubilized under acid and basic conditions, and converted to cyanide ions by reaction with an oxidizing agent, it has been considered to be less adaptable to acid or alkali hydrolysis techniques.

  The present inventors have found that the subsequent enzymatic saccharification efficiency is greatly improved by simply contacting a polysaccharide with an aqueous calcium thiocyanate solution at various temperatures, particularly from low to room temperature for a short time. Moreover, even if most of the calcium thiocyanate was removed by washing, the effect of the change in the structure of the polysaccharide remained, and the enzymatic saccharification efficiency was kept high. Furthermore, the pH after calcium thiocyanate has been removed to some extent by washing with water is around 4, and considering that there is a pH on the weakly acidic side for the action of many carbohydrate hydrolases, There is a great advantage that the labor of adjustment can be saved. In addition, the antibacterial property of high concentration of calcium thiocyanate can be used to increase the storage stability of highly septic samples.

Calcium thiocyanate not only decomposes at 160 ° C. or more, but also easily decomposes in the presence of an acid or an oxidizing agent, and has a drawback in handling that calcium is easily insoluble on the alkali side.
However, the inventors have shown that these disadvantages can be minimized and can be a practical new technology when using mild biological methods. The inhibitory effect of enzyme activity by calcium thiocyanate and the growth inhibitory effect of fermenting microorganisms such as yeast vary depending on the type and conditions, so the effects of calcium thiocyanate on the enzymatic reaction or microbial fermentation reactivity are well known. It is necessary to consider using a method. For example, as a result of measurement with a cellulolytic enzyme (Celluclast 1.5L FG, Novozymes), activity was observed even at a calcium thiocyanate concentration of about 5%. As a result, the growth of 70% of the amount of cells cultured in the absence of calcium thiocyanate was observed.

The recovery of chemicals is a very important process for process development. The recovery of calcium thiocyanate is extremely important not only from a cost reduction perspective, but also from the perspective of controlling thiocyanate release to the environment.
In the present invention, calcium thiocyanate is believed to act by polysaccharide and non-covalent bond. When interacting with non-covalent bonds, calcium thiocyanate does not decompose, so it is recovered after contact with a high-concentration calcium thiocyanate solution, and concentrated, purified (impurities removed), and concentration as necessary. After making adjustments, all of this can be reused. Assuming that one residue of calcium thiocyanate binds to one glucose residue, the amount adsorbed on the polysaccharide is considered to be on the order of about the same as the percentage of polysaccharide present.

Further, in the present invention, it has been clarified that even if the polysaccharide-containing material is washed with water after contacting with calcium thiocyanate, the effect on the efficiency of the subsequent enzymatic hydrolysis reaction is maintained. In this step, the recovery rate of calcium thiocyanate is further improved.
Furthermore, it is considered that calcium thiocyanate non-covalently bound to the polysaccharide is liberated by the subsequent enzymatic hydrolysis reaction.
The calcium thiocyanate in these steps can be recovered by a known method such as an electrodialysis method or an ion exchange method. For example, using an acylator S1 (Asahi Kasei Kogyo Co., Ltd., cartridge AC120-10), a mixture of glucose and calcium thiocyanate can be separated by electrodialysis. Such a step of reducing the concentration of calcium thiocyanate in the saccharified solution is also desirable in terms of avoiding fermentation inhibition by calcium thiocyanate. In addition, in order to avoid the release of calcium thiocyanate, which is difficult to recover, to the environment, a known bioconversion technique or the like can be used.

Many biomass exist in a wet state after sufficiently washing and removing the water-soluble fraction. Examples include starch squeeze from corn or potatoes, paper sludge, sugar squeeze from bagasse or beet. When such samples (polysaccharide-containing materials) are reacted with calcium thiocyanate for solid-liquid separation, the purity of the recovered calcium thiocyanate is relatively high because there are few substances extracted into the liquid phase. It is expected to sag.
On the other hand, about the biomass which contains many water-soluble fractions, the purity at the time of collect | recovering calcium thiocyanate will become high by performing the pre-processing process including a water washing process prior to a calcium thiocyanate process.
When considering the cascade use of the method of the present invention, in this pretreatment step, extraction of useful substances such as antioxidant components, enzyme treatment with hemicellulase etc. for decomposing and recovering components derived from hemicellulose etc. first, etc. It is possible to devise measures to reduce the total cost, for example. Since such a pretreatment step is expected to increase the recovery rate of low molecular carbohydrates, it is necessary to evaluate the merit as a total process.

  In the present invention, the reaction between the polysaccharide-containing material and calcium thiocyanate can be carried out by bringing a dry or wet sample (a material containing polysaccharide) into contact with a solution containing calcium thiocyanate. In this case, the concentration of calcium thiocyanate is set to a high concentration, specifically 20% (w / v) or more, preferably 50% (w / v) or more as described in claim 5. The reaction proceeds efficiently. A solution having the same activity as a high-concentration calcium thiocyanate solution is a solution of calcium thiocyanate hydrate, a solution of thiocyanate and calcium salt, or a solution of calcium thiocyanate and another compound dissolved therein. And those containing polar organic solvents such as acetone and methanol. In addition, when a high concentration calcium thiocyanate solution is used at a low concentration, the molecular structure of some calcium thiocyanate and hydrate is maintained at a high concentration, and the action on the sample is high. It is easily conceived that it will remain. When the moisture content of the material containing the polysaccharide is very high, the average concentration when 60% (w / v) calcium thiocyanate is added may be practically 40% (w / v) or less. However, even in that case, the reaction proceeds and the efficiency is improved in the subsequent enzymatic hydrolysis. In addition, when there is a process of freezing and thawing at least a part of the whole reaction solution, a phenomenon in which the enzymatic degradability of the sample is improved by locally increasing the concentration of calcium thiocyanate has been found. It is included in the present invention.

  Furthermore, to stabilize solids such as calcium thiocyanate and tetrahydrated water directly on the sample and to stabilize the activated higher-order structure of calcium thiocyanate formed in a high concentration state even at low concentrations. The present invention also includes a method using calcium thiocyanate activated at a low concentration and a method of mixing a plurality of salts, for example, a method of mixing calcium thiocyanate and sodium thiocyanate. .

  The temperature at which the polysaccharide-containing material and calcium thiocyanate are reacted may be 160 degrees Celsius or lower, at which calcium thiocyanate is decomposed. However, in order to reduce costs, 100 degrees Celsius or lower is desirable. . Since the reaction occurs even at minus 20 degrees Celsius and room temperature, the temperature can be set to an optimum temperature in consideration of the temperature and energy efficiency of the pretreatment process.

  In addition, the present inventors have found that in the case of microcrystalline cellulose, the effect appears even if it is removed immediately after mixing, in the reaction time when the polysaccharide-containing material is reacted with calcium thiocyanate. The effect of the long-term reaction varies depending on the higher-order structure of the material containing the polysaccharide, the composition of subcomponents, the size, and the like. The reaction time should be optimized according to the characteristics of the sample, taking into account the possibility that water-soluble components in the sample will gradually elute and the recovery efficiency of calcium thiocyanate.

  In order to improve the reactivity between the material sample containing polysaccharide and calcium thiocyanate, it is necessary to infiltrate the calcium thiocyanate into the sample. Use a micronized sample, pre-define the sample, or perform pretreatment on the sample side, or stir and shear the sample after adding calcium thiocyanate. It is desirable to appropriately perform a process for strengthening.

As described above, the polysaccharide crystal structure is modified while minimizing the low molecular weight and solubilization of the polysaccharide .

In the above description, calcium thiocyanate has been described as an example of a substance having an activity of modifying the crystal structure of the polysaccharide, but sodium thiocyanate, copper ammonia, zinc chloride, calcium chloride, magnesium chloride, sodium sulfate. The same applies to salts such as ammonium sulfate and urea. For example, when a calcium chloride dihydrate aqueous solution or a solution having the same activity is used, the concentration is high, specifically 20% as described in claim 6. By setting it to (w / v) or more, preferably 50% (w / v) or more, the reaction proceeds efficiently.
Furthermore, as described in Claim 12, polar organic solvents, such as acetone and methanol, can also be included.

As described above, substances other than calcium thiocyanate, for example, various reagents such as calcium chloride, sodium thiocyanate, zinc chloride, magnesium chloride, sodium sulfate, ammonium sulfate, and urea are also effective in the production of low molecular weight carbohydrates and the like. Sex can be found.
For example, calcium chloride has been known in the fiber processing field as a swelling agent for cellulose or chitin. In accordance with the method described for calcium thiocyanate, the enzymatic hydrolysis step is made more efficient by allowing a calcium chloride solution having a concentration of 20% (w / v) or more in terms of dihydrate to act on the cellulosic material. It also shows high effectiveness for starch-based materials.

  In addition, with respect to the treatment time when using calcium chloride, when a 100% (w / v) calcium chloride dihydrate solution is used, a high effect can be obtained by performing the treatment at room temperature for 1 to 6 hours. Appears and the effect improves according to the processing time. In addition, although a slight effect is seen at a treatment temperature of 4 degrees Celsius, a remarkable treatment effect appears by treating at a room temperature or higher, and the effect is improved according to the temperature.

Compared with calcium thiocyanate, calcium thiocyanate and zinc chloride, calcium chloride has advantages such as higher work safety, less environmental impact, and lower raw material costs.
In addition, calcium chloride is recognized as a food additive in Japan at a low concentration. Therefore, in the saccharification / fermentation process using starch pomace as a raw material, the product is used. There is a possibility of expanding to food applications. Since these reagents can find an effect even when used at room temperature, they suppress the release and solubilization of starch, pectin and various hot water extracts, and after the enzyme hydrolysis step, the reagents, enzymes, and This is convenient for collecting and reusing the remaining insoluble fraction. On the other hand, in order to increase the permeability of reagents to polysaccharides and efficiently carry out enzymatic hydrolysis reactions, it is sometimes effective to perform heat treatment to break hydrogen bonds that exist between or within insoluble polysaccharide molecules. Exists. Calcium chloride with high thermal stability exhibits effectiveness in such treatment.

  Similar to calcium thiocyanate, the property of returning to a neutral pH by washing with water even after the action of substances such as calcium chloride, sodium thiocyanate, zinc chloride, magnesium chloride, urea is the chemical hydrolysis of the sample. This is a great advantage in smoothly performing the enzyme hydrolysis step.

After the reaction between the polysaccharide-containing material and a swelling substance such as calcium thiocyanate, it is desirable to appropriately collect a swelling substance such as calcium thiocyanate and perform a washing step to prevent enzyme reaction inhibition. In this case, as described above, it is desirable to evaluate in advance the sensitivity of the enzyme used to a swelling substance such as calcium thiocyanate.
That is, as described in claim 13, it is desirable to wash the material containing the polysaccharide after modifying the crystal structure of the polysaccharide. Washing may be performed by a conventional method using water or the like.

It is possible to efficiently obtain a low-molecular-weight carbohydrate by washing the sample in which the reaction between the polysaccharide-containing material and the swelling substance such as calcium thiocyanate is completed as appropriate, followed by enzymatic hydrolysis.
The enzymatic hydrolysis reaction can be performed by a known method using a commercially available enzyme preparation or an enzyme obtained from a natural microorganism, plant or animal. Compared with conventional methods, the enzyme reaction time is significantly shortened, so it is possible to control microbial contamination and increase the amount of enzyme that can avoid deactivation, thereby improving the recovery rate and reducing costs. It becomes.

  Examples of the polysaccharide used in the present invention include cellulose, starch, xylan, pectin, chitin, chitosan, mannan, glucomannan, galactomannan, xyloglucan, fructan, arabinogalactan, filamentous fungal polysaccharide, yeast polysaccharide, and seaweed polysaccharide. And bacterial polysaccharides, viral polysaccharides, animal polysaccharides, and the like.

Examples of low molecular weight carbohydrates obtained by enzymatic hydrolysis of polysaccharides include cellulose, starch, xylan, pectin, chitin, chitosan, mannan, glucomannan, galactomannan, xyloglucan, fructan, arabinogalactan, and filamentous fungal polysaccharide. Various monosaccharides or oligosaccharides that can be produced from yeast polysaccharides, seaweed polysaccharides, bacterial polysaccharides, viral polysaccharides, animal polysaccharides, and the like are conceivable.
Among these, as described in claim 9, particularly glucose is extremely important as a key compound in the production of various useful substances such as ethanol and polylactic acid by fermentation.

In addition to agriculture, forestry and fishery products themselves, sugar cane residue, starch extraction residue, okara, fruit juice residues, agricultural and forestry fishery products discarded for production adjustment, unused plants, Resource crops, unused algae, bagasse, rice husks, straw, crop stover, corn cob, tea grounds, coffee grounds, pruned branches, sawdust, wood fragments, driftwood, thinned wood, used chopsticks, construction waste, paper sludge, waste paper, old cloth , Fermentation residues, crustacean shells, and the like.
These materials are preferably subjected to pretreatment such as pulverization, delignification, demineralized salt, and protein removal as appropriate to improve the permeability of swelling substances such as calcium thiocyanate solution.
As a raw material containing polysaccharide, as described in claim 10, starch extracted from starch or sugar accumulation site in starch or sugar, or starch or sugar accumulation site in the plant What is obtained from squeezed residue can be used.
Moreover, as a raw material containing polysaccharide, as described in Claim 11, the raw material containing the cellulose byproduced at the time of pulp manufacture, a pulp, waste paper, or the fiber containing a cellulose can also be used.

Enzymes required for carrying out the enzymatic hydrolysis reaction are mainly carbohydrate-related enzymes such as cellulose hydrolase, starch hydrolase, hemicellulose hydrolase, pectin hydrolase, and chitin hydrolase. However, the present invention is not limited to this. For example, it is easily conceived to add lignin hydrolase to promote the production of low molecular weight carbohydrates. Artificial enzymes whose functions have been increased by genetic recombination techniques, partial hydrolysis, etc. can also be used in the present invention.
As shown in claim 7, when at least a part of the polysaccharide contained in the polysaccharide-containing material is cellulose, at least a part of the enzyme has an activity to hydrolyze a cellulose hydrolase or a partial hydrolyzate of cellulose. Can be used. Further, as described in claim 8, when at least a part of the polysaccharide contained in the polysaccharide-containing material is starch, at least a part of the enzyme has an activity to hydrolyze starch hydrolase or a partial hydrolyzate of starch. Enzymes possessed can be used.

  It is easily conceived that polysaccharides having a modified crystal structure are improved in reactivity with an enzyme from which a substrate binding site has been removed. Alternatively, conversely, it is considered that a region where the role of the substrate binding site is increased is also generated. Furthermore, the step of assimilating the microorganism or the polysaccharide whose structure has been modified using a highly functionalized microorganism directly includes the step of decomposing the substrate using an enzyme produced by the microorganism. Included in the content.

After completion of the enzyme reaction, it contains a swelling material such as an enzyme, a buffer solution component, calcium thiocyanate, in addition to the residue / unreacted material of the original material and low molecular weight carbohydrate. In order to reduce the amount of residues and unreacted materials, it is necessary to carry out pretreatment and extend the enzyme reaction time, etc., but taking into account the effects of reaction efficiency, enzyme recovery efficiency and microbial contamination, etc. There is a need to. Residues and unreacted substances can be removed by methods such as filtration and centrifugation.
In addition, in order to remove swelling substances such as enzymes, buffer components and calcium thiocyanate and recover only low molecular weight carbohydrates, use ultrafiltration membranes or electrodialysis, separation with ion exchange resins, enzyme purification tags It is possible to use a publicly known method such as use.

  In this manner, the low molecular weight carbohydrate obtained after removing the contaminating components can be used for various fermentation processes including ethanol fermentation as it is or after being appropriately concentrated.

As described above, the present inventor has found a method for efficiently producing a low molecular weight carbohydrate from a material containing a hardly degradable polysaccharide by enzymatic saccharification. The inventors have focused on modifying the structure of a material further containing a polysaccharide while considering the swellability in the present invention by using it together with an oxidizing agent and a strong acid.
The present inventor has found that by adding a polysaccharide and an acid to a solution in which a salt of a high-concentration strong acid is dissolved, the hydrolysis and swelling of the acid are greatly improved. The invention has been completed.

That is, the present invention according to claim 14 is a salt of a strong acid or a hydrate thereof, a solution thereof, a solution having a functionally equivalent activity thereto, a material containing a polysaccharide and a reaction solution containing a strong acid. Among them, a method for producing a low molecular weight carbohydrate or a material imparted with solubility / dispersibility, characterized by including a step of chemically lowering the molecular weight or solubilizing / dispersing reaction.
Here, the strong acid salt used in the present invention according to claim 14 is desirably a highly stable substance. For example, calcium chloride, magnesium chloride, ammonium sulfate, sodium sulfate, sodium nitrate, sodium chloride and the like can be mentioned, and any salt of a strong acid that shows water solubility and high stability by acid can be used in the present invention.

  The strong acid used in the present invention according to claim 14 includes, for example, hydrochloric acid, sulfuric acid, nitric acid and the like. That is, the present invention according to claim 15 is imparted with the low molecular weight carbohydrate or the solubility / dispersibility according to claim 14, characterized in that the strong acid is composed of at least one of sulfuric acid, hydrochloric acid or nitric acid. It is a manufacturing method of the material.

  In the reaction solution in the presence of a high-concentration salt according to claims 14 and 15, the acid is likely to be in the form of a molecule and the fact that ion hydration does not sufficiently occur, thereby affecting the acid hydrolysis of the polysaccharide. It is considered that decomposition and swelling are promoted.

Next, the present invention according to claim 16 is characterized in that the salt of the strong acid is sodium sulfate and / or ammonium sulfate, and the strong acid is sulfuric acid, This is a method for producing a material imparted with dispersibility.
That is, the present invention according to claim 16 includes sodium sulfate and / or ammonium sulfate or a hydrate thereof, a solution thereof, a solution having a functionally equivalent activity thereto, a polysaccharide-containing material, and sulfuric acid. A method for producing a low-molecular weight carbohydrate or a material imparted with solubility / dispersibility, characterized by comprising a step of chemically reducing or solubilizing / dispersing in a reaction solution containing is there.

  As described above, in the reaction solution containing sodium sulfate and / or ammonium sulfate and sulfuric acid, the mechanism for promoting the low molecular weight reaction or the solubilization / dispersion reaction of the polysaccharide is high. Therefore, it is considered important that sulfuric acid is likely to be in a molecular state and that hydration of hydrogen ions and sulfate ions is insufficient.

Next, the present invention according to claim 17 provides the low-molecular-weight carbohydrate or solubility / dispersibility according to claim 14 or 15, characterized in that the salt of the strong acid is calcium chloride and the strong acid is hydrochloric acid. It is a manufacturing method of the made material.
That is, the present invention according to claim 17 includes a calcium chloride or a hydrate thereof, a solution thereof, a solution having a functionally equivalent activity thereto, a material containing polysaccharide, and a reaction solution containing hydrochloric acid. And a method for producing a low molecular weight carbohydrate or a material imparted with solubility / dispersibility, characterized by including a step of chemically lowering the molecular weight or solubilizing / dispersing reaction.

  In addition, as described above, the mechanism of promoting the low molecular weight reaction or solubilization / dispersion reaction of polysaccharides in the reaction solution containing calcium chloride and hydrochloric acid is because of the high concentration of chloride ions. It is thought that it is important to be easily in a molecular state and that hydration of hydrogen ions and chloride ions is insufficient.

  In the present invention according to claims 14 to 17, the concentration of the acid in the reaction solution when the low molecular weight of the hardly degradable polysaccharide and the provision of solubility / dispersibility proceed efficiently is 0.3 N or more, Preferably it is 0.3 to 4 N, more preferably 0.3 to 3 N. However, even in the presence of thin sulfuric acid or hydrochloric acid of around 0.3 to 1 N in particular, low molecular weight and solubility / dispersibility Can be efficiently advanced.

  Further, in the present invention according to claims 14 to 17, the concentration of the strong acid salt in the reaction solution when the low molecular weight of the hardly degradable polysaccharide and the provision of solubility and dispersibility proceed efficiently is as follows: 20% (w / v) or more, preferably 50% (w / v) or more, more preferably 62% (w / v) or more, most preferably the saturated solution, but the higher the concentration, the lower the concentration. It is possible to efficiently promote molecularization and the provision of solubility and dispersibility.

In addition, examples of the polysaccharide that can be used in the present invention according to claims 14 to 17 include the same polysaccharides as those used in the enzymatic saccharification.
For example, cellulose, starch, xylan, pectin, chitin, chitosan, mannan, glucomannan, galactomannan, xyloglucan, fructan, arabinogalactan, filamentous fungal polysaccharide, yeast polysaccharide, seaweed polysaccharide, bacterial polysaccharide, viral polysaccharide, animal polysaccharide Etc.
Among these, especially when cellulose, starch, chitin, etc. are used, the low molecular weight of the hardly degradable polysaccharide and the provision of solubility / dispersibility proceed, but especially for starch, Conversion proceeds very efficiently.
That is, the present invention according to claim 18 is characterized in that at least a part of the polysaccharide contained in the polysaccharide-containing material is at least one selected from starch, cellulose and chitin. It is a method for producing a low molecular carbohydrate or a material imparted with solubility / dispersibility described in any one of the above.

  In the present invention according to claims 14 to 18, the low-molecular-weight carbohydrate is efficiently produced from a material containing the hardly-degradable polysaccharide by a technique for swelling the insoluble hardly-degradable polysaccharide using the strong acid salt and acid. In addition to being able to produce, it is also possible to convert an insoluble, hardly degradable polysaccharide material into a novel polysaccharide material imparted with solubility and dispersibility.

As a method for improving the swelling property of the insoluble hardly degradable polysaccharide, it is expected that the permeability of the swelling substance is improved by once raising the temperature of the reaction solution.
In practice, however, the water molecules are activated as a result of increasing the solubility of the swelling substance due to the temperature rise of the solution. As a result, the interaction between water molecules and the swelling substance is diversified, and the titer of the swelling substance is lowered, so that the entry of the swelling substance into the polysaccharide is prevented, and the structural change of the polysaccharide is unlikely to occur.
In order to avoid this problem, it is expected that the activation of water molecules is suppressed by increasing the temperature once and then decreasing the temperature, thereby suppressing the swelling efficiency.
When the polysaccharide is treated with an aqueous calcium chloride / dihydrate solution, the present inventor performs the low temperature treatment after the high temperature treatment, and the polysaccharide swellability becomes extremely high, and the polysaccharide is in the solution. It is found to be highly dispersed. The present invention includes a technique for combining a plurality of such optimum temperature conditions in each process.

That is, the present invention according to claim 19 uses a calcium chloride or a hydrate thereof, a solution thereof, or a solution having a functionally equivalent activity to the material containing polysaccharide and subjecting it to a high temperature treatment. The method for producing a low molecular carbohydrate or a material imparted with solubility / dispersibility according to any one of claims 14 to 18, further comprising a step of performing a low temperature treatment.
The low molecular weight according to any one of claims 14 to 18, wherein the dispersible and soluble imparted polysaccharide material produced through the step of "low temperature treatment after high temperature treatment" in claim 19 is then applied. It can be converted into a highly swelled polysaccharide material by using it in a method for producing a saccharide or a material imparted with solubility and dispersibility.

The present invention according to claim 20 uses calcium chloride or a hydrate thereof, a solution thereof, or a solution having a functionally equivalent activity to the material containing polysaccharide and subjecting the material containing polysaccharide to high temperature treatment. The method according to claim 1, further comprising a step of performing a low temperature treatment.
The enzymatically saccharified enzyme according to any one of claims 1 to 13, wherein the dispersible and soluble imparted polysaccharide material produced through the "step of applying a low temperature treatment after performing a high temperature treatment" in claim 20 is then applied. By using the method for producing a low molecular weight carbohydrate including a step, glucose or low molecular weight sugar can be efficiently produced from a polysaccharide material.

The high temperature treatment in the present invention according to claims 19 and 20 is a treatment for promoting the invasion of the swelling substance into the crystal by increasing the mobility of the crystal structure of the polysaccharide. Depending on the process temperature. However, in general, the temperature is high enough not to cause thermal decomposition of the polymer, and it is desirable for handling to be not more than the boiling point of the solution used.
Specifically, it is suitable that the temperature is higher than 50 degrees Celsius, preferably 60 to 100 degrees Celsius. Moreover, as time to perform a high temperature process, time longer than 5 minutes, Preferably it is 10 minutes or more, More preferably, 15 minutes-2 hours are suitable.

In addition, the low temperature treatment in the present invention according to claims 19 and 20 is a treatment for increasing the saturation of the swelling substance by accelerating the activation of the swelling substance by lowering the temperature of the solution. The treatment temperature varies depending on the swelling substance, but in general, it is desirable that the temperature reaches a saturation solubility or a temperature that reaches a supersaturated state.
Specifically, it is suitable to carry out at a temperature of 37 degrees centigrade or less, preferably lower than 20 degrees centigrade, more preferably 4 to 16 degrees centigrade. Moreover, there is no restriction | limiting in particular as time to perform a low-temperature process, However, 30 minutes or more are suitable.
It is conceivable that the low-temperature treatment temperature can be increased at a higher temperature by adding a large excess amount of a swelling substance to the solution to increase the temperature at which saturation solubility is reached.

  Here, the present invention according to claim 27 is a starch and / or cellulose and / or chitin imparted with solubility and dispersibility obtained by the method according to any one of claims 14 to 19. For example, when chitin is used as a polysaccharide, it becomes possible to impart solubility and dispersibility to chitin by setting reaction conditions. Moreover, when starch, cellulose, and chitin are used as polysaccharides, it becomes possible to impart solubility and dispersibility to starch, cellulose, and chitin by setting reaction conditions. The reaction product after the treatment can be purified by using a known desalting / separation technique such as ion exchange chromatography or separation by a dialysis membrane.

  The polysaccharide material imparted with solubility and dispersibility thus obtained is expected to improve the suitability of the derivative and the regenerative property, and is expected to lead to the development of new uses for polysaccharide resources. In addition to the possibility of partial molecular reduction due to the acid treatment, it is expected that the utilization is improved by modifying the supramolecular structure of the polysaccharide.

Furthermore, the present inventor has found that further enzymatic saccharification can be performed using the polysaccharide material and / or its residue produced after the chemical solubilization / dispersion reaction as described above.
That is, the present invention according to claim 21 uses the polysaccharide material and / or the residue after the reaction of degrading or solubilizing / dispersing the hardly degradable polysaccharide according to claims 14-19. Furthermore, the present invention relates to a method for producing a low-molecular weight carbohydrate that performs enzymatic saccharification using various polysaccharide hydrolases.

Next, the present inventor performs the swelling of the polysaccharide while suppressing the decay and the degradation of the polysaccharide by storing the aqueous solution of the strong acid salt in a bacteriostatic property and storing the highly decaying material in the aqueous solution. As a result, the present invention according to claim 22 was reached.
In the present invention according to claim 22, since the preservation step can be a step that also serves as a swelling treatment (pretreatment), it is particularly effective when using a highly septic material such as starch squeezed.
Effective salts include calcium chloride, magnesium chloride, ammonium sulfate, sodium sulfate, sodium nitrate, sodium chloride and the like. In the storage / swelling treatment step, calcium chloride and the like have high hygroscopicity, and therefore it is necessary to pay attention to humidity control when performing long-term storage.

  The present invention according to claim 23 is the step of bacteriostatically preserving the polysaccharide-containing material according to claim 22, wherein the strong acid salt is calcium chloride and the solution is converted to calcium chloride dihydrate. The concentration is 10% (w / v) or more.

  In addition, as described in claim 23, the concentration of calcium chloride contained in the solution used for the storage / swelling treatment is 10% (w / v) or more, preferably 25 in terms of calcium chloride / dihydrate. Under conditions of% (w / v) or more, the growth of general microorganisms can be greatly suppressed.

Next, when using plant-derived materials as polysaccharide-containing materials, the present inventor uses a material containing polysaccharides using alkali and hydrogen peroxide as a method for removing lignin or hemicellulose that binds to cellulose. As a result, the present inventors have found a method for performing an oxidation treatment of the present invention.
In addition, as a method for removing lignin, hemicellulose and the like contained in plant-derived polysaccharide-containing materials, an oxidation treatment using an alkali and an oxidizing agent is effective.

Calcium hydroxide, potassium hydroxide and sodium hydroxide are preferably used as the alkali reagent used in the oxidation treatment step in which an alkali and an oxidizing agent are used in combination.
That is, in the oxidation treatment step, the pH of the reaction solution is shifted to the alkali side to activate an oxidation reaction such as a delignification reaction of hydrogen peroxide, thereby enabling removal of lignin, hemicellulose, and the like.
When calcium hydroxide is used as the alkali, there is an advantage when a calcium salt or hydrochloric acid is subsequently used. Similarly, when sodium hydroxide is used, there is an advantage when sodium salt, hydrochloric acid, sulfuric acid or the like is used thereafter.

For example, calcium hydroxide used in the oxidation treatment step according to claim 25 is converted into calcium chloride by hydrochloric acid treatment performed thereafter.
At that time, calcium chloride itself is used as a swelling agent for polysaccharides before treatment with hydrochloric acid, and is therefore suitable for reaction control and reuse because it does not contain other salts.

The low molecular weight carbohydrate obtained from the present invention according to claims 1 to 25 can be subjected to various fermentation processes including ethanol fermentation as it is or as appropriate as described above.
That is, as described in claim 26, ethanol can be produced by fermenting the low molecular weight carbohydrate produced as described above.
What is necessary is just to perform ethanol fermentation in accordance with a conventional method.

Thus, according to the present invention, a salt having an activity of modifying the crystal structure of a polysaccharide or an aqueous solution thereof, a substance having substantially the same activity as the above, or an aqueous solution of urea, or substantially equivalent thereto. Using a substance with the following activity, modify the crystal structure of the polysaccharide-containing material while minimizing the low molecular weight and solubilization of the polysaccharide, and then wash the polysaccharide-containing material as necessary. After that, by carrying out enzymatic hydrolysis and / or hydrolysis with hydrochloric acid to lower the molecular weight and solubilize the polysaccharide, to produce a low molecular weight carbohydrate, and then to adopt this process of ethanol fermentation It is possible to produce ethanol efficiently.
In the present invention, swelling substances such as zinc chloride, calcium thiocyanate, calcium chloride, magnesium chloride, sodium sulfate, and ammonium sulfate work effectively at normal temperature and pressure, thereby increasing the speed of enzymatic saccharification and / or hydrolysis by acid. It can be significantly improved. Moreover, since an effect is recognized on cellulose, starch, and chitin, it can be a breakthrough in a wide variety of biomass conversion technologies.

  Based on the present invention, it is possible to construct a saccharification treatment system and an ethanol production system for materials containing polysaccharides. For example, an apparatus for treating a sample (a material containing polysaccharide) with a swelling substance such as a calcium thiocyanate aqueous solution, a subsequent water washing / dehydrating apparatus, an agitation / temperature control apparatus for performing an enzyme reaction, an enzyme reaction tank, insoluble Of the apparatus for removing fractions, the apparatus for recovering used reagents such as enzymes and calcium thiocyanate, the apparatus for purifying sugar liquid, the apparatus for concentrating sugar liquid, the ethanol fermentation apparatus, the solid-liquid separator, and the distillation apparatus A saccharification treatment system and an ethanol production system characterized by including at least several types can be newly designed.

For example, FIG. 1 is a drawing showing an example of such a saccharification processing system (saccharification processing apparatus).
In FIG. 1, reference numeral 1 is an outer container (made of glass), reference numeral 2 is an inner container (made of plastic; for pretreatment and saccharification), reference numeral 3 is an overflow / stirring slit, reference numeral 4 is a stirrer, and reference numeral 5 is a stirrer. Reference numeral 6 denotes a weight for the stirrer cover (ring).

  As a device required for a saccharification treatment system, a material part (sample) containing a polysaccharide is placed, and a part for processing by adding a reagent that modifies the crystal structure of the polysaccharide (symbol 2: internal container), a sample depending on the reagent After the treatment, the water washing device (reference 2: water is added while stirring in the inner container, overflow: overflowing through the overflow / stirring slit, the insoluble fraction is washed without taking out from the container of reference 2 ), An apparatus portion for performing enzyme hydrolysis (symbol 1: add an enzyme solution into an external container, and stir with a stirrer under constant temperature conditions so that the liquid volume is high enough to communicate with the liquid container and the inner container of symbol 2 Enzyme reaction was carried out while the insoluble matter derived from the sample was reacted without taking it out of the inner container), and the device part for collecting the enzyme reaction solution (reference numeral 3: overflow, stirring slit through the enzyme solution / sugar) The product is equilibrated in the inner and outer containers), and the part of the device that collects the saccharification reaction product (with stirring by the lower stirrer, drains from the drain port at the lower part of the device. Reference 2: Insoluble matter remaining in the inner container The remaining saccharified product is squeezed from the inner container, and the insoluble matter in the inner container is collected separately). The stirrer cover indicated by reference numeral 5 allows the lower stirrer to rotate while the inner container is stably installed. The weight of reference numeral 6 is used for stably installing the stirrer cover indicated by reference numeral 5. Is.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) is weighed into a plastic tube, and at room temperature, 60% (w / v) calcium thiocyanate aqueous solution, 60% (w / v) sodium thiocyanate aqueous solution, 70 % (W / v) zinc chloride aqueous solution, 100% (w / v) calcium chloride dihydrate aqueous solution, 8 M urea or water (1 mL) were added and dispersed by stirring with a vortex mixer for 1 hour. I put it. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer solution (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) and 1 mg previously prepared and maintained at 37 ° C. 1 mL of / mL Novozyme 188 (SIGMA)) was added, and the mixture was stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rpm. After 24 hours, 20 μL was sampled, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, 8.75 mM glucose was produced in the sample pretreated with water.
In contrast, the sample pretreated with 60% (w / v) aqueous calcium thiocyanate solution was 18.8 mM, and the sample pretreated with 60% (w / v) aqueous sodium thiocyanate solution was 17.0 mM, 70% (w / V) 13.3 mM for samples pretreated with aqueous zinc chloride, 17.1 mM for samples pretreated with 100% (w / v) aqueous calcium chloride dihydrate, and for samples pretreated with 8 M urea. 11.1 mM glucose was produced respectively, and all of them were able to greatly improve the saccharification efficiency of the sample.

Example 2
Weigh 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) into a plastic tube, and at room temperature, 60% (w / v) calcium thiocyanate aqueous solution, 100% (w / v) calcium chloride dihydrate 1 mL of the product or water was added, and the mixture was stirred and dispersed with a vortex mixer, and then allowed to stand for 1 hour. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer solution (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) and 1 mg previously prepared and maintained at 37 ° C. 1 mL of / mL Novozyme 188 (SIGMA)) was added, and the mixture was stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rpm. In the middle, 20 μL was sampled and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 2, the decomposition of the one treated with calcium thiocyanate rapidly progressed, and about 15.6 mM glucose was generated in 3 hours, and the one treated with calcium chloride treated with 8.24 mM. Glucose was produced, but the water treatment product remained at about 3.51 mM.
In FIG. 2, ▪ represents a water-treated product, ▲ represents a product subjected to calcium chloride treatment, and ▼ represents a product treated with calcium thiocyanate.

Example 3
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and 1 mL of 50% (w / v) calcium thiocyanate aqueous solution was added at room temperature. After stirring and dispersing with a vortex mixer, the mixture was allowed to stand for a predetermined time as shown in FIG. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. After 24 hours, 20 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared.
As a result, as shown in FIG. 3, after mixing with the calcium thiocyanate aqueous solution, the sample was immediately centrifuged and washed, and the treatment effect was obtained, and a high effect was obtained with a short treatment. It has been suggested.

Example 4
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube and 1 mL of 50% (w / v) calcium thiocyanate at each temperature shown in FIG. The aqueous solution was added and dispersed by stirring with a vortex mixer, and then allowed to stand for 1 hour. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. After 24 hours, 20 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared.
As a result, as shown in FIG. 4, in addition to the effect obtained at room temperature, a high effect was also obtained for a sample subjected to treatment at 0 ° C. which is the lowest temperature among the tested treatment temperatures.

Example 5
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and a calcium thiocyanate aqueous solution (1 mL) or water having a predetermined concentration shown in FIG. 5 at room temperature. After stirring and dispersing with a vortex mixer, the mixture was allowed to stand for 1 hour. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. After 24 hours, 20 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared.
As a result, as shown in FIG. 5, the reaction rate was slightly improved by treatment with 20% (w / v) calcium thiocyanate, and a high effect was observed at 50% or more.

Example 6
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, 30% (w / v) calcium thiocyanate aqueous solution (1 mL), 30% at room temperature. (W / v) sodium thiocyanate (1 mL), 30% (w / v) 0.5 mL of calcium thiocyanate aqueous solution mixed with 0.5 mL of 30% (w / v) sodium thiocyanate, or water was added . They were immediately stirred and dispersed with a vortex mixer and allowed to stand at room temperature for 1 hour. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. After 24 hours, 20 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared.
As a result, the sample pretreated with 30% (w / v) calcium thiocyanate aqueous solution was 10.2 mM, and the sample pretreated with 30% (w / v) sodium thiocyanate was 10.1 mM. In the sample pretreated with a mixture of 0.5 mL of 30% (w / v) aqueous calcium thiocyanate solution and 0.5 mL of 30% (w / v) sodium thiocyanate, 17.1 mM glucose was released.

Example 7
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and an aqueous solution of calcium chloride dihydrate having a predetermined concentration shown in FIG. 6 at room temperature. (1 mL) or water was added, and the mixture was stirred and dispersed with a vortex mixer, and then allowed to stand for 1 hour. Moreover, about 1 thing which added 1 mL of 100% (w / v) calcium chloride dihydrate aqueous solution, it stirred and disperse | distributed with the vortex mixer, Then, it left still at 100 degreeC for 1 hour. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. After 24 hours, 20 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared.
As a result, as shown in FIG. 6, the reaction rate was slightly improved by treatment with 20% (w / v) calcium chloride dihydrate aqueous solution, and a high effect was observed at 80% or more. Moreover, about the data which left still at 100 degreeC for 1 hour, 19.1 mM glucose was released.

Example 8
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and 1 mL of 50% (w / v) calcium thiocyanate aqueous solution was added at room temperature. After stirring and dispersing with a vortex mixer, the mixture was allowed to stand for 1 hour. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated a predetermined number of times.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. After 24 hours, 20 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared. As a result, no decrease in efficiency due to washing was observed.

Example 9
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and 1 mL of 60% (w / v) calcium thiocyanate aqueous solution was added at room temperature. After stirring and dispersing with a vortex mixer, the mixture was allowed to stand. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was stirred and dispersed with a vortex mixer, and allowed to stand for a predetermined time. This was centrifuged to remove the supernatant, and after adding 1 mL of water and stirring and dispersing with a vortex mixer, this was again centrifuged and washed by removing the supernatant.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. One hour later, 50 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared. As a result, when the liberation rate of the 0-hour stationary sample (sample immediately subjected to the second washing) is 100%, the liberation rate is 94.9% for 1 hour and 94.7% for 2 hours. It was 85.2% after standing for 3 hours.

Example 10
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) is weighed into a plastic tube, and 1 mL of water or 5% (w / v) aqueous calcium thiocyanate solution is added at room temperature. After stirring and dispersing with a vortex mixer, the mixture was allowed to stand overnight at minus 20 degrees Celsius. After returning them to room temperature and allowing them to thaw naturally, they were stirred with a vortex mixer to disperse the cellulose powder. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) prepared in advance and maintained at 37 degrees Celsius was added to the resulting precipitate. After adding 1 mL and stirring with a vortex mixer, the enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. Two hours later, 20 μL was sampled, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rates were compared. As a result, when the glucose release rate of the water-treated sample was 100%, the 5% (w / v) calcium thiocyanate aqueous solution-treated sample was improved to 226%.

Example 11
The effects of thiocyanate treatment were examined using samples containing various polysaccharides. (1) Bagasse powder (air dry weight 5 mg), (2) Rice husk pulverized product (air dry weight 5 mg), (3) Okara dry product (air dry weight 5 mg), (4) Cracked chopsticks (air dry weight 5 mg), (5) Cassava starch pomace (wet weight 20 mg), (6) sweet potato starch pomace (wet weight 25 mg), (7) potato starch pomace (wet weight 25 mg), (8) crushed corn cob (wet weight 22 mg) or (9 ) Using potato starch (air-dried weight 5 mg) as a substrate, following the method of Example 2, 1 mL of 60% (w / v) aqueous calcium thiocyanate solution or water was added and dispersed by stirring with a vortex mixer. (1) to (4) were left at 100 degrees Celsius, and the others were left at room temperature for 1 hour. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice.
A cellulase-degrading enzyme (16.5 mg / mL Celluclast 1.5L FG (Novozymes) containing 20 mM sodium acetate buffer (pH 5.5) previously adjusted and maintained at 37 degrees Celsius was added to the obtained precipitate. 1 mg / mL Novozyme 188 (SIGMA)) or amylase-degrading enzyme (2.84 mg / mL Bacillus-derived Type II-A α-amylase (SIGMA) and 1.39 mg / mL Rhizopus glucoamylase (Oriental yeast stock) After adding 1 mL of a solution containing at least one of the companies)) and stirring with a vortex mixer, an enzyme reaction was carried out at 37 degrees Celsius at 12 rpm. 20 μL was sampled after a predetermined time, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rates were compared. The results are shown in Table 1.
As a result, the effect of the treatment with calcium thiocyanate was confirmed. In particular, high starch production was observed for starch pomace from moss.

Example 12
Following the method of Example 2, with bagasse powder (air dried, 5 mg) as substrate, (1) 1 mL of 60% (w / v) aqueous calcium thiocyanate, (2) 1 mL of 60% (w / v) thiocyanic acid 50 μL of methanol or (3) water was added to the aqueous calcium solution, and the mixture was stirred and dispersed with a vortex mixer, and then allowed to stand at room temperature for 1 hour. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice.
A cellulase-degrading enzyme (16.5 mg / mL Celluclast 1.5L FG (Novozymes) containing 20 mM sodium acetate buffer (pH 5.5) previously adjusted and maintained at 37 degrees Celsius was added to the obtained precipitate. After adding 1 mL of a solution containing 1 mg / mL Novozyme 188 (SIGMA)) and stirring with a vortex mixer, an enzyme reaction was performed at 37 degrees Celsius and 12 rotations / minute. After 5 hours, 20 μL was sampled, the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose release rate was compared. As a result, 0.3 mM glucose was produced in (1), 0.67 mM in (2) and 0.195 mM in (3), and the effect of promoting glucose release by adding an organic solvent was observed.

Example 13
Weigh 25 mg of cassava starch pomace into a plastic tube, add 1% each of 60% (w / v) calcium thiocyanate aqueous solution, 100% (w / v) calcium chloride dihydrate or water at room temperature, After stirring and dispersing with a vortex mixer, the mixture was allowed to stand for 1 hour. Thereafter, the supernatant was removed by centrifugation, and 1 mL of water was added to the precipitate and dispersed by stirring with a vortex mixer. Immediately after centrifugation, washing was performed, and this washing operation was repeated once more.
In the resulting precipitate portion, an amylase-based degrading enzyme (0.71 mg / mL Bacillus-derived Type II-A α-amylase (SIGMA) and 0.348 mg / mL Rhizopus-derived glucos) prepared in advance and maintained at 37 ° C 1 mL of amylase (Oriental Yeast Co., Ltd.) was added and stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rotations / minute. 20 hours later
μl was sampled and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, about 18.1 mM glucose was produced in one hour with the calcium thiocyanate treatment, and 24.9 mM glucose was produced with the calcium chloride treatment. It remained at about 3 mM.

Example 14
Place cassava starch pomace (wet weight 32 g) on the bottom of the plastic inner container 2 of the apparatus shown in FIG. 1, and soak it directly with 16 mL of 60% (w / v) aqueous calcium thiocyanate solution. The mixture was allowed to stand at room temperature for 5 minutes. Thereafter, while stirring with the stirrer 4, 700 mL of water was gently added, and washing was performed by overflowing from the overflow / stirring slit 3. Enzyme solution preliminarily heated at 37 degrees Celsius (cellulase-degrading enzyme (0.516 mg / mL Celluclast 1.5L FG (Novozymes)) containing 10 mM sodium acetate buffer (pH 5.5), 0.125 mg / mL Novozyme 188 (SIGMA), 0.102 mg / mL Bacillus-derived Type II-A α-amylase (SIGMA) and 0.174 mg / mL Rhizopus-derived glucoamylase (Oriental Yeast Co., Ltd.) The enzymatic decomposition reaction was carried out while stirring at 37 degrees C. 10 μL was sampled over time, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 7, the liberation of glucose rapidly proceeded immediately after the reaction, and 8 g or more of glucose was produced in about 3 hours after the reaction.

Example 15
In Example 14, the reaction solution obtained after 6 hours was centrifuged (5,830 × g, 10 minutes), and the supernatant was recovered. Bacto-Tryptone (Difco) powder and Yeast Extract (Difco) powder are added to the supernatant to a final concentration of 1% and 0.5%, respectively, and then filtered through a 0.22 μm filter. 5 mL was dispensed into a 15 mL sterile plastic tube. Thereafter, a colony of Saccharomyces cerevisiae NFRI 3053 (IAM 4125) was inoculated, and after capping, it was tilted sideways and cultured with shaking at 30 degrees Celsius and 64 rpm for 15 hours. A medium containing Bacto-Tryptone and Yeast Extract was prepared using water instead of the reaction supernatant, and inoculated and cultured in the same manner. Thereafter, the amount of ethanol produced in the medium was quantified with F-kit ethanol (JK International).
As a result, 0.44% ethanol was produced in the medium containing the enzyme reaction product in Example 14, whereas ethanol was not produced in the medium containing Bacto-Tryptone and Yeast Extract without adding the enzyme reaction product. Did not produce.

Example 16
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and 100% (w / v) calcium chloride dihydrate or 1 mL of water at room temperature. Each was added and dispersed by stirring with a vortex mixer and allowed to stand for 12 hours. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer solution (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) and 1 mg previously prepared and maintained at 37 ° C. 1 mL of / mL Novozyme 188 (SIGMA)) was added, and the mixture was stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rpm. In the middle, 20 μL was sampled and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 8, about 14 mM glucose was produced after 5 hours in the case of the calcium chloride treatment.
In FIG. 8, ■ indicates the one subjected to calcium chloride treatment, and ▲ indicates the one subjected to water treatment.

Example 17
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and 100% (w / v) calcium chloride dihydrate or 1 mL of water at room temperature. Each was added and dispersed by stirring with a vortex mixer and allowed to stand for a predetermined time. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer solution (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) and 1 mg previously prepared and maintained at 37 ° C. 1 mL of / mL Novozyme 188 (SIGMA)) was added, and the mixture was stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rpm. After 24 hours, 20 μL was sampled, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 9, according to the calcium chloride treatment time, the glucose production rate after 24 hours of the enzyme reaction was improved, and the production rate was almost saturated after about 5 hours of treatment.

Example 18
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and 100% (w / v) calcium chloride dihydrate was added to each. After stirring and dispersing with a vortex mixer, the mixture was allowed to stand at a predetermined temperature. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer solution (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) and 1 mg previously prepared and maintained at 37 ° C. 1 mL of / mL Novozyme 188 (SIGMA)) was added, and the mixture was stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rpm. 20 μL was sampled after a certain time, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 10, the calcium chloride treatment temperature conditions, changes in the enzyme reaction rate was observed. Samples that were treated for 24 hours continuously showed higher enzyme reaction rates in the order of 25, 37, 50, and 4 degrees Celsius, especially the sample enzyme treatment efficiency treated at 25 degrees Celsius for 24 hours. Shows the highest value in the treatment section of Example 18.
In contrast, the samples that were treated at 100 degrees Celsius for the first 2 hours and then subjected to each temperature treatment for 22 hours showed a significantly lower enzyme reaction rate than the corresponding 24-hour continuous treatment sample. In particular, the sample treated at 100 degrees Celsius for 2 hours and then treated at 4 degrees Celsius for 22 hours showed the lowest enzyme reaction rate value in the treatment section of Example 18.
However, in the sample treated at 100 degrees Celsius for 2 hours and then treated at 4 degrees Celsius for 22 hours, the cellulose became transparent after the low temperature treatment, and the cellulose recovered as a precipitate by centrifugation was translucent and highly swollen. It was.
From this result, when cellulose is treated with calcium chloride dihydrate aqueous solution, low temperature treatment after high temperature treatment makes the cellulose highly swellable and highly dispersed in the solution. It was revealed.
In FIG. 10, ■ is treated at 100 degrees Celsius for 2 hours and then treated at 4 degrees Celsius for 22 hours, ▲ is treated at 100 degrees Celsius for 2 hours and then treated at 25 degrees Celsius for 22 hours, and ▼ is 100 degrees Celsius Treated for 2 hours at 37 degrees Celsius and then treated for 22 hours at 37 degrees Celsius, ◆ treated for 2 hours at 100 degrees Celsius and then treated for 22 hours at 50 degrees Celsius, ● treated for 4 hours at 4 degrees Celsius, □ indicates that processed for 24 hours at 25 degrees Celsius, Δ indicates that processed for 24 hours at 37 degrees Celsius, and ▽ indicates that processed for 24 hours at 50 degrees Celsius.

Example 19
Weigh 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) into a plastic tube, add 1 mL each of 100% (w / v) calcium chloride dihydrate, and stir with a vortex mixer to disperse. Then, it was allowed to stand at a predetermined temperature (temperature condition 1) for a predetermined time (reaction time 1). Thereafter, the mixture was stirred with a vortex mixer, and immediately left at a predetermined temperature (temperature condition 2) for 30 minutes. The subsequent change of the powder was visually confirmed, and the dispersibility of the cellulose powder was evaluated in the following five stages.
In this evaluation system, if “++++” and “++”, it can be said that it was confirmed to be converted into cellulose having high dispersibility. The results are shown in Table 2.
In the nineteenth embodiment, the temperature condition 1 can be regarded as a “high temperature condition”, and the temperature condition 2 can be regarded as a “low temperature condition”.

[Evaluation of cellulose dispersibility]
+++: Translucent particles are dispersed and transparency is high.
-++: Translucent particles are dispersed.
+: Translucent particles are dispersed but slightly white.
・ ±: Slightly translucent white fine particles are almost precipitated.
-: White particles are almost precipitated.

As a result, as shown in Table 2, it was confirmed that insoluble cellulose powder was converted into highly dispersible cellulose under a plurality of treatment conditions.
Specifically, as shown in Samples 1 to 4, in a sample that was allowed to stand at a temperature of 100 degrees Celsius for “longer than 5 minutes” as a high-temperature condition and was left to stand at a temperature of 4 degrees Celsius for 30 minutes as a low-temperature condition, Although it was observed that the cellulose powder was dispersed as translucent particles having no white turbidity, in the sample in which the treatment time under the high temperature condition was performed only for 5 minutes, the impartment of dispersibility was insufficient.
In addition, as shown in Samples 4 to 9, insoluble cellulose powder in a sample that was allowed to stand for 20 minutes at “temperature higher than 50 degrees Celsius” as a high-temperature condition, and left for 30 minutes at 4 degrees Celsius as a low-temperature condition, Although it was observed that the particles were dispersed as translucent particles without white turbidity, the dispersibility was not sufficiently imparted in the sample subjected to the high temperature condition at 50 degrees.
Furthermore, as shown in Sample 4 and Samples 10-13, the sample that was allowed to stand at 100 degrees Celsius for 20 minutes as a high temperature condition and that was allowed to stand at "temperature lower than 20 degrees Celsius" for 30 minutes as a low temperature condition was insoluble. It was observed that the cellulose powder was dispersed as translucent particles without white turbidity, but white precipitation was observed in the sample subjected to the low temperature condition at 20 degrees.

Example 20
100 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Co., Ltd.) is placed in a 50 mL Erlenmeyer flask together with a magnetic stirrer, and 10 mL of 100% (w / v) calcium chloride dihydrate or water is added thereto. After the addition, 500 μl of concentrated hydrochloric acid was added with stirring at 60 degrees Celsius. Thereafter, 20 μl was sampled over time, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 11, the release of glucose was confirmed only in the sample reacted with the acid in the presence of 100% (w / v) calcium chloride dihydrate. In FIG. 11, ▲ indicates a sample treated with hydrochloric acid in the presence of 100% (w / v) calcium chloride dihydrate, and ■ indicates a sample treated with hydrochloric acid with water.

Example 21
400 mg of cassava pomace (wet weight, as used in Example 11) was placed in a 50 mL Erlenmeyer flask with a magnetic stirrer and 10 mL of 100% (w / v) calcium chloride dihydrate or water. Then, 500 μl of concentrated hydrochloric acid was added with stirring at 60 degrees Celsius. Thereafter, 20 μl was sampled over time, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 12, the release of glucose was confirmed only in the sample reacted with the acid in the presence of 100% (w / v) calcium chloride dihydrate. In FIG. 12, ▲ indicates a sample treated with hydrochloric acid in the presence of 100% (w / v) calcium chloride dihydrate, and ■ indicates a sample treated with hydrochloric acid with water.

Example 22
Place 1.02 g of paper sludge (dark gray, wet weight, water content 52.4%) in a 50 mL Erlenmeyer flask with a magnetic stirrer, and add 10 mL of 100% (w / v) calcium chloride / dihydrate. After adding a Japanese product or water, the mixture was stirred at room temperature for 10 minutes and then allowed to stand overnight at room temperature. It was placed in a 60 ° C. water bath and 3 mL of concentrated hydrochloric acid was added. Thereafter, 20 μl was sampled over time, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 13, high glucose release was confirmed in the sample reacted with acid in the presence of 100% (w / v) calcium chloride dihydrate. In FIG. 13, ▲ indicates a sample treated with hydrochloric acid in the presence of 100% (w / v) calcium chloride dihydrate, and ■ indicates a sample treated with hydrochloric acid with water.

Example 23
Three 200 mg bagasse fibers (used in Example 11) were prepared in a 50 mL Erlenmeyer flask together with a magnetic stirrer (hereinafter referred to as samples (1), (2) and (3)). Samples (1) and (2) were each added with 7 mL of calcium hydroxide saturated water (adjusted by standing at room temperature overnight), and then added with 1 mL of hydrogen peroxide solution. Sample (3) For, 8 mL of water was added and stirred at 37 degrees Celsius for 10 hours. For samples (1) and (2), the ocherous bagasse fiber was greatly decolorized and converted to a slightly yellowish white fiber. The fiber was suction filtered using filter paper and it was washed with a sufficient amount of water.
The fiber was transferred to a 50 mL plastic tube, 5 mL of 100% (w / v) calcium chloride dihydrate was added for sample (1), and water was added for samples (2) and (3). Then, it was left still for 1 hour at 60 degrees Celsius. From there, the fiber was again collected by filtration and washed with a sufficient amount of water. This was put into a 50 mL Erlenmeyer flask together with a magnetic stirrer, and the cellulolytic enzyme reaction solution (20 mM sodium acetate buffer (pH 5.5), 3.30 mg / mL Celluclast 1.5 L) prepared in advance and maintained at 37 degrees Celsius. 10 mL of FG (Novozymes) and 0.2 mg / mL Novozyme 188 (SIGMA)) were added and enzymatic reaction was performed at 37 degrees Celsius. Thereafter, 20 μl was sampled over time, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 14, high release of glucose was confirmed in the sample (1) pretreated with 100% (w / v) calcium chloride dihydrate. Further, comparing the samples (2) and (3), the effect of pretreatment with calcium hydroxide-hydrogen peroxide solution was observed. In FIG. 14, ▲ is a sample (1) pretreated with calcium hydroxide-hydrogen peroxide solution and 100% (w / v) calcium chloride dihydrate, ■ is calcium hydroxide-hydrogen peroxide solution and water The sample pretreated with (2) and ▼ indicates the sample pretreated with water only (3).

Example 24
Prepare two 100mg chitin powder (Kitin EX, Funakoshi Co., Ltd.) in a 50mL Erlenmeyer flask together with a magnetic stirrer, and 10mL each of 100% (w / v) calcium chloride dihydrate or water Then, 1.5 g of calcium chloride dihydrate powder was further added to the former, and 1 mL of concentrated hydrochloric acid was added with stirring at 60 degrees Celsius. Thereafter, 30 μl was sampled over time, 20 μL or 40 μL of 1M aqueous sodium carbonate solution was added, respectively, and the amount of reducing sugar was measured by the Somogy Nelson method. N-acetyl-D-glucosamine was used as a standard substance.
As a result, as shown in FIG. 15, the release of reducing sugar was confirmed only in the sample reacted with an acid in the presence of 100% (w / v) calcium chloride dihydrate. In FIG. 15, ▪ represents a sample treated with hydrochloric acid in the presence of 100% (w / v) calcium chloride dihydrate, and ◆ represents a sample treated with hydrochloric acid with water.

Example 25
Prepare two 100mg chitin powder (Kitin EX, Funakoshi Co., Ltd.) in a 50mL Erlenmeyer flask together with a magnetic stirrer, and 10mL each of 100% (w / v) calcium chloride dihydrate or water Was added, and the mixture was stirred at room temperature for 1 hour and then allowed to stand overnight. Thereafter, 1.5 g of calcium chloride dihydrate powder was further added only to the former. 3 mL of concentrated hydrochloric acid was added thereto, and the mixture was stirred at 37 degrees Celsius for 4 hours. As a result, in the sample treated with acid in 100% (w / v) calcium chloride dihydrate, the suspension became clear. The sample acid-treated in water remained as the original suspension and became a precipitate when stirring was stopped. When the former sample was diluted twice with water and further twice as much ethanol was added, a white fine powder derived from chitin was produced.

Example 26
1 g of chitin powder (Chitin EX, Funakoshi Co., Ltd.) is placed in a 50 mL Erlenmeyer flask together with a magnetic stirrer, and 10 mL of 100% (w / v) calcium chloride dihydrate is added thereto, and then at room temperature. Stir for 2 minutes and then let stand overnight. Thereafter, 3 mL of concentrated hydrochloric acid was added and stirred at 37 degrees Celsius for 1 hour. As a result, the suspension changed to a paste. The white paste recovered by centrifuging this sample (6,850 × g, 4 ° C., 10 minutes) was washed twice with about 60 mL of water, and then suspended in about 20 mL of water. A colloidal chitin-like suspension with high dispersibility was obtained.

Example 27
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, and 62.5% (w / w) magnesium chloride hexahydrate aqueous solution at room temperature. Alternatively, 1 mL of water was added, and the mixture was stirred and dispersed with a vortex mixer, and then allowed to stand overnight. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer solution (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) and 1 mg previously prepared and maintained at 37 ° C. 1 mL of / mL Novozyme 188 (SIGMA)) was added, and the mixture was stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rpm. After 4 hours, 20 μL was sampled, and the amount of glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, in the case of 62.5% (w / w) magnesium chloride hexahydrate treatment, about 6.4 mM glucose was formed after 4 hours, but in the water treatment product, it was 3.5 mM. It was.

Example 28
LB agar medium (10 g / L Bacto-tryptone, 5 g / L Yeast Extract, 10 g / L NaCl in water) containing 0%, 10% or 25% (each w / v) calcium chloride dihydrate. After adjusting the pH to between 6 and 7, add 20 g / L agar, heat, dispense into a plastic petri dish, cool and solidify), scrape a small amount of cells growing on the rot cassava surface, This was suspended in 5 mL of water, and then diluted with water by a factor of 100,000. One day later, the number of colonies present on the plate was 170, 0, and 0, respectively. Further, after 15 days, 1 to several thin colonies grew even in a medium containing 10% calcium chloride dihydrate, but the growth was not good.

Example 29
Following the method of Example 2, 5 mg of cellulose powder (Avicel TYPE FD-101, Asahi Kasei Chemicals Corporation) was weighed into a plastic tube, 500 μL of water (hereinafter referred to as sample (1)), 500 μL of water and concentrated sulfuric acid. 50 μL (hereinafter referred to as sample (2)), 500 μL of sodium sulfate aqueous solution saturated at room temperature (hereinafter referred to as sample (3)), 500 μL of sodium sulfate aqueous solution saturated at room temperature, and 50 μL of concentrated sulfuric acid (hereinafter referred to as “sample”). Sample (4)), 500 μL of aqueous ammonium sulfate saturated at room temperature (hereinafter referred to as sample (5)), 500 μL of aqueous ammonium sulfate saturated at room temperature and 50 μL of concentrated sulfuric acid (hereinafter referred to as sample (6)) .) Were added and stirred by a vortex mixer to disperse, and then allowed to stand for 42 hours. Thereafter, the supernatant was removed by centrifugation, 1 mL of water was added to the precipitate, and the mixture was dispersed by stirring with a vortex mixer, and immediately washed by centrifugation, and this washing operation was repeated twice more.
Cellulose-degrading enzyme reaction solution (20 mM sodium acetate buffer solution (pH 5.5), 16.5 mg / mL Celluclast 1.5L FG (Novozymes)) and 1 mg previously prepared and maintained at 37 ° C. 1 mL of / mL Novozyme 188 (SIGMA)) was added, and the mixture was stirred with a vortex mixer, and then each enzyme reaction was performed at 37 degrees Celsius and 12 rpm. After 1, 2 and 3 hours, 20 μL was sampled, and the glucose level was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.).
As a result, as shown in FIG. 16, the swelling effect by the salt treatment and the swelling effect under the condition where the sulfate aqueous solution and sulfuric acid coexist were observed. In FIG. 16, ■ indicates the sample (1), ▲ indicates the sample (2), ▼ indicates the sample (3), ◆ indicates the sample (4), ○ indicates the sample (5), and □ indicates the sample (6).

According to the present invention, by providing a technique for producing a low molecular weight carbohydrate from a material containing a polysaccharide, in particular, a hardly degradable polysaccharide, it is possible to lead to advanced utilization of carbohydrate biomass targeting ethanol or petroleum substitute products. it can.
Furthermore, according to the present invention, it is possible to greatly accelerate the conversion of biomass into a wide variety of useful materials such as agricultural, forestry and fishery waste, food manufacturing waste, and resource crops. It is expected to contribute to alleviating the problem. The scope of use includes domestic and overseas areas where sugarcane, corn, and moss are used as large-scale plantations as resource crops, and business / living systems such as waste paper, pulp waste liquid and paper sludge, building waste, and distribution waste such as convenience store lunches. It covers urban areas where waste is generated, and areas where agricultural, forestry and fishery waste and community-based food manufacturing waste are generated.

It is explanatory drawing which shows an example of the saccharification processing apparatus of the raw material containing polysaccharide. 3 is a graph showing the enzymatic saccharification rate of cellulose in Example 2. It is a graph which shows the relationship between the time of the pre-processing by the calcium thiocyanate solution in Example 3, and glucose free concentration. It is a graph which shows the relationship between the temperature of the pre-processing by the calcium thiocyanate solution in Example 4, and glucose free concentration. It is a graph which shows the relationship between the calcium thiocyanate density | concentration at the time of the pre-processing by the calcium thiocyanate solution in Example 5, and glucose free density | concentration. It is a graph which shows the relationship between the calcium chloride dihydrate aqueous solution density | concentration at the time of the pre-processing by the calcium chloride dihydrate aqueous solution in Example 7, and glucose free concentration. It is a graph which shows a glucose free concentration change at the time of the enzymatic saccharification of cassava starch pomace in Example 14. 14 is a graph showing the effect of calcium chloride dihydrate aqueous solution treatment on the enzymatic saccharification rate of cellulose in Example 16. It is a graph which shows the relationship between the time of the pre-processing by the calcium chloride dihydrate aqueous solution in Example 17, and glucose free concentration. It is a graph which shows the relationship between the temperature of the pre-processing by the calcium chloride dihydrate aqueous solution in Example 18, and glucose free concentration. 14 is a graph showing the effect of calcium chloride dihydrate aqueous solution on the acid saccharification rate of cellulose in Example 20. 10 is a graph showing the effect of an aqueous calcium chloride dihydrate solution on the acid saccharification rate of cassava pomace in Example 21. 10 is a graph showing the effect of an aqueous calcium chloride dihydrate solution on the acid saccharification rate of papermaking sludge in Example 22. It is a graph which shows the effect of the pretreatment by the calcium hydroxide-hydrogen peroxide solution treatment and the calcium chloride dihydrate aqueous solution on the enzymatic saccharification rate of bagasse fiber in Example 23. 10 is a graph showing the effect of calcium chloride dihydrate aqueous solution on the acid saccharification rate of chitin in Example 24. 10 is a graph showing the effect of sulfate or sulfate containing sulfuric acid on the acid saccharification rate of cellulose in Example 29.

Claims (15)

Calcium thiocyanate, sodium thiocyanate, a hydrate thereof or their aqueous solution at 40 degrees Celsius minus 20 degrees Celsius, by acting against the material including polysaccharides, are included in the material containing the polysaccharide A low-molecular-weight carbohydrate characterized by reducing the molecular structure and solubilization of polysaccharides while minimizing the crystal structure and then performing an enzymatic hydrolysis reaction to lower the molecular weight and solubilize the polysaccharide. Manufacturing method. Using calcium thiocyanate or a hydrate thereof, or an aqueous solution thereof , and acting on a material containing a polysaccharide so that its concentration is 20% (w / v) or more in terms of calcium thiocyanate. The method according to claim 1 . The polysaccharide according to claim 1 or 2 , wherein at least a part of the polysaccharide contained in the polysaccharide-containing material is cellulose, and at least a part of the enzyme is an enzyme having an activity of hydrolyzing a cellulose hydrolase or a partial hydrolyzate of cellulose. The method described. The polysaccharide according to claim 1 or 2 , wherein at least a part of the polysaccharide contained in the polysaccharide-containing material is starch, and at least a part of the enzyme is a starch hydrolase or an enzyme having an activity to hydrolyze a partial hydrolyzate of starch. The method described. The method according to any one of claims 1 to 4 , wherein at least a part of the low molecular weight / solubilized carbohydrate is glucose. The polysaccharide-containing material is obtained from starch extraction residue or sugar juice residue from at least a part of an organ of a plant body in which starch or sugar is accumulated, or a starch or sugar accumulation site in the plant body, the method of any of claims 1-5. The method according to any one of claims 1 to 6 , wherein the material containing polysaccharide is a material containing cellulose by-produced during pulp production, pulp, waste paper, or fiber containing cellulose. Calcium thiocyanate, sodium thiocyanate, their hydrates or their aqueous solution, characterized in that it comprises a polar organic solvent, A method according to any one of claims 1-7. The method according to any one of claims 1 to 8 , wherein the material containing the polysaccharide is washed after modifying the crystal structure of the polysaccharide. A step of adding calcium chloride or a hydrate thereof or a solution in which they are dissolved to a polysaccharide-containing material and subjecting the material to a high temperature treatment at a temperature higher than 50 degrees Celsius , followed by a low temperature treatment at 37 degrees Celsius or less. The method according to any one of claims 1 to 9 , wherein the method for producing a low molecular weight carbohydrate according to any one of claims 1 to 9 is performed . In the step of modifying the crystalline structure of the polysaccharide, salt of a strong acid or a hydrate or in the dissolved solution, characterized by bacteriostatic and store materials containing polysaccharide of claim 1-10 The method according to any one. The method according to claim 11 , wherein the salt of the strong acid is calcium chloride, and the concentration of the strong acid is 10% (w / v) or more in terms of calcium chloride dihydrate in the solution. Performs the step of oxidizing the material comprising the polysaccharide in a solution containing an alkali and hydrogen peroxide, characterized in that a step of subsequently modifying the crystal structure of the polysaccharide, in any one of claims 1 to 12 The method described. The process according to claim 13 , characterized in that the alkali is calcium hydroxide. Preparation of ethanol, characterized in that to produce a low molecular carbohydrate, fermenting the low molecular carbohydrates by the method according to any one of claims 1-14.

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