JP2011010597A - Sugar and method for producing sugar, method for producing ethanol and method for producing lactic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing sugar, a method for producing ethanol and a method for producing lactic acid, in which enzymatic saccharification is efficiently carried out and hence the efficiencies of producing sugar, ethanol and lactic acid are improved, and to provide sugar produced by the method for producing sugar.SOLUTION: The method for producing sugar includes: a process for alkali treatment in which a cellulose fraction whose crystallized type is changed is obtained from the alkali treated products of at least either leaf and stem part of herb material or broad leaf tree material; and a process for saccharification in which the cellulose included in the cellulose fraction is saccharified by enzymes.

Description

  The present invention relates to a sugar and a method for producing sugar, a method for producing ethanol, and a method for producing lactic acid.

  In response to the increasing global needs for bio-combustion, competition for the development of bioethanol production technology has been developed on a global scale. In particular, it is expected that development of utilization technology of cellulosic biomass that does not compete with food resources will be the most important breakthrough not only in Europe and the United States but also in Japan.

  Conventionally, sugars such as sugar cane and starches such as cereals were mainly used as raw materials for bioethanol, but these raw materials are often used as food and feed. Increasing the production of bioethanol used is a concern for affecting the supply of food and feed.

On the other hand, grass and trees do not compete with food production and are useful biomass materials for energy use. Cellulosic biomass materials derived from plants contain cellulose, hemicellulose, and lignin as main components. The cellulose is a fibrous polysaccharide in which glucose is formed in a straight chain by β-1,4-glucoside bonding, and is a main component constituting a cell wall of a plant. The said hemicellulose is a polysaccharide which comprises the cell wall etc. of a plant with the said cellulose. The lignin is a polymer material polymerized using hydroxyphenylpropane as a basic unit. Among these, the cellulose is a natural polymer having the largest abundance on the earth.
Therefore, in recent years, attempts have been widely made to produce ethanol from raw materials containing cellulose such as herbaceous biomass and woody biomass and use them as various fuels and chemical raw materials.

Production of ethanol from cellulosic biomass material can be performed, for example, by decomposing the collected biomass material into sugar by enzymatic saccharification, then fermenting it with a microorganism such as yeast and converting it to ethanol.
In order to extract sugar from a cellulosic biomass raw material, it is necessary to solubilize cellulose and hemicellulose existing in a macromolecule in the plant body and simultaneously decompose them into monosaccharides such as glucose. However, saccharides in cellulosic biomass raw materials are embedded in cell walls having a complex structure, and are pretreated using harsh conditions such as strong acid such as concentrated sulfuric acid and concentrated hydrochloric acid before saccharification. Although the method of separating the quality has been used, it is desired to avoid the use of these strong acids from the viewpoint of reducing the environmental load.

Therefore, in recent years, saccharification of biomass raw materials using enzymes has been widely studied as an alternative to saccharification with strong acids such as concentrated sulfuric acid and concentrated hydrochloric acid. Enzymatic saccharification is a desirable means from the viewpoints of reducing environmental burden, product selectivity, and high yield. Enzymes used for saccharification of cellulosic biomass raw materials are starches that have already been put to practical use. Compared with the amylase addition amount required for enzymatic saccharification, the addition amount of cellulase necessary for saccharifying cellulose is remarkably large, and therefore the increase in sugar conversion cost is a problem.
Moreover, about the enzymatic saccharification of biomass raw material, it is assumed that reaction time will take 24 hours-100 hours or more, and the present condition is that practical bioethanol production technology is hardly developed.

  Furthermore, even if this enzymatic saccharification is directly performed on a biomass raw material, efficient enzymatic saccharification cannot be performed. Cellulose existing in nature has a parallel chain structure called cellulose type I. Adjacent cellulose molecules are aligned in the same direction, and a strong structure is constructed by intramolecular and intermolecular hydrogen bonding. When enzymatic saccharification is performed, it is necessary to guide cellulose molecules to the enzyme active center part while breaking these hydrogen bonds, and since the rate is slow, it is considered that the enzymatic saccharification efficiency is lowered.

  Therefore, it is necessary to pre-process the biomass raw material in advance for the purpose of facilitating the action of the enzyme for enzymatic saccharification. Various methods are known as pretreatment methods for biomass raw materials, but steaming treatment with dilute sulfuric acid, pressurized hot water, and the like are common (see Patent Documents 1 to 4). However, as described above, the use of sulfuric acid is not preferable. Further, when these pretreatments are performed on the biomass raw material and the obtained processed product is subjected to enzymatic saccharification, it is necessary to perform the pretreatment in multiple stages in order to obtain a desired degree of enzymatic saccharification efficiency, This is a problem in that the temperature must be higher than 200 ° C.

  It is also known that chemical and biochemical reactivity can be improved by finely pulverizing biomass raw materials by physical means. However, if sufficient enzymatic saccharification efficiency is obtained by pulverization alone, This is a problem in that it requires a lot of energy and may lose economic rationality.

  In addition, it has been reported that the chemical and biochemical reactivity of the biomass raw material is improved by pretreatment using organic amine or ammonia (see Patent Document 5). Even in the case of biomass, the enzymatic saccharification efficiency is a problem in that it is not yet sufficient.

Furthermore, it is also reported that the biomass raw material is pretreated by supercritical ammonia fluid treatment to change the crystal form of cellulose from cellulose type I to cellulose III type _I to promote enzyme hydrolysis (Patent Document 6). However, with this method, it is necessary to sufficiently reduce the moisture content of the biomass raw material, increase the amount of heat energy used, and develop a combination of expensive ammonia recovery and reuse technologies. In addition, lignin and hemicellulose in the biomass raw material remain, so the waste liquid treatment efficiency is poor, it is difficult to secure the slurry concentration, and the enzyme reuse rate is low to promote nonspecific adsorption of the enzyme. This is a problem in that it is considered that the load on the waste liquid treatment which becomes a stage increases.

  Therefore, it is still desired to develop an enzyme saccharification technology that can further increase the enzyme saccharification efficiency and to develop a pretreatment technology for herbaceous stem and leaf material and hardwood material suitable for the enzyme saccharification.

JP 2006-075007 A JP 2004-121055 A JP 2002-541355 A JP 2002-159954 A European Patent Publication No. 77287 JP 2008-161125 A

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention can efficiently carry out enzymatic saccharification, and therefore can improve sugar production efficiency, ethanol production efficiency, and lactic acid production efficiency. It is an object to provide a method, a method for producing lactic acid, and a saccharide obtained by the method for producing saccharide.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, the cellulose fraction obtained from the processed material of at least one of the herbaceous stem and leaf material and the hardwood material has a very high enzymatic saccharification efficiency. The cellulose fraction includes the herbaceous stem and leaf material. And at least one of cellulose type II and alkali cellulose type IV, which are modified from cellulose type I contained in at least one of the hardwood raw materials and have a crystal density lower than that of cellulose type I, and modification of this crystalline type Can be performed by immersing in an alkaline aqueous solution of a certain concentration or more for several minutes to several hours, the crystal form does not change even after washing with water after the modification, the moisture content of the cellulose fraction When the rate is lowered, modification to cellulose type II occurs, and a cellulose fraction containing a large amount of cellulose type II is obtained. That it is possible to obtain a treated product containing a large amount of alkaline cellulose type IV by maintaining the wet state without drying the cellulose fraction, and the cellulose type II is more enzymatically saccharified than the cellulose type I. The efficiency is improved, and the alkaline cellulose type IV further improves the enzymatic saccharification efficiency as compared with the cellulose type II, and thus the sugar production efficiency, ethanol production efficiency, and lactic acid production efficiency are greatly improved. It is a knowledge that can be made.

  By subjecting at least one of the herbaceous stem and leaf material and the broad-leaved tree material to alkali treatment, the cellulose type I contained in at least one of the herbaceous stem and leaf material and the broad-leaved tree material becomes the alkaline cellulose IV type. It is a new finding according to the present invention that it is modified and irreversibly modified to the cellulose type II after drying.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> An alkali treatment step of obtaining a cellulose fraction containing at least one of cellulose II type and alkali cellulose IV type from at least any one of an alkali-treated product of a herbaceous stem and leaf material and a hardwood material; and the cellulose fraction And a saccharification step in which at least one of the cellulose type II and the alkali cellulose type IV contained is saccharified with an enzyme.
<2> The method for producing a sugar according to <1>, wherein the alkali treatment step includes a washing treatment for washing the alkali-treated product.
<3> The method for producing a sugar according to any one of <1> to <2>, wherein the alkali treatment step includes a separation treatment for solid-liquid separation of the alkali-treated product.
<4> The sugar production method according to any one of <1> to <3>, wherein at least one of cellulose II type and alkali cellulose IV type is an insoluble fraction.
<5> Any of <1> to <4>, wherein the alkali treatment step is a step of obtaining a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV without drying the alkali-treated product. It is a manufacturing method of the described sugar.
<6> The method for producing a sugar according to any one of <1> to <5>, wherein the content of alkali cellulose IV type is 90% by mass or more based on the total cellulose content.
<7> The above <1> to <4>, wherein the alkali treatment step is a step of obtaining a cellulose fraction containing at least one of cellulose II type and alkali cellulose IV type by reducing the water content of the alkali-treated product. Or a sugar production method according to any one of the above.
<8> The method for producing a sugar according to <7>, wherein the content of alkali cellulose type II is 90% by mass or more based on the total cellulose content.
<9> The method for producing a sugar according to any one of <1> to <8>, wherein the alkali treatment step includes a pH adjustment treatment for adjusting a pH of the cellulose fraction.
<10> The sugar production method according to any one of <1> to <9>, wherein the enzyme is an enzyme group containing at least one of cellulase and β-glucosidase.
<11> A saccharide obtained by the method for producing a saccharide according to any one of <1> to <10>.
<12> A method for producing ethanol, wherein the sugar according to <11> is fermented to obtain ethanol.
<13> A method for producing lactic acid, wherein lactic acid is obtained by fermenting the sugar according to <11>.

  According to the present invention, the conventional problems can be solved, the object can be achieved, and enzymatic saccharification can be efficiently performed. Therefore, sugar production efficiency, ethanol production efficiency, and lactic acid production The sugar obtained by the manufacturing method of saccharide | sugar, the manufacturing method of ethanol, the manufacturing method of lactic acid, and the manufacturing method of the said saccharide | sugar which can improve production efficiency can be provided.

FIG. 1 is a diagram showing the saccharification rate of alkali-treated microcrystalline cellulose. FIG. 2 is a graph showing the change over time of the saccharification rate after alkali treatment of microcrystalline cellulose. FIG. 3 is a graph showing the saccharification rate of the sugarcane bagasse pulverized product and the rice straw pulverized product after alkali treatment. FIG. 4 is a graph showing the change over time of the saccharification rate after alkali treatment of the pulverized sugarcane bagasse. FIG. 5 is a graph showing the saccharification rate after alkali treatment of sugarcane bagasse pulverized material (herbaceous stem and leaf material), quercus pulverized material (hardwood material) and cedar pulverized material (coniferous material). FIG. 6 is a diagram showing the saccharification rate after alkali treatment of various plants that are herbaceous stem and leaf material. FIG. 7A is a diagram showing an X-ray diffraction pattern of microcrystalline cellulose (cellulose type I). FIG. 7B is a diagram showing an X-ray diffraction pattern of cellulose II obtained by subjecting microcrystalline cellulose to alkali treatment and then drying. FIG. 7C is a diagram showing an X-ray diffraction pattern of alkali cellulose type IV obtained by subjecting microcrystalline cellulose to an alkali treatment and then maintaining a wet state without drying. FIG. 8 is a diagram in which projected views of the molecular chain axes of the molecular chain arrangement in the cellulose II type and alkaline cellulose IV type unit cells are superimposed. FIG. 9A is a diagram showing an X-ray diffraction pattern of an untreated product (cellulose type I) of pulverized sugarcane bagasse. FIG. 9B is a diagram showing an X-ray diffraction pattern of cellulose II obtained by subjecting a pulverized sugarcane bagasse to an alkali treatment and then drying. FIG. 9C is a diagram showing an X-ray diffraction pattern of alkali cellulose type IV obtained by maintaining a wet state without drying after pulverized sugarcane bagasse, after alkali treatment.

(Method for producing sugar)
The method for producing sugar according to the present invention includes an alkali treatment step for obtaining a cellulose fraction containing at least one of cellulose II type and alkali cellulose IV type from at least any one of an alkali-treated product of a herbaceous stem and leaf material and a hardwood material. A saccharification step in which at least one of the cellulose type II and the alkali cellulose type IV contained in the cellulose fraction is saccharified with an enzyme, and further includes other steps as necessary.

<Alkali treatment process>
The alkali treatment step is a step of obtaining a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV from an alkali-treated product of at least one of a herbaceous stem and leaf part raw material and a hardwood raw material. In addition, other processing is included. The alkali treatment step is advantageous in that it can directly treat at least one of the herbaceous stem and leaf material and the hardwood material.

<< Raw material >>
The raw material used in the alkali treatment step is not particularly limited as long as it is at least one of a herbaceous stem and leaf part raw material and a hardwood raw material, and can be appropriately selected according to the purpose, but a raw material containing a large amount of cellulose is preferable. A raw material containing cellulose type I is more preferred. Use of the raw material containing cellulose type I is advantageous in that a large amount of sugar can be obtained.

-Herbaceous stem and leaf material-
The herbaceous stem and leaf material is not particularly limited and may be appropriately selected according to the purpose.For example, sugarcane bagasse, sorghum bagasse, rice straw, wheat straw, barley, suki, johnsongrass, napiergrass, switchgrass, area Nsass, corn stover, etc.

-Raw material for hardwood-
The hardwood raw material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Japanese oak, beech, drill, maple, mulberry, cus, cypress, oak, wig, and hippo.

−Use−
The herbaceous stem and leaf material and the broad-leaved tree material may be a residue obtained by squeezing or separating a valuable material such as sugar from the various herbaceous stem and leaf material and the various broad-leaved tree material. The cellulose I type obtained by the above may be used. The herbaceous stem and leaf material and the hardwood material may be used alone or in combination of two or more.
Cellulose type I, which is a natural type cellulose, is classified into cellulose type I _α type and cellulose type I _β type. The cellulose type I contained in the herbaceous stem and leaf material and the hardwood material is any of these. Or both of them may be used.

  The state of the herbaceous stem and leaf material and the hardwood material is not particularly limited and may be appropriately selected according to the purpose. The collected material may be used as it is, or may be used to some extent by cutting, pulverizing, or the like. You may use it after making it the following magnitude | sizes, and you may use the residue after squeezing.

The size of at least one of the herbaceous stem and leaf material and the hardwood material that has been cut is not particularly limited and may be appropriately selected depending on the intended purpose. For example, 20 cm ³ or less is preferable, and 1 cm ³ or less is preferable. Is more preferable. If the size exceeds 20 cm ³ , the treatment may be insufficient, or fibers softened during washing may be entangled. On the other hand, when the size is within the preferable range, it is advantageous in that the treatment time can be shortened and the capacity of the aqueous alkaline solution used can be reduced.
The size of at least one of the pulverized herbaceous stem and leaf material and the hardwood material is not particularly limited and may be appropriately selected depending on the purpose. For example, the diameter is preferably 10 mm or less, and preferably 2 mm or less. Is more preferable. When the size is within the preferred range, it is advantageous in that the treatment time can be shortened and the volume of the aqueous alkaline solution used can be reduced.
When the alkali-treated product is solid-liquid separated by filtration to obtain the cellulose fraction as an insoluble fraction, the mesh size of the screen used for the filtration may be about 0.5 mm. In such a case, it is preferable that the insoluble fraction has a size that does not pass through the mesh size.

<< Processing method >>
A method for obtaining an alkali-treated product of at least one of the herbaceous stem and leaf material and the broadleaf tree material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples include a method in which an alkaline aqueous solution described later is added to at least one of the raw materials and the desired temperature and time are allowed to act.

  There is no restriction | limiting in particular as the frequency | count of adding the said alkaline aqueous solution, According to the objective, it can select suitably, For example, the method of making alkaline aqueous solution of a desired density | concentration act once, the method of acting multiple times, etc. Can be mentioned. There is no restriction | limiting in particular as the method of making it act multiple times, According to the objective, it can select suitably, For example, after making a dilute alkaline aqueous solution act, a concentrated alkaline aqueous solution is made to act, and alkaline aqueous solution is made to act in two steps. (Hereinafter, sometimes referred to as “two-stage alkali treatment method”). The two-stage alkali treatment method is advantageous in that the alkaline aqueous solution can be efficiently reused. In addition, the two-stage alkali treatment method is effective when treating a broad-leaved tree material whose degree of lignification has progressed.

  The method of allowing the alkaline aqueous solution to act is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include a method of standing and a method of stirring. Among these, a method of stirring is included. , Which is preferable in terms of improving processing efficiency.

-Alkaline aqueous solution-
The alkaline aqueous solution is not particularly limited as long as it is at least an alkaline aqueous solution, and can be appropriately selected according to the purpose, and further contains other components as necessary.
There is no restriction | limiting in particular as a kind of said alkaline aqueous solution, According to the objective, it can select suitably, For example, sodium hydroxide, potassium hydroxide, etc. are mentioned. The said alkaline aqueous solution may be used individually by 1 type, and may use 2 types together. Among these, sodium hydroxide is preferable because it is inexpensive and has a low environmental load.

  Other components in the alkaline aqueous solution are not particularly limited and may be appropriately selected depending on the purpose. For example, an oxidizing agent such as oxygen, an alkali such as calcium hydroxide, and an organic substance such as a surfactant. Etc. When the alkaline aqueous solution contains the other components, it is advantageous in that the treatment efficiency and the recovery effect of the alkaline aqueous solution can be improved.

  The amount of the alkaline aqueous solution used is not particularly limited and may be appropriately selected according to the amount of use, the size, etc. of at least one of the herbaceous stem and leaf material and the hardwood material. At least one of the raw material and the hardwood raw material is preferably 50 parts by mass to 3,000 parts by mass, more preferably 100 parts by mass to 2,000 parts by mass with respect to 100 parts by mass. If the amount used is less than 50 parts by mass, at least one of the herbaceous stem and leaf material and the hardwood material may not be sufficiently immersed, resulting in insufficient treatment and poor treatment efficiency. On the other hand, when the amount used is within the preferred range, it is advantageous in that the processing time can be shortened. In addition, when the water content of at least one of the herbaceous stem and leaf material and the hardwood material is extremely high, it is preferable to add a minimal amount of concentrated alkaline aqueous solution so as not to increase the amount of liquid.

There is no restriction | limiting in particular as a density | concentration of the said aqueous alkali solution, Although it can select suitably according to the objective, When performing the said alkali treatment by 1 time, 2N-5N are preferable and 3N-5N are more preferable. If the concentration is less than 2N, the treatment may be insufficient, and if it exceeds 5N, the efficiency of the treatment may deteriorate. On the other hand, when the concentration is within the preferable range, it is advantageous in that the treatment time can be shortened and the amount of the alkaline aqueous solution used can be reduced. In addition, when the moisture content of at least one of the herbaceous stem and leaf material and the hardwood material is extremely high, it is possible to maintain the efficiency of the treatment without increasing the liquid volume by adding a minimal amount of a concentrated alkaline aqueous solution. preferable.
There is no restriction | limiting in particular as a density | concentration of the said dilute alkali aqueous solution in the case of performing the said 2 step | paragraph alkali treatment method, Although it can select suitably according to the objective, 0.1N-5N are preferable, and 0.3N-2N are More preferred. There is no restriction | limiting in particular as a density | concentration of the said concentrated alkaline aqueous solution in the case of performing the said 2 step | paragraph alkali treatment method, According to the objective, it can select suitably, For example, the alkaline aqueous solution in the case of performing the said alkali treatment once Examples include the same concentration.

In the alkali treatment step, the alkaline aqueous solution is used after burning the hemicellulose, lignin, and other components eluted from at least one of the herbaceous stem and leaf material and the hardwood material in the alkali treatment step. This is advantageous in that it can be reused and release of substances such as lignin into the environment can be avoided.
About the technique which reuses the said aqueous alkali solution, there exists abundant knowledge in the pulp industry, for example, a conventional method can be used.

-Processing temperature-
In the alkali treatment step, the temperature at which the aqueous alkali solution acts is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably −40 ° C. to 100 ° C., more preferably 0 ° C. to 50 ° C. . When the temperature is lower than −40 ° C., the processing cost may increase. When the temperature is higher than 100 ° C., side reactions are liable to occur and the processing cost may increase. On the other hand, when the concentration is within the preferred range, it is advantageous in that the treatment time can be shortened and the amount of the alkaline aqueous solution used can be reduced.

-Processing time-
In the alkali treatment step, the time for which the alkaline aqueous solution is allowed to act is not particularly limited, and a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV is obtained from the alkali-treated product of a desired degree. Can be appropriately selected within the range in which the temperature can be reduced, but is preferably 10 minutes to 24 hours, more preferably 30 minutes to 4 hours. When the time is less than 10 minutes, it may be impossible to obtain a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV from a desired degree of the alkali-treated product. As a whole, it may become inefficient. On the other hand, when the time is within the preferable range, it is advantageous in that a cellulose fraction containing at least one of cellulose type II and alkali cellulose IV type can be efficiently obtained from the alkali-treated product.

<< Alkali-treated product >>
The alkali-treated product is a treated product obtained by adding the alkaline aqueous solution to at least one of the herbaceous stem and leaf material and the broad-leaved tree material and allowing the above-mentioned desired temperature and time to act.
There is no restriction | limiting in particular as a state of the said alkali processed material, According to the objective, it can select suitably, For example, a liquid state, a solid state, etc. are mentioned. Among these, in order to obtain a high concentration of a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV, a solid state is preferable.

<< Other processing >>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, a washing process, a separation process, a pH adjustment process etc. are mentioned.

-Cleaning treatment-
The alkali treatment step preferably includes a washing treatment for washing the alkali-treated product. The washing treatment is advantageous in that unnecessary substances such as lignin contained in the alkali-treated product can be removed and the pH of the alkali-treated product can be neutralized.
There is no restriction | limiting in particular as a solution used for the said washing | cleaning, According to the objective, it can select suitably, For example, water, dilute alkaline aqueous solution, etc. are mentioned, Among these, water can adjust pH to neutrality. This is preferable.
As the solution used for the washing, water already used for washing, dilute alkaline aqueous solution, or the like can be used. It is advantageous in that the amount of water used can be reduced by using the water, dilute alkaline aqueous solution, or the like. In addition, when using the said water repeatedly, it is preferable to remove and use the alkali which melt | dissolved in water by washing | cleaning, the lignin contained in the said alkali processed material. The method for removing the alkali or lignin is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a method of separation by membrane separation, an ion exchange method, an adsorption method, and combustion to collect water vapor. The method etc. are mentioned. Among these, the method of burning and collecting water vapor is advantageous in that alkali can be regenerated.
The number of times of washing is not particularly limited as long as the alkali-treated product can be made near neutral after washing, and can be appropriately selected according to the purpose.

-Separation process-
The alkali treatment step preferably includes a separation treatment for solid-liquid separation of the alkali aqueous solution and the alkali-treated product. The separation treatment is advantageous in that a high concentration of a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV can be secured.
The solid-liquid separation method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a centrifugal separation method and a filtration method.
There is no restriction | limiting in particular as an order which performs the said isolation | separation process, According to the objective, it can select suitably, For example, before the said washing | cleaning process, simultaneously with the said washing | cleaning process, after the said washing | cleaning process etc. are mentioned.
When the solid-liquid separation is performed by the separation treatment, the alkali-treated product is separated as an insoluble fraction in the solid phase, and the unnecessary substance such as lignin is separated as a water-soluble fraction in the liquid phase.

In the separation step, the insoluble cellulose type I in the vicinity interacts inside at least one of the herbaceous stem and leaf material and the hardwood material, thereby converting the crystal form between adjacent celluloses. This is advantageous in that most of the cellulose can be recovered as an insoluble fraction.
In addition, since the liquid phase can be combusted as a waste liquid, the processing cost of lignin, which is difficult to treat the waste liquid, can be suppressed, the discharge of the waste liquid into the environment can be avoided, and the load on the environment is small. This is advantageous in that a high concentration of a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV can be secured. It is also advantageous in that the recovery rate of the enzyme used in the saccharification step described later is increased.

<< Cellulose fraction >>
The cellulose fraction is a fraction containing at least one of cellulose type II and alkali cellulose type IV.
At least one of the cellulose type II and the alkaline cellulose type IV is obtained by applying the alkaline aqueous solution to at least one of a herbaceous stem and leaf part raw material and a hardwood raw material containing the cellulose type I. The cellulose I type contained in the herbaceous stem and leaf material and the broad-leaved tree material by reducing the water content of the alkali-treated product, if necessary, by washing treatment and separation treatment. It is preferable that at least a part is modified to at least one of the cellulose type II and the alkali cellulose type IV.

-Content-
The content of at least one of the cellulose type II and the alkali cellulose type IV in the cellulose fraction is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferable that at least one of the types of cellulose IV is large, and among these, it is more preferable that the amount of the alkali cellulose IV type is large in that excellent saccharification efficiency can be exhibited.
Further, the cellulose fraction may be, for example, cellulose I type (cellulose I _α type, cellulose I _β type) or other components other than at least one of the cellulose II type and the alkali cellulose IV type, for example, Hemicellulose, lignin, etc. may be contained. However, from the viewpoint of improving the efficiency of enzymatic saccharification, it is preferable that lignin is not contained or the content thereof is small.

The alkali cellulose type IV can be suitably obtained by maintaining a wet state without drying the cellulose fraction.
The wet state is not particularly limited as long as the cellulose fraction contains moisture, and can be appropriately selected according to the purpose. However, the moisture content of the cellulose fraction is 5% by mass or more. Preferably, 20 mass% or more is more preferable. When the water content is within the preferable range, it is advantageous in that the alkali fraction IV can be contained at a high concentration in the cellulose fraction.
When the cellulose fraction contains the alkali cellulose type IV in a high concentration, the content of the alkali cellulose type IV with respect to the total cellulose is not particularly limited and can be appropriately selected according to the purpose. The mass% or more is preferable in that excellent saccharification efficiency can be exhibited. Moreover, not drying the cellulose fraction is advantageous in that cost and labor are not required.

The cellulose type II can be suitably obtained by reducing the water content of the cellulose fraction, and the cellulose type II can be further contained at a higher concentration by completely drying.
The method for reducing the water content is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a heat drying method, a wind drying method, a freeze drying method, a vacuum drying method, and a spraying method. The method of drying, the method of combining these, etc. are mentioned.
When the cellulose fraction contains the alkali cellulose type II in a high concentration, the content of the alkali cellulose type II with respect to the total cellulose is not particularly limited and may be appropriately selected according to the purpose. The mass% or more is preferable.

-PH adjustment treatment-
It is preferable that the alkali treatment step includes a pH adjustment treatment for adjusting the pH of the cellulose fraction because saccharification described later can be efficiently performed.
There is no restriction | limiting in particular as said pH, Although it can select suitably according to the objective, pH3-pH10 is preferable and pH5-pH8 is more preferable.
There is no restriction | limiting in particular as a solution used for the said pH adjustment, According to the objective, it can select suitably, For example, hydrochloric acid, an acetic acid, a sulfuric acid, a citric acid, phosphoric acid, a carbon dioxide etc. are mentioned.

-Storage method-
The method for storing the cellulose fraction is not particularly limited and may be appropriately selected depending on the intended purpose. However, a method of storing in a sodium azide aqueous solution is preferable. The cellulose type II can be stored even in a dried state.
There is no restriction | limiting in particular as a density | concentration of the said sodium azide aqueous solution, According to the objective, it can select suitably.

-Confirmation of modification-
In the cellulose fraction, at least a part of the cellulose type I contained in the alkali-treated product of at least one of the herbaceous stem and leaf material and the hardwood material is at least one of the cellulose type II and the alkaline cellulose type IV. There is no restriction | limiting in particular as a method of confirming that it changed into the key, According to the objective, it can select suitably, For example, it can confirm by X-ray diffraction, FT-IR, solid state NMR etc.

  The cellulose type I X-ray diffraction peak has one peak at 2θ of 22 ° (eg, FIG. 7A), while the cellulose II type X-ray diffraction peak has 2θ of 12 °, 20 °. It has peaks at ° and 22 ° (eg, FIG. 7B). In addition, the alkali cellulose type IV has a peak at 2 ° of 20 ° and 22 ° as in the case of the cellulose type II, and a large peak at a position of 27 ° that the cellulose type I and the cellulose type II do not have. (For example, FIG. 7C). The modified cellulose can be confirmed by the difference in these peaks.

<Saccharification process>
The saccharification step is a step of saccharifying at least one of the cellulose type II and the alkali cellulose type IV contained in the cellulose fraction with an enzyme. It is advantageous in that an excellent saccharification efficiency can be exhibited by using a cellulose fraction containing at least one of the cellulose type II and the alkali cellulose type IV for saccharification with an enzyme in a wet state. The cellulose fraction can be added again in the saccharification step.

<< Enzyme >>
There is no restriction | limiting in particular as an enzyme used for the said saccharification process, Although it can select suitably according to the objective, It is an enzyme group containing at least any one of a cellulase and (beta) -glucosidase, and exhibits outstanding saccharification efficiency It is preferable in that it can be performed.
Cellulase is a general term for enzymes that hydrolyze cellulose. Broadly speaking, exo-type cellobiohydrolase that liberates cellobiose from the end of cellulose, crystalline cellulose cannot be decomposed, but amorphous cellulose (amorphous cellulose) chains. There are three types of endo-type endoglucanase that cleaves randomly, and exo-type β-glucosidase that produces glucose from the ends of cellobiose and short chains (cello-oligosaccharides), and there are various types of enzymes.

-How to obtain-
The method for obtaining an enzyme group containing at least one of the cellulase and the β-glucosidase is not particularly limited and can be appropriately selected depending on the purpose. At least one of the cellulase and the β-glucosidase can be selected. Examples thereof include a method of cultivating and obtaining the produced bacteria, a method of obtaining it from a commercial product, and the like.
There is no restriction | limiting in particular as a microbe which produces at least any one of the said cellulase and the said (beta) -glucosidase, According to the objective, it can select suitably, For example, filamentous fungi, bacteria, basidiomycetes, etc. are mentioned.

-Usage-
There is no restriction | limiting in particular as the usage-amount of the enzyme group containing at least any one of the said cellulase and the said (beta) -glucosidase, According to the objective, it can select suitably, For example, it is 0 with respect to 100 mass parts of said cellulose fractions. 0.01 parts by mass to 20 parts by mass is preferable, and 0.1 parts by mass to 5 parts by mass is more preferable. If the amount of the enzyme used is less than 0.01 parts by mass, enzyme saccharification may be insufficient, and if it exceeds 20 parts by mass, the amount of enzyme used increases and the efficiency deteriorates. On the other hand, when the amount of the enzyme used is within the preferable range, it is advantageous in that the amount of sugar obtained is larger than the amount of enzyme added.

-Temperature-
There is no restriction | limiting in particular as temperature in the said saccharification process, Although it can select suitably according to the objective, For example, 4 to 100 degreeC is preferable and 25 to 80 degreeC is more preferable. If the temperature is less than 4 ° C, enzyme saccharification may not proceed sufficiently, and if it exceeds 100 ° C, the enzyme may be deactivated. On the other hand, when the temperature is within the preferable range, it is advantageous in that a large amount of sugar is obtained with respect to the amount of enzyme added.

-PH-
There is no restriction | limiting in particular as pH in the said saccharification process, Although it can select suitably according to the objective, For example, pH3-pH10 is preferable and pH5-pH8 is more preferable. If the pH is less than 3 or exceeds 10, the enzyme may be deactivated. On the other hand, when the pH is within the preferable range, it is advantageous in that a large amount of sugar is obtained relative to the amount of enzyme added.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, the process etc. which cut and grind | pulverize at least any one of the said herbaceous stem and leaf part raw material and the said hardwood raw material.

<< Cutting and grinding process >>
In the alkali treatment step, it is preferable that at least one of the herbaceous stem and leaf part raw material and the hardwood raw material containing the cellulose type I is cut and pulverized in advance in that the treatment with the aqueous alkali solution proceeds efficiently.
There is no restriction | limiting in particular as an apparatus used for the said cutting and grinding | pulverization, According to the objective, it can select suitably, For example, a wheelie mill, a grinder, a cutter mill, a hammer mill, a pin mill etc. can be used.

<Application>
Since the method for producing sugar can efficiently carry out enzymatic saccharification, it can be suitably used for the method for producing ethanol and the method for producing lactic acid of the present invention described later.

(sugar)
The sugar of the present invention is a sugar obtained by the sugar production method. The sugar is a substance obtained by reducing the molecular weight of a polysaccharide with an enzyme or acid. The sugar obtained by the sugar production method is not particularly limited as long as it uses cellulose as a substrate, and can be appropriately selected according to the purpose. Examples thereof include glucose, cellobiose, and cellooligosaccharide. .

<Application>
The sugar can be suitably used, for example, in the ethanol production method and lactic acid production method of the present invention described later.

(Ethanol production method)
The method for producing ethanol of the present invention includes a step of fermenting the sugar obtained by the above-described method for producing sugar of the present invention to obtain ethanol (hereinafter sometimes referred to as “alcohol fermentation step”). If necessary, other steps are further included.

<Alcohol fermentation process>
In the method for producing ethanol, the method for fermenting the sugar is not particularly limited and may be appropriately selected depending on the purpose. However, alcohol fermentation microorganisms such as yeast are added to the sugar to perform alcohol fermentation. The method to be performed is preferable.

<< Yeast >>
There is no restriction | limiting in particular as said yeast, According to the objective, it can select suitably, For example, Saccharomyces genus yeast etc. are mentioned. The yeast may be natural yeast or genetically modified yeast. Specific examples of the ethanol-fermenting microorganism include Saccharomyces cerevisiae , Kluyveromyces fragilis , Kluyveromyces lactis ( K. lactis ) and Kluyveromyces. K. marxianus), Pichia Sutipitisu (Pichia stipitis), Pichia pastoris (P. pastoris), Pachisoren-Tan'nofirusu (Pachysolen tannophilus), Candida Gurabirata (Candida glabrata) yeast or of genes recombinants such as, Zaimomonazu mobilis (Zymomonas mobilis), Saimo Compactors Palme (Zymobacter palmae), Clostridium thermocellum (Clostridium thermocellum), Clostridium Rujungudari (C.ljungdahlii) may be used bacteria or these genes recombinant such.

  In the alcohol fermentation step, the amount of yeast used, fermentation temperature, pH, fermentation time and the like are not particularly limited, and are appropriately selected according to, for example, the amount of sugar to be used for alcohol fermentation, the type of yeast to be used, etc. can do.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, the process etc. which isolate | separate and refine | purify ethanol obtained by the said alcohol fermentation process are mentioned. The method for separation and purification is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include distillation.

<Application>
Ethanol obtained by the ethanol production method can be suitably used as, for example, fuel ethanol, industrial ethanol, and the like. Since the ethanol can be obtained from at least one of the herbaceous stem and leaf material and broad-leaved tree material, it can be reproduced as long as it can produce a plant that becomes at least one of the herbaceous stem and leaf material and broad-leaved tree material, In addition, since the plant absorbs carbon dioxide in the atmosphere at the time of cultivation, even if the ethanol is burned to generate carbon dioxide, the concentration of carbon dioxide in the atmosphere is not increased. Therefore, it can be said that ethanol is a desirable energy source for preventing global warming. In recent years, such ethanol is particularly expected to be mixed with gasoline and used as an environmentally friendly automobile fuel.

  An alcohol other than ethanol is produced by fermenting the sugar obtained by the sugar production method of the present invention with a microorganism that produces the desired alcohol in place of the above-mentioned yeast that produces ethanol. You can also For example, butanol can be produced by fermentation using acetone / butanol bacteria.

(Production method of lactic acid)
The method for producing lactic acid of the present invention includes a step of fermenting the saccharide obtained by the above-described method for producing saccharide of the present invention to obtain lactic acid (hereinafter sometimes referred to as “lactic acid fermentation step”). If necessary, other steps are further included.

<Lactic acid fermentation process>
In the method for producing lactic acid, the method for fermenting the sugar is not particularly limited and may be appropriately selected depending on the purpose. However, a lactic acid-fermenting microorganism such as lactic acid bacteria is added to the solution containing the sugar, A method of performing lactic acid fermentation is preferred.

<< Lactic acid bacteria >>
The lactic acid bacterium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Lactobacillus manihotolans ( Lactobacillus manihot IV type orans ), Lactobacillus plantarum ( Lactobacillus plantarum ), Streptococcus thermos Examples thereof include Streptococcus thermophilus and Lactobacillus bulgaricus . The lactic acid bacterium may be a natural lactic acid bacterium or a genetically modified lactic acid bacterium.

  In the lactic acid fermentation step, the amount of lactic acid bacteria used, fermentation temperature, pH, fermentation time, etc. are not particularly limited, and are appropriately selected according to, for example, the amount of sugar to be used for lactic acid fermentation, the type of lactic acid bacteria to be used, etc. can do.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, the process etc. which isolate | separate and refine | purify the lactic acid obtained by the said lactic acid fermentation process. The separation / purification method is not particularly limited and may be appropriately selected depending on the intended purpose.

<Application>
The lactic acid obtained by the lactic acid production method can be suitably used for producing polylactic acid by chemical polymerization, for example. At present, it is desirable that lactic acid, which is often produced from starch such as corn, can be produced from at least one of the herbaceous stem and leaf material and the hardwood material containing cellulose that cannot be used for food. According to this production method, it is possible to efficiently produce polylactic acid from at least one of the herbaceous stem and leaf part raw materials and hardwood raw materials containing such cellulose.

  The saccharide obtained by the method for producing saccharides of the present invention is fermented by using microorganisms that produce the desired organic acid in place of the lactic acid bacteria, so that organic acids other than lactic acid, such as citric acid and succinic acid, can be obtained. Acid, malic acid, oxalic acid and the like can also be produced. In addition to organic acids, various fermented industrial products such as food materials such as vitamins, amino acids and sugar alcohols, and chemical raw materials such as industrial alcohol raw materials can be produced.

  EXAMPLES The present invention will be specifically described below with reference to examples of the present invention, but the present invention is not limited to these examples.

(Test Example 1: Improvement of saccharification efficiency by modification of cellulose crystal type)
<Preparation of alkali cellulose type IV>
1 g of microcrystalline cellulose (Funacel; manufactured by Funakoshi Co., Ltd.), which is cellulose type I, was dispersed in 150 mL of 5N aqueous sodium hydroxide solution and gently stirred at room temperature for 1 hour to obtain an alkali-treated product. The alkali-treated product was centrifuged at 5,000 × g for 3 minutes, and then the supernatant was removed. The residue was dispersed by adding 150 mL of water, washed with water, and centrifuged at 5,000 × g for 3 minutes. This water washing was repeated until the alkali-treated product became neutral, and then a cellulose fraction was prepared, and an alkali cellulose type IV was prepared by maintaining a wet state without drying.
Using a portion of the cellulose fraction, freeze-drying, and measuring the mass of the obtained solid content, the solid content concentration in the cellulose fraction is determined, and the concentration is determined as the concentration of alkaline cellulose IV. did. In addition, the solid content mass of the cellulose type II and the alkali cellulose type IV is the same (see Hisao Nishimura and Anatole Sarko, Macromolecules, 1991, 24, p. 771-778).

<Preparation of cellulose type II>
Cellulose type II was prepared by freeze-drying the cellulose fraction prepared in the same manner as described above.

<Enzymatic saccharification>
10 mg each of alkali cellulose type IV, cellulose type II, and microcrystalline cellulose (cellulose type I) prepared as described above were used as a comparative control, and acetate buffer (final concentration 50 mM acetic acid, 0.02 mass% sodium azide). , PH 4.5) was added to a final concentration of 1.0% by mass. Next, cellulase (Celluclast 1.5L; manufactured by Novozymes Japan) and β-glucosidase (Novozyme 188; manufactured by Novozymes Japan) were respectively added to a final concentration of 0.1 mg / mL or 0.5 mg / mL. Enzymatic saccharification was performed by adding to each cellulose and reacting at 37 ° C. and 12 rpm for 24 hours using a thermoblock rotating machine (SN-48BN, manufactured by Nisshin Rika Co., Ltd.). After the reaction, the enzyme reaction was stopped by keeping the thermoblock at 100 ° C. and holding for 15 minutes.

<Calculation of saccharification rate>
<< Measurement of glucose concentration >>
As described above, alkaline cellulose type IV, cellulose type II, and microcrystalline cellulose (cellulose type I), which are substrates for enzymatic saccharification, are enzymatically saccharified, then centrifuged at 10,000 × g for 5 minutes, and in the supernatant. The glucose was colored with a glucose test CII Wako (manufactured by Wako Pure Chemical Industries, Ltd.), and the glucose concentration was measured with an ultraviolet-visible spectrophotometer (UVmini-1240; manufactured by Shimadzu Corporation). From this glucose concentration, the amount of glucose liberated from 1 g of enzyme saccharification substrate was calculated.

<< Measurement of glucan content in enzyme saccharification substrate >>
The glucan content contained in alkaline cellulose type IV, cellulose type II, and microcrystalline cellulose (cellulose type I), which are substrates for enzymatic saccharification, was examined by the following method. That is, 20 mg of each cellulose before enzymatic saccharification was used, dispersed in 1 mL of 72 mass% sulfuric acid, treated at 30 ° C. for 1 hour, diluted 8 times with pure water, and further at 100 ° C. for 2 hours. This was treated to obtain a hydrolyzed solution (hereinafter sometimes referred to as “two-stage sulfuric acid treatment”). Calcium carbonate powder was added to a part of this hydrolyzed solution to neutralize it. Subsequently, centrifugation was performed at 10,000 × g for 5 minutes, and the glucose concentration in the supernatant was measured by the same method as the glucose measurement method. From this glucose concentration, the glucan content per gram of enzyme saccharification substrate was calculated.

<< Calculation method of saccharification rate >>
Using the amount of glucose released from 1 g of the enzyme saccharification substrate and the glucan content per g of the enzyme saccharification substrate, the saccharification rate was calculated from the following calculation formula (1).
Saccharification rate (mass%) = 100 × (amount of glucose liberated from 1 g of enzyme saccharification substrate × 0.9) / glucan content per g of enzyme saccharification substrate—calculation formula (1)
In the calculation formula (1), “0.9” is a conversion factor considering the number of glycosidic bonds of the polysaccharide.

<Result>
The saccharification rate is shown in FIG. From FIG. 1, cellulose type I, cellulose type II, and alkaline cellulose type IV were all saccharified depending on the enzyme concentration, but cellulose type II and alkaline cellulose type IV had a saccharification rate as compared with cellulose type I. The saccharification rate of alkali cellulose type IV was particularly high.
Moreover, the time-dependent change of the saccharification rate at the time of using 0.5 mg / mL of enzyme groups is shown in FIG. The saccharification rate of cellulose type I for the first hour was as low as 9% by mass. On the other hand, the saccharification rate of cellulose type II for the first hour was 13% by mass, and the saccharification rate of alkali cellulose type IV for the first hour was 31% by mass. From these results, cellulose type II had a faster saccharification rate than cellulose type I, and alkaline cellulose type IV was even faster.

(Test Example 2: Examination of saccharification efficiency by alkali treatment of herbaceous stem and leaf material)
<Preparation of alkali cellulose type IV>
Sugarcane bagasse and rice straw were used as herbaceous stem and leaf material. Sugarcane bagasse and rice straw were crushed and passed through a 2 mm mesh filter. Alkaline cellulose type IV was prepared in the same manner as in Test Example 1, except that 6 g each of this sugarcane bagasse pulverized product and rice straw pulverized product were used, and 150 mL of 5N aqueous sodium hydroxide was allowed to act for 3 hours. .

<Preparation of cellulose type II>
Cellulose type II was prepared in the same manner as in Test Example 1, except that 6 g each of the sugar cane bagasse pulverized product and the rice straw pulverized product were used.

<Preparation of cellulose III type _I by supercritical ammonia fluid treatment>
Use 1g each of sugarcane bagasse pulverized product and rice straw pulverized product, put it in a 30mL pressure vessel, seal it, and cool the container to -13 ° C with a cooling device, and inject ammonia at a constant pressure of 0.5MPa for 30 minutes. The sugarcane bagasse pulverized product and the rice straw pulverized product were each completely immersed in liquid ammonia. Subsequently, the heating thru | or pressurization process for 1 hour were performed at 140 degreeC. At this time, it was confirmed that the pressure in the container was 11 MPa or more. After the supercritical ammonia fluid treatment, ammonia gas was leaked. By drying the treated cellulose, cellulose III type _I was prepared by the supercritical ammonia fluid treatment.

<Enzymatic saccharification>
Using alkaline cellulose type IV and cellulose type II prepared as described above, enzymatic saccharification was performed in the same manner as in the case where the enzyme group in Test Example 1 was 0.5 mg / mL. Further, as a comparative control, cellulose III type _I obtained as described above, untreated sugarcane bagasse ground product (cellulose type I), and untreated rice bran ground product (cellulose type I) were used in the same manner. Enzymatic saccharification was performed.

<Measurement of glucose concentration and calculation of saccharification efficiency>
After enzymatic saccharification of alkali cellulose type IV, cellulose type II, cellulose III type _I , untreated sugarcane bagasse pulverized product (cellulose type I) and untreated rice bran pulverized product (cellulose type I), the same as in Test Example 1 The amount of glucose released from 1 g of enzyme saccharification substrate and the glucan content per gram of enzyme saccharification substrate were calculated by the above method, and the saccharification rate was calculated from the above formula (1).

<Result>
The saccharification rate is shown in FIG. From FIG. 3, saccharification was hardly recognized in the untreated sugarcane bagasse pulverized product (cellulose type I) and the untreated rice bran pulverized product (cellulose type I). Cellulose III type _I which was treated with supercritical ammonia fluid had a low saccharification rate of about 25% by mass in both sugarcane bagasse pulverized product and rice straw pulverized product. On the other hand, the saccharification rate of the cellulose type II subjected to the alkali treatment was as good as about 55% by mass. Furthermore, the saccharification rate of alkali cellulose type IV was about 80% by mass in the pulverized sugarcane bagasse, which was a particularly good value.
FIG. 4 shows the change over time in the saccharification rate of the pulverized sugarcane bagasse. Similar to the results of Test Example 1, cellulose type II had a faster saccharification rate than cellulose type I, and alkaline cellulose type IV was even faster.

(Test Example 3: Examination of carbohydrate recovery rate by alkali treatment of sugarcane bagasse ground product)
<Method>
In the alkali treatment process, washing out of low-molecular sugars and solids may occur when washing is performed. Therefore, the recovery rate of sugars using the cellulose fraction prepared from the ground sugarcane bagasse in Test Example 2 was examined. Went.
10 mg each of the cellulose fraction and untreated sugarcane bagasse pulverized product (cellulose type I) were used and subjected to a two-step sulfuric acid treatment in the same manner as in Test Example 1 to obtain a hydrolyzed solution. Calcium carbonate powder was added to a part of the sample to neutralize it. Subsequently, centrifugation was performed at 10,000 × g for 5 minutes, and glucose and xylose in the supernatant were measured by the same method as described above. The glucan content per gram of each cellulose was calculated from the measured glucose concentration. Moreover, the xylan content per 1 g of each cellulose was calculated from the measured xylose concentration.

<< Calculation method of carbohydrate recovery rate >>
The glucan recovery rate of the cellulose fraction was calculated from the following calculation formula (2), and the xylan recovery rate of the cellulose fraction was calculated from the following calculation formula (3).
Glucan recovery rate (mass%) = 100 × glucan content per gram of cellulose fraction / glucan content per gram of untreated sugarcane bagasse pulverized product: Formula (2)
Xylan recovery rate (mass%) = 100 × xylan content per gram of cellulose fraction / xylan content per gram of untreated sugarcane bagasse crushed product (Formula 3)

<Result>
The results are shown in Table 1 below. From Table 1, although most of xylan was flowing out in the cellulose fraction obtained through the alkali treatment step, there was almost no glucan outflow. From this, it was shown that since the alkali treatment step does not flow out glucan, the crystal form of cellulose can be efficiently modified from cellulose type I to cellulose type II and alkaline cellulose type IV.

(Test Example 4: Comparison of alkali treatment for herbaceous stem and leaf material, hardwood material, and coniferous material)
<Preparation of alkali cellulose type IV>
Test Example 1 except that 1 g of each of the herbaceous shoots of sugarcane bagasse, crushed crickets of hardwood, and cedar that is crushed softwood is used, and the 5N aqueous sodium hydroxide solution is 200 mL. In the same manner, alkali cellulose type IV of each raw material was prepared.

<Enzymatic saccharification>
Enzymatic saccharification was performed in the same manner as in the case where the enzyme group of Test Example 1 was 0.5 mg / mL, using the alkali cellulose type IV of each raw material obtained as described above. In addition, enzymatic saccharification was performed in the same manner using untreated sugarcane bagasse pulverized product, corn pulverized product, and cedar pulverized product as a comparative control.

<Measurement of glucose concentration and calculation of saccharification efficiency>
About each processed material after the reaction in enzyme saccharification, the glucose concentration was measured by the same method as in Test Example 1, and the saccharification rate was calculated.

<Result>
The saccharification rate is shown in FIG. From FIG. 5, the saccharification rate of the cedar (the coniferous raw material) after the alkali treatment was about 40% by mass. On the other hand, sugarcane bagasse (herbaceous stem and leaf material) and quercus (hardwood material) are treated with alkali and modified to alkaline cellulose IV type, so that the saccharification rate is around 90% by mass or more, which is a breakthrough. Improved. From these results, it was confirmed that the alkali treatment showed a marked improvement in saccharification efficiency with respect to herbaceous stem and leaf material and hardwood material.

(Test Example 5: Examination of two-step alkali treatment method for hardwood raw materials)
<Preparation of alkali cellulose type IV>
20 g of a 0.3N sodium hydroxide aqueous solution was added to 1 g of the pulverized Japanese oak (hardwood material) prepared in Test Example 4 and treated in a pressure vessel at 100 ° C. for 1 hour (first-stage alkali treatment). Next, after centrifuging at 5,000 × g for 3 minutes and removing the supernatant, 200 mL of 5N aqueous sodium hydroxide (concentrated alkali) and NaBH ₄ were added to a final concentration of 0.01% by mass, and the mixture was brought to room temperature. The mixture was gently stirred for 1 hour (second stage alkali treatment). After the second-stage alkali treatment, the mixture was centrifuged at 5,000 × g for 3 minutes, and 400 mL of water was added to the residue, dispersed and washed, and centrifuged at 5,000 × g for 3 minutes. This washing with water was repeated until the treated product became neutral.
The first-stage alkali treatment and the second-stage alkali treatment may be hereinafter referred to as “two-stage alkali treatment method”.

<Enzymatic saccharification>
Enzymatic saccharification was carried out in the same manner as in Example 1 where the enzyme group in Test Example 1 was 0.5 mg / mL, using each raw material alkaline cellulose type IV obtained by the two-step alkali treatment method. In addition, enzymatic saccharification was performed in the same manner using untreated pulverized materials of each raw material as a comparative control.

<Measurement of glucose concentration and calculation of saccharification efficiency>
About each processed material after the reaction in enzyme saccharification, the glucose concentration was measured by the same method as in Test Example 1, and the saccharification rate was calculated.

<Result>
The results are shown in Table 2 below. From Table 2, when the two-step alkali treatment method was performed, the saccharification rate in the enzyme saccharification reaction for 24 hours was about 90% by mass in Quercus (a hardwood raw material). On the other hand, the saccharification rate in the case of only the first-stage alkali treatment was about 40% by mass.

  From Test Examples 4 to 5, it was shown that the modification of cellulose crystal type by treatment with 5N sodium hydroxide is effective in the hardwood raw material having a degree of lignification. In the two-step alkali treatment method, the saccharification rate is good, and further, a part of lignin is removed by treatment with 0.3N sodium hydroxide in the first step, so that it is used in the second step due to an elution substance such as lignin. Contamination of concentrated alkali (5N sodium hydroxide aqueous solution) is prevented, and it is advantageous in that concentrated alkali can be used repeatedly. In addition, it is advantageous in that the amount of the alkaline aqueous solution can be reduced by diluting the waste liquid in the treatment with 5N sodium hydroxide and using it in the first stage.

(Test Example 6: Improvement of saccharification rate by alkali treatment on herbaceous stem and leaf material)
<Preparation of alkali cellulose type IV>
Barley (Cultivar: Silky Snow), Wheat (Cultivar: Yumesihou), Susuki (Cultivar: Yatsushiro), Switchgrass (Cultivar: Konlow), Johnson Grass, Napiergrass (Cultivar: Merker), Eliansus ( The pulverized product of 8 kinds of herbaceous stem and leaf material of cultivar line: KO1 and KO2) was thoroughly washed with pure water, dried at 65 ° C. for 3 days, and then pulverized again. Alkaline cellulose IV of each raw material was tested in the same manner as in Test Example 1, except that 140 mg of each of the above 8 kinds of herbaceous stem and leaf material was used and 2.8 mL of 5N sodium hydroxide aqueous solution was used. A mold was prepared.

<Enzymatic saccharification>
Enzymatic saccharification was performed in the same manner as in the case where the enzyme group of Test Example 1 was 0.5 mg / mL using each raw material alkaline cellulose type IV prepared as described above. In addition, enzymatic saccharification was performed in the same manner using untreated pulverized materials of each raw material as a comparative control.

<Measurement of glucose concentration and calculation of saccharification efficiency>
About each processed material after the reaction in a saccharification process, the glucose concentration was measured by the method similar to Test Example 1, and the saccharification rate was computed.

<Result>
The saccharification rate is shown in FIG. From FIG. 6, the glucose recovery after the enzymatic saccharification reaction was improved to 80% by mass to 90% by mass by performing alkali treatment on any herbaceous stem and leaf material.
From these results, even when alkaline treatment is performed as a pretreatment for the enzymatic saccharification reaction of the herbaceous stem and leaf material, there is almost no loss of glucan and the efficiency of the enzymatic saccharification reaction is dramatically improved. It was found that most of the glucan in the raw material can be recovered as glucose.

(Test Example 7: Measurement of X-ray diffraction of cellulose type II and alkali cellulose type IV)
<Method>
In Test Example 1, 100 mg each of cellulose type II and alkali cellulose type IV prepared from microcrystalline cellulose (funacell) and untreated microcrystalline cellulose was used as a comparative control, and the X-ray diffraction pattern was measured under the following conditions. I did it.
Measuring conditions Measuring device: RINT2000 (manufactured by Rigaku Corporation)
X-ray output (Cu Kα): 38 kV, 50 mA
X-ray wavelength: λ = 0.15418 nm
Scanning range: 2θ = 6 ° -30 °, Δ2θ = 0.1 °
Measurement time at each step: 20 seconds Slit: DS = 0.5 °, SS = 0.5 °, RS = 0.15 mm

  7A shows an X-ray diffraction pattern of untreated microcrystalline cellulose (cellulose type I), FIG. 7B shows an X-ray diffraction pattern of cellulose type II after alkali treatment, and FIG. 7C shows an X-ray diffraction pattern of alkali cellulose type IV. Indicates.

The three equator peaks (lattice spacing (d)) characteristic of cellulose appearing in FIG. 7B were determined using the Bragg equation. The crystallite size (L) is a surface interval in a direction perpendicular to each surface, and was obtained using a Scherrer equation represented by the following calculation equation (4).
L = 0.9λ / (H cos θ) (4)
In the calculation formula (4), “L” represents the crystal size, “λ” represents the X-ray wavelength, “H” represents the full width at half maximum, and “θ” represents the Bragg angle. The degree of crystallinity (CrI) was calculated from the ratio of the area based on crystals to the total area. The results are shown in Table 3 below.

<Result>
From FIGS. 7A to 7C and Table 3, in cellulose type II and alkali cellulose type IV, a large peak on the low angle side as in cellulose type I was not observed.
Cellulose type II showed lattice spacing (d) (three peaks on the low angle side) (FIG. 7B), but the crystal size (L) was comparable to cellulose type I (Table 3). .
The X-ray diffraction pattern of alkali cellulose type IV, which has been kept wet without being dried after alkali treatment, is greatly different from cellulose type II which has been dried after alkali treatment, and has a large broad peak at 2θ around 27 °. It was recognized (indicated by a dashed ellipse in FIG. 7C). This is a diffuse scattering due to excess water. Further, the peak at the lowest angle side of the lattice spacing (d) was broader than that of cellulose type II and was shifted by 1 ° to the low angle side (indicated by an arrow in FIG. 7C). Furthermore, the crystal size (L) was 1.8 nm, which was significantly different from cellulose type I and cellulose type II.

From the results of X-ray diffraction, FIG. 8 shows a projection of the molecular chain axis of the molecular chain arrangement in the unit cell of cellulose type II and alkali cellulose type IV. In FIG. 8, the c-axis indicates upward from the paper surface. The cellulose molecular chain sheet deposited by hydrophobic bonding (indicated by an ellipse in FIG. 8) was held in both the crystal form of cellulose type II and alkali cellulose type IV. However, since alkali cellulose type IV has water molecules inserted between them, the interval between the (1-10) planes of cellulose type II was 0.726 nm, whereas alkali cellulose type IV (1 The interval between the (10) planes was 0.787 nm (Table 3). Alkaline cellulose type IV swells 8.4% in the [1-10] direction (indicated by an arrow in FIG. 8) when the interval between (1-10) planes of cellulose type II is 100%. It was. This molecular chain sequence is described in Hisao Nishimura and Anatolite Sarko, Macromolecules, 1991, 24, p. 771-778, which is considered to be consistent with alkali cellulose type IV.
In Table 3, the crystal size (L) of the alkaline cellulose type IV in the [1-10] direction was 1.8 nm, and the cellulose type II was 4.2 nm. This is presumably because the cellulose type II was dried, so that water molecules were released from the hydrophobic binding sheet and shrunk in the [1-10] direction, so that the crystal size was twice or more that of the alkali cellulose type IV.

(Test Example 8: Measurement of X-ray diffraction of sugarcane bagasse alkali treated product)
<Method>
Measurement of X-ray diffraction pattern under the same conditions as in Test Example 2 using 100 mg of cellulose II and alkaline cellulose IV prepared from pulverized sugarcane bagasse and 100 mg of untreated crushed sugarcane bagasse as a comparative control in Test Example 2 Was done.

<Result>
FIG. 9A shows an X-ray diffraction pattern of an untreated sugarcane bagasse pulverized product, FIG. 9B shows an X-ray diffraction pattern of cellulose II after alkali treatment, and FIG. 9C shows an X-ray diffraction pattern of alkali cellulose IV type.
From these results, even when the sugarcane bagasse pulverized product was alkali-treated, three peaks were observed in cellulose type II, which was dried after alkali treatment, as in Test Example 7, but without drying after alkali treatment. In alkali cellulose IV maintained in a wet state, a large broad peak is observed at 2θ of around 27 ° (indicated by a dashed ellipse in FIG. 9C), and the peak on the lowest angle side of the lattice spacing (d) is Compared with cellulose type II, it was broad and shifted by 1 ° to the low angle side (indicated by an arrow in FIG. 9C). Alkaline cellulose type IV that promotes saccharification efficiency showed a pattern different from cellulose type II in X-ray diffraction, and it was recognized that cellulose type II and alkaline cellulose type IV were clearly different.

  The sugar production method of the present invention can efficiently carry out enzymatic saccharification, and therefore, a sugar production method capable of improving sugar production efficiency, ethanol production efficiency, and lactic acid production efficiency, It can use suitably for the manufacturing method of ethanol, and the manufacturing method of lactic acid.

Claims (11)

An alkali treatment step of obtaining a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV from at least any one of the herbaceous stem material and the hardwood material;
A saccharification step of saccharifying at least one of the cellulose type II and the alkali cellulose type IV contained in the cellulose fraction with an enzyme;
A method for producing sugar, comprising:   The method for producing sugar according to claim 1, wherein the alkali treatment step includes a washing treatment for washing the alkali-treated product.   The method for producing sugar according to any one of claims 1 to 2, wherein the alkali treatment step is a step of obtaining a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV without drying the alkali-treated product. .   The method for producing a sugar according to any one of claims 1 to 3, wherein the content of alkali cellulose IV type is 90% by mass or more based on the content of total cellulose.   The alkali treatment step is a step of obtaining a cellulose fraction containing at least one of cellulose type II and alkali cellulose type IV by reducing the water content of the alkali-treated product. A method for producing sugar.   The method for producing sugar according to claim 5, wherein the content of cellulose type II is 90% by mass or more based on the total cellulose content.   The method for producing sugar according to any one of claims 1 to 6, wherein the alkali treatment step includes a pH adjustment treatment for adjusting the pH of the cellulose fraction.   The method for producing sugar according to any one of claims 1 to 7, wherein the enzyme is an enzyme group containing at least one of cellulase and β-glucosidase.   A sugar obtained by the method for producing a sugar according to any one of claims 1 to 8.   A method for producing ethanol comprising fermenting the sugar according to claim 9 to obtain ethanol.   A method for producing lactic acid, wherein the saccharide is fermented to obtain lactic acid.