JP2010035431A - Method for saccharifying rice - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently and stably recovering saccharides in a rice straw by considering saccharification ability and crushing ability of each position of the rice straw. <P>SOLUTION: The method for saccharifying rice includes cutting the stem and leaf parts of the rice so that the fragments of 70% or more of the whole by weight may have a length of not more than 10 cm, separating culms, and stem and leaf parts except the culms from the cut stem and leaf parts, and enzymically saccharifying the fraction in which the culms are concentrated in a condition containing at least one of an enzyme selected from a cellulase, an amylase, a β-(1-3) or (1-4) glucanase, and a hemicellulase. The method for producing ethanol uses the method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for recovering sugar from rice.

  In response to the growing global needs for biofuels, competition for the development of bioethanol production technology derived from carbohydrate-based biomass is taking place on a global scale. In particular, it is considered that the development of utilization technology of lignocellulosic biomass that does not compete with food resources can be the most important breakthrough not only in Europe and the United States but also in Japan.

  The development of saccharification technology for lignocellulosic biomass has a history of 200 years, but is now active again. In particular, enzyme saccharification technology centered on cellulase is attracting high expectations instead of saccharification technology centered on acid saccharification. However, in general, carbohydrates in lignocellulosic biomass are embedded in cell walls having a complex structure, and it is necessary to separate carbohydrates by pretreatment under severe conditions prior to saccharification. For this reason, the conversion cost is increased, and the process of saccharifying the sugar in the cell wall into a raw material for processing is not economically independent at the present time.

  Rice straw is a major herbaceous biomass raw material for agricultural waste. With regard to the development of efficient saccharification technology using rice straw as raw material, it is possible to consider agricultural policy and biomass policy in Japan, the United States, China, South Korea, Vietnam, Thailand, etc. We are gathering expectations. In Japan, the development of domestic biofuel production technology using rice straw, the most abundant agricultural waste resource, has become an urgent issue. Moreover, when the saccharification technology of the whole rice plant body as a whole plant raw material is completed, it is expected to exhibit high effectiveness in agricultural structural reform.

  When saccharifying rice, what is harvested from the field is used as a raw material, but in general, after harvesting the above-ground part of the plant body, it is threshed appropriately, and further to reduce the bulk during collection and packing The cut material is used for preprocessing and conversion.

  As an agricultural machine used for recovering herbaceous plant cell walls such as rice straw, there is a knotter (binding device). This is attached to the rice combine, and it has a device that binds without cutting and drops it backwards for later use of rice straw. Also known is a type of system in which rice straw is scooped up with a compact baler and formed into a square shape while being pulled by a tractor, and a roll baler string is wound around and released. Similarly, there is a machine called a roll baler which is molded and packed into a cylindrical shape, and a work machine that is sealed with a film that wraps the roll bale with a bale wrapper is used (see Non-Patent Document 1). In addition, after harvesting rice, methods such as sun-drying, roll bale treatment, ammonia treatment, urea treatment, and drying treatment, which are performed after bundling the straw, have been developed in order to collect and preserve the straw part or the whole above-ground part of rice.・ It is being considered.

  Examples of the herbaceous plant cell wall crusher include a hammer shredder, a planter, a two-shaft low-speed crusher, a KDS Micronex technology, a vibration mill, a cutter mill, and a refiner. A hammer shredder is a device that crushes long wood wastes and the like by rotating a large hammer (see Non-Patent Document 2). A planting machine is a swelling crushing machine, and is an apparatus that easily changes a strong woody structure and obtains a soft material while having properties close to blasting (see Non-Patent Document 3). The biaxial low-speed crusher has a structure in which biaxial teeth mesh with each other, and is a device that cuts by a strong shear force generated on the outer peripheral side surface of the rotary blade (see Non-Patent Document 4). KDS Micronex technology is a technology introduced from Canada FASC, and is a technology that crushes biomass and dries the pulverized product. A vibration mill is an impact force of a rod or a ball, a cutter mill is a cutting force by a cutter inside the device, and a refiner is a grinding device that uses frictional force (see Non-Patent Document 5).

  However, these technologies are stable for easily degradable carbohydrates in rice stalks and leaves such as starch, free sugar (sucrose, fructose, glucose), β- (1 → 3), (1 → 4) -glucan. This is a method that does not take recovery into consideration at all, and a technique for efficiently recovering these substances has not been studied at all. In addition, when the rice stalks and leaves such as rice straw are crushed and pre-treated and saccharified, the whole rice stalks and leaves are crushed and combined, and there are both highly pulverizable and low pulverized parts. In consideration of the structure of rice shoots and leaves, no technology has been developed for saccharification by adding a fractionation and grinding process. Considering the stable recovery of readily degradable carbohydrates, there is a concern that the wear and tear will be severe when left under conditions of high moisture content at room temperature. It becomes expensive.

Hitoshi Shigeta: Current situation of rice straw collection and packing machine and future development direction, No. 18, pp.23-28, 2006 Nobuhiro Tsumura: Introduction of Wood Crusher “Hama Shredder” Introduction of Recycling Crusher, Environmental Purification Technology, Vol. 3, No. 6, pp.51-53,73, 2004 Kazuo Hirata: Utilization of Biomass in Textile Machines-Dream Material Fluffy-, Construction Planning, 667, pp.27-31, 2005 Shojiro Yano: Biomass utilization and solid handling Biomass crushing and grinding technology, powder and industry, Vol. 38, No. 12, pp. 47-54, 2006 Shinsuke Kobayashi et al .: Journal of the Japan Institute of Energy, Vol. 86, No. 9, pp.730-735, 2007

  Optimum flexibility and adaptability while keeping the easily degradable sugar mass in the straw stable and finely modifying the separation technology of straw and other parts taking into account the variety of rice straw varieties and cultivation conditions It is important to make the separation technology possible. It is also important to optimize the enzymatic saccharification efficiency by improving the enzymatic saccharification efficiency of koji, improving the saccharification efficiency of cellulose and hemicellulose, and elucidating the mechanism of improving the saccharification efficiency.

  As a result of detailed analysis of the accumulation sites of readily degradable carbohydrates in rice stalks and leaves, the present inventor has confirmed that these exist mainly in the cocoon part. In addition, the cocoon part has higher mechanical strength than the other parts in the foliage, and it has been found that the crushing efficiency of the remaining part is improved by removing this part. Based on these findings, the present invention has been completed.

That is, the present invention according to claim 1, after cutting the rice shoots and leaves so that 70% or more of the whole fragments have a length of 10 cm or less by weight ratio, A rice saccharification method characterized by enzymatically saccharifying a fraction enriched with cocoons after separating the shoots from the shoots and leaves and separating the shoots and other shoots and leaves.
The present invention according to claim 2, after subjecting “the fraction enriched with koji” to pulverization and / or heat treatment at 80 ° C. to 130 ° C., cellulolytic enzyme, starch degrading enzyme, β- ( 2. The rice saccharification according to claim 1, wherein enzymatic saccharification is performed under conditions containing at least one kind of enzyme selected from the group consisting of 1 → 3), (1 → 4) glucan-degrading enzyme and hemicellulose-degrading enzyme. Saccharification method.
In the present invention according to claim 3, the fraction other than the “fraction enriched with cocoons” obtained by separating the cocoon and other stems and leaves is pulverized, then acid-treated, The rice saccharification method according to claim 1 or 2, wherein at least one kind of pretreatment selected from the group consisting of alkali treatment and hydrothermal treatment is performed, and enzymatic saccharification is performed.
The present invention according to claim 4 is characterized in that the rice shoots and leaves are the above-ground part of the plant body after cutting the above-ground part of rice and separating the straw or a cut product thereof. This is a method for saccharification of rice.
The present invention according to claim 5 is characterized in that enzymatic saccharification is carried out after storing “the fraction enriched with koji” in a state where the water content is lowered to 20% (w / w) or less. Item 5. The method for saccharification of rice according to any one of Items 1 to 4.
The present invention according to claim 6 is the rice according to any one of claims 1 to 5, characterized in that the method of dividing the binding portion between the straw and the other foliage is a method by grinding. This is a saccharification method.
The present invention according to claim 7 is characterized in that the method for separating the cocoon and the other foliage part is a method of separating by the specific gravity difference between the cocoon and the other foliage part. The saccharification method for rice according to any one of the above.
The present invention according to claim 8 is characterized in that, before the step of separating the cocoon and the other stem and leaves, the cocoon is pressed and crushed to reduce the void size of the cocoon. 7. The method for saccharification of rice according to any one of 7 above.
The present invention according to claim 9 is characterized in that the method for separating the cocoon and the other foliage part is a method for separating the two by the difference in behavior when being blown by the wind. The saccharification method for rice according to any one of the above.
The present invention according to claim 10 is a method for producing ethanol, characterized in that the rice saccharification method according to any one of claims 1 to 9 is used.

The present invention relates to a technique for improving the efficiency of a saccharification process for extracting a saccharide useful as a fermentation source from rice.
Carbohydrates useful as a fermentation source are mainly present in rice stems and leaves (mainly cocoons, leaf sheaths and leaves (body)), cocoons (mainly brown rice and rice husks), and roots. In normal rice cultivation for harvesting rice, the above-ground part is cut in the harvesting process, and after threshing, the rice foliage part (so-called “rice straw”) is appropriately cut and bundled for separation. By this operation, the rice is separated into the root and a few rice leaves and leaves, the straw as a harvest, and the rice leaves and leaves. The foliage may be trapped in the field, but after being appropriately bundled or collected by a roll baler or the like, it is stored as silage under high-humidity / anaerobic conditions or supplied as litter. Therefore, it is dried by a method such as sun drying or indoor natural drying. In many cases, “rice straw”, which is highly expected as a raw material for saccharification, is considered to be supplied in a form close to the rice shoots and leaves collected to be supplied as litter.

  The harvesting process for handling the whole above-ground part of rice (called whole plant or whole crop) as biomass is being studied mainly for the purpose of preparing whole crop silage. Whole crop harvesting methods are roughly classified into methods using a forage harvester or a roll baler. Forage harvesters, just like corn harvesting, are harvested and shredded directly and packed into stationary silos to prepare silage. Roll balers are mainly used for pre-drying silage preparation. (Atsushi Sawamura: Improvement of paddy cultivation autumn harvesting technology for forage rice harvesting with forage harvester) Expected to reduce costs in large-scale crops, mechanized agriculture, No. 3039, pp.8-12, 2004).

  There have been several reports on saccharification technology for rice shoots. Most studies aimed at saccharification of rice straw are carried out for the purpose of saccharifying cellulose and hemicellulose, which are the main components of cell walls, to obtain glucose, xylose and the like. Currently, there are two main saccharification methods being investigated in various places: the sulfuric acid method or the enzymatic method. In the sulfuric acid method, hemicellulose or amorphous glucan is appropriately decomposed with dilute sulfuric acid, and then all glucan is extracted with concentrated sulfuric acid, and after dilution, it is hydrolyzed to decompose into monosaccharides. The enzyme method is a method in which rice straw is appropriately pretreated with acid, alkali, high-temperature water, biological treatment, or the like, and then saccharified by adding cell wall degrading enzymes such as cellulase and xylanase.

As a result of detailed investigation of the rice stem and leaves, the present inventors have found rice straw starch, sucrose, fructose, glucose, β- (1 → 3), (1 → 4) -glucan, etc. The need to develop a pretreatment / saccharification process that takes these into consideration was found (see Japanese Patent Application No. 2008-045766). These carbohydrates are appropriately hydrolyzed into glucose and fructose, and become a source of fermented sugar for many microorganisms including Saccharomyces yeasts, and their recovery is extremely important.
However, since these sugars are easily degradable, they are easily subjected to microbial degradation by leaving them under high humidity conditions at room temperature, such as outdoor rain conditions after harvesting. In addition, when high-temperature treatment is used in the pretreatment process, these easily degradable carbohydrates are eluted, so that these carbohydrates are washed away by washing and collecting the insoluble fraction after the pretreatment reaction. It becomes. In addition, since fructose is susceptible to excessive decomposition in the acid hydrolysis step, it must be separated before pretreatment and saccharification using an acid. Since reducing sugars such as fructose and glucose cause Maillard reaction in the presence of alkali, it is necessary to separate them before carrying out ammonia water or ammonia treatment.
Thus, the present inventor has discovered that many factors need to be taken into consideration in order to develop a saccharification process that includes easy-degradable carbohydrate recovery from herbaceous raw materials.

Although the risk of microbial degradation of readily degradable carbohydrates by collecting rice shoots and leaving them at room temperature under high humidity conditions at room temperature has been described above, the rice straw bundled after harvesting is exposed to rain in the field. In many cases, it is considered that a considerable part of the easily degradable carbohydrate is decomposed when the sun is dried later. In addition, it is considered that a considerable amount of easily degradable carbohydrate is assimilated when silaged.
In order to solve this problem, it is extremely effective to store the rice stem leaves after drying. However, if the entire rice shoots and leaves are dried, the drying cost becomes extremely high, which increases the cost of supplying the saccharified raw material.

Under such circumstances, as a result of further studies, the present inventors have found that most of these easily degradable carbohydrates are unevenly distributed in the cocoon part and hardly exist in the leaf sheath and leaves. By separating only sugar-rich cocoons from other parts, a method for recovering and saccharifying most readily degradable carbohydrates has been completed.
In rice straw, the weight ratio of straw and other parts is about 1: 2, and the weight of the straw is about 1/3 of the whole. Separating only the portion of the cocoon that contains abundantly degradable carbohydrates reduces the amount of fractions that should not be left under high humidity conditions such as raindrops to around 1/3, thus reducing drying costs. can do. Parts other than cocoons have less easily degradable carbohydrates and less loss of glucan, xylan, etc. even under high humidity conditions such as raindrops, so that time can be taken for recovery and drying after separation.

  According to the present invention, pulverization efficiency and saccharification efficiency when saccharifying biomass can be improved by separating rice straws from rice shoots and others (parts centering on leaf sheaths and leaves). This technology will lead to the development of low-cost, low-environmental saccharification technology using rice straw and rice plant. In particular, it is highly important as a new innovation in the development of domestic bioethanol production technology, which has become an urgent issue not only in Japan but around the world.

Hereinafter, the present invention will be described in detail.
The present invention is characterized in that after separating the straw and other parts constituting the rice foliage part, each is separately treated.
Rice shoots and leaves are harvested from the above-ground part from which rice was harvested at the maturity stage or from its cuts (so-called "rice straw"), and from rice plants before the maturity stage. The entire above-ground part or the cut piece is shown. In addition, when the whole rice plant body is used as a whole plant, the portion of the rice plant body above the vine that is attached after harvest is mainly composed of cocoons, leaf sheaths and leaves (body) other than the cocoon. It is defined as the foliage. When performing mechanical harvesting of rice, it is usually cut from a portion several cm to several tens of cm higher than the ground, but the rice stem and leaf portion in the present invention includes the leaf and leaf portion when cutting the above-ground portion near the ground. . In addition, when harvesting rice with a combine, after appropriate threshing, the remaining rice foliage (so-called “rice straw”) can be discharged as harvested length, or several cm long. It can be discharged after cutting.

  The rice (Orizae sativa) in the present invention is not particularly limited, and japonica varieties, indica varieties, or cultivar lines obtained by crossing both can be used. For example, cultivar lines such as “Koshihikari”, “Milky Queen”, “Dream Aoba” can be used, but are not limited thereto. In addition, the harvesting time of the rice plant body is not limited to the maturity period in which both rice foliage and straw can be collected, and the heading period when starch accumulation in the foliage becomes high or after is desirable.

  The rice shoots and leaves used in the present invention are stored under conditions of a humidity of 20% or less or a temperature of 60 ° C. or more immediately after harvesting from the field to within 24 hours after harvesting, or after harvesting rice plants or rice shoots and leaves. However, it is desirable to suppress the degradation of easily degradable carbohydrates by plants and microorganisms. Although it depends on temperature conditions, it is considered that the risk that the loss of easily degradable carbohydrates increases due to the proliferation of microorganisms that have invaded from the cut surface or the like will occur after 24 hours have passed since harvesting. Moreover, in order to suppress the microbial contamination of the rice stem and leaves after harvesting, it is effective to keep the water content low. As mentioned above, most readily degradable carbohydrates are present in the cocoon. When considering room temperature storage, the water content of the rice bran part is not less than 20% (w / w), preferably less than 10% (w / w) in rice shoots and leaves that are not separated from other parts. It is desirable to do.

  When separating the rice shoots and leaves into straw and other fractions, it is first necessary to separate the straw from the leaf sheath and leaves, and then to separate and collect them. As defined in the present invention, the heel includes a “section” that exists between the ridges of the inter-node portion. The pods and leaf sheaths are connected to each node, but by cutting the stem and leaves containing both in a direction that is close to the long axis and in the direction perpendicular to the long axis (the direction in which the stem is shredded), the length of the cut leaves and leaves is shortened. can do. As a result, a break is made between the nodes, the connection between the cocoon in the cut material and the other foliage part is cut off, the part where the cocoon and the leaf sheath are separated is exposed, and the subsequent mechanical separation becomes easy. When cutting the stem, it is important that there is a break between the nodes. As a guideline, the length of the fragment should be 70% or more by weight, 1mm-10cm, preferably 5mm-4cm, more preferably about 2cm. It is. The smaller the fragment length, the easier the separation of the cocoon and the leaf sheath and the separation of the cocoon and the leaf are promoted, but care is required since the cost for coarse grinding increases.

  It is important not to increase the moisture content of the straw after storage, after the cross-section of the straw is exposed by cutting the rice stover. Some leek peelers, etc., use water pressure to separate the skin from the leek. From the aspect of humidity control, the present invention should not be used to separate the cocoon from other parts. Absent. Wind pressure can be used to separate or separate the other parts of the straw and rice shoots, but is not limited thereto. However, even at that time, it is necessary to pay attention to the risk that the starch granules fall from the cross section of the cocoon and affect the recovery rate of easily degradable carbohydrates. Particles containing starch granules produced as a by-product in the separation step can be recovered and saccharified as a part of the koji fraction.

  There is no particular limitation on the method of separating the scissors from other parts, and there is a method of grinding by hand, but there is also a method of cutting with a sharp blade or grinding with a mortar grinder (grinding machine). It is particularly effective to use a method that separates them. The cocoons have higher mechanical strength than the leaf sheaths and leaves, and the cocoons are hardly crushed by grinding, whereas the leaf sheaths and leaves are easily crushed by grinding. In particular, the leaf sheath can be easily separated by a force perpendicular to the axis, and the cocoon and other parts can be roughly separated by the size after cutting and grinding. In addition, wax may be observed to deposit wax, and has a higher density (specific gravity) than other parts. However, in the case where the hollow structure is highly maintained, the apparent density is low due to the high porosity. For this reason, when separating the two using a density difference (specific gravity difference), the apparent density is reduced by compressing and crushing the hollow structure of the ridge and reducing the void size of the ridge. It is important to increase it. This operation not only reduces the separation efficiency due to the air gap structure that is susceptible to wind, but also prevents the starch from falling due to the intrusion of wind in the air gap when using wind pressure to separate the two. it can.

  In addition to visual separation and mechanical sorting using mechanical sensors, fractionation using size differences and behavioral differences during vibration, etc. Although it can be divided, the means is not particularly limited. In the separation by wind force, the difference in behavior when being blown by the wind, such as when the other part is blown away farther than the dense cocoon fraction, or when it is quickly blown, makes use of the difference in behavior. Separate parts. Various wind sorters have been developed, such as a method in which a sample is placed in a cylinder and blown up from below using a blower. In addition, in the vibration type sorter using the density difference, a machine has been developed in which a sample is separated from each other by placing the sample on a slightly tilted table and vibrating it.

  The separation efficiency between sputum and other sites can be evaluated using the concentration of readily degradable carbohydrates. Here, the easily degradable saccharide refers to glucose, fructose, sucrose, starch, and β- (1 → 3), (1 → 4) -glucan. It can be calculated as the recovery rate of easily degradable carbohydrates by grinding both without separation, evaluating the content of easily degradable carbohydrates in the powder, and comparing the value with the data of the separated samples. . If it is possible to collect the starch granules falling from the straw, it is desirable to calculate this fraction as the straw fraction.

  The cocoons separated from the rice shoots and leaves can be stored as appropriate before enzymatic saccharification. At this time, in order to avoid degradation of easily degradable carbohydrates during storage, the moisture content of the heel portion is preferably 20% (w / w) or less, preferably 10% (w / w) or less. .

  Separated rice cake fraction (the fraction in which rice cake is concentrated, that is, the portion in which the ratio of rice cake is improved as compared to the whole rice stalk and leaf portion before separation, and refers to the fraction in which the starch grains dropped from the rice cake are combined. ), Most of the readily degradable carbohydrates exist, including fructose and sucrose, which may be excessively decomposed due to dilute acid decomposition or concentrated acid decomposition. It also contains reducing sugars that are altered by. Starch solubilized by heat treatment, β- (1 → 3), (1 → 4) -glucan and low-molecular-weight carbohydrates are high. It is necessary to avoid pretreatment such as pulp treatment or dilute sulfuric acid explosion treatment. With respect to the koji fraction of the present invention, as shown in the prior invention (see Japanese Patent Application No. 2008-045766), at least one of pulverization and heat treatment at 80 ° C. or higher and 130 ° C. or lower as necessary. An enzyme saccharification method after one treatment is effective.

  Enzymatic saccharification in the present invention includes starch-degrading enzymes such as amyloglucosidase and α-amylase, cellulolytic enzymes such as cellulase and β-glucosidase, xylanase, β-D-xylosidase, acetyl xylan esterase, α-L-arabinofuranosidase At least one of hemicellulose degrading enzymes such as feruloyl esterase and β- (1 → 3), (1 → 4) glucan degrading enzymes such as lichenase can be used. In order to improve the enzymatic degradability of the koji fraction and improve the degradation efficiency of non-degradable β-glucan other than easily degradable carbohydrates, weak acid, weak alkaline conditions, oxidant treatment such as ozone, organic solvent It is possible to perform at least one simple and gentle pretreatment such as treatment. It was obtained in this way because it was not subjected to severe pretreatment such as alkaline pulp treatment, dilute sulfuric acid explosion treatment, strong acid, strong alkali, hydrothermal treatment under conditions above 130 ° C, sulfuric acid treatment, ammonia water treatment, etc. It is considered that cellulose and hemicellulose remain in the insoluble fraction as the enzyme saccharification residue of the koji fraction without being partially decomposed. When the portion of the leaf sheath and leaves separated from the rice bran fraction from the rice stalk leaves is subjected to a pretreatment and saccharification process different from the method for the rice cake fraction, an insoluble fraction is added as a saccharification residue of the koji fraction. In addition, it can be appropriately mixed with fermentation residue and used as feed or methane fermentation raw material, or used as boiler fuel.

  In the fraction mainly from leaf sheath and leaves, separated from the straw fraction from rice shoots and leaves, there are few easily degradable carbohydrates, and components that can be saccharified by β-glucan and hemicellulose, mainly cellulose. It becomes. In that case, since the direct saccharification efficiency is not high, it is considered that the amount of the saccharification raw material hardly decreases even if strict humidity control is not performed. For such lignocellulosic raw materials, in accordance with known methods, harsh treatments such as strong acid treatment at pH 1 or lower, strong alkali treatment at pH 11 or higher, hydrothermal treatment at conditions above 130 ° C., dilute sulfuric acid explosion treatment, etc. It is effective to carry out enzymatic saccharification after performing. In that case, it is possible to use sulfuric acid treatment, aqueous ammonia treatment, alkaline pulping treatment, dilute sulfuric acid explosion treatment, etc., which cannot be applied to the koji fraction.

  The rice bran fraction has a relatively high strength, and when crushing the whole rice foliage part, not only does the koji remain in the crushing chamber, reducing the crushing efficiency, but also depending on the processing conditions of the crusher. May get stuck in the gap of the blade and cause machine stoppage or failure. The present invention solves such problems and makes the grinding process for saccharification more efficient. By separating the straw and other parts of the rice shoots and leaves, it is possible to prepare two types of fractions having a difference in crushing characteristics, and to perform crushing separately using the respective optimum conditions. The portion having low grindability can be pulverized quickly under conditions specific to the portion, and the portion having high grindability can be pulverized with a low energy input. In addition, it is not necessary to saccharify the fraction separated from the rice bran fraction from the rice stalk and leaves using all types of enzymes used for saccharification of the rice straw fraction. It is possible to construct a simple saccharification process.

Thus, the rice saccharified solution recovered by the saccharification method of the present invention can be used as a fermentation raw material in ethanol production as it is or as a concentrated / purified product.
In addition, in applying the saccharification method of the present invention to a method for producing ethanol, not only a method of performing ethanol fermentation using a saccharified solution as a raw material as described above, but also ethanol fermentation before the saccharification step is completed. It is also possible to adopt a “parallel multi-fermentation” system in which a saccharifying step and an ethanol fermentation step are performed in parallel by adding microorganisms that perform the above.

Ethanol fermentation using the saccharified solution obtained by the saccharification method of the present invention can be performed using a microorganism that performs ethanol fermentation by assimilating saccharides having a low polymerization degree, particularly glucose, such as yeast and Zymomonas bacteria. it can. Specifically, it can be performed using yeast Saccharomyces cerevisiae (NBRC 0224).
Although it can be performed using microorganisms that have improved ethanol fermentation efficiency by genetic recombination technology such as recombinant Escherichia coli, focusing only on hexoses such as glucose and fructose, such as yeast and Zymomonas bacteria By using non-recombinant microorganisms, it is possible to reduce facility costs and management costs for measures for preventing the spread of genetically modified microorganisms.

Ethanol fermentation using the saccharified solution obtained by the saccharification method of the present invention can be performed using a known fermentation method.
In the present invention, the saccharification treatment step and the fermentation step can be separated, and the obtained sugar solution can be transferred to a batch-type or continuous fermentation tank. “Parallel double fermentation” is particularly desirable in terms of effects such as space saving and shortening of processing time.

The concentration of saccharides in the saccharified solution used for the ethanol fermentation is in the range of about 0.5% to 30%, preferably in the range of 5% to 30%.
The higher the saccharide concentration of the saccharified solution, the better the fermentation efficiency and the distillation efficiency, but when the operability is taken into consideration, the above range is preferable. For example, when a lignocellulosic raw material is suspended, it becomes bulky and the operability such as stirring is lowered.
Moreover, when the saccharide | sugar density | concentration of a saccharified liquid is lower than about 0.5% (preferably 5%), sufficient ethanol amount cannot be produced and it is not desirable.
In addition, when performing parallel double fermentation in which saccharification reaction and ethanol fermentation are performed simultaneously, the amount of rice straw as a raw material is adjusted so that the concentration of sugars expected to be produced falls within the predetermined range. Should. For example, when production of 0.25 gram of glucose from 1 gram of dry raw material (dry rice straw) can be expected by the saccharification method in the present invention, 2% or more of the dry raw material (2% or more with respect to the reaction solution for parallel double fermentation By using dried rice straw, it is expected that 0.5% or more of glucose is produced.

Although the reaction temperature in the said ethanol fermentation changes with microorganisms to be used, in the case of yeast, it is the range of 25 to 50 degreeC, and in the range of 25 to 60 degreeC in bacteria. The reaction time largely depends on the yeast cell concentration, the amount of saccharifying enzyme, the reaction temperature, the stirring speed, etc., but in the batch method, about 10 to 48 hours is appropriate.
Moreover, when parallel double fermentation which performs saccharification reaction and ethanol fermentation simultaneously is performed, the saccharification enzyme and yeast contained in a liquid are moved by moving the liquid used for parallel double fermentation to the fermentor containing the following raw material after fermentation. Can be made to act again on the next raw material as it is.

  The present invention will be specifically described below with reference to examples and test examples.

Test Example 1 [Milky Queen grinding data]
(1) Physical strength test of straw, leaf sheath and leaf blades 20 g of rice straw [Cultivar name: Milky Queen, mature rice straw (moisture content of 10% or less) was cut and stored in a low-temperature room at 4 ° C. ], 3 mm above and 3 mm below were cut from each node, and the wrinkles and other stems and leaves (leaf sheath and leaf blades) were separated manually. The separated cocoon, leaf sheath and leaf blades are further cut to a length of 1 cm and each 2 g is weighed and used for 1 minute under maximum rotation conditions using New Power Mill PM-2002 (Osaka Chemical Co., Ltd.). Grinding was performed. After standing for 10 minutes, the crushed particle size distribution was examined using sieves (Tokyo Screen) with mesh sizes of 4 mm, 2 mm, 1 mm, 500 μm, 250 μm, 125 μm, 63 μm, and 20 μm.

  The results are shown in FIG. Compared to the fact that the average crushed particle size of the leaf sheath / leaf blade (● in Fig. 1) is about 100 µm, the buttocks (○ in Fig. 1) has a strong physical strength and doubles the average crushed particle of 200 µm. Shown the size.

(2) Carbohydrate content measurement of the pulverized groin The whole pulverized product of the buttock (average particle size 200 μm) is separated with the above mesh size 250 μm sieve, and each pulverized product with a particle size of 250 μm or less is 100 mg each. Weighed out. This was subjected to two-step sulfuric acid treatment (72% sulfuric acid, 1 ml, treated at 30 ° C for 1 hour, then diluted to a sulfuric acid concentration of 9% and treated at 100 ° C for 2 hours). After neutralization with an aqueous NaOH solution, the amount of glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

  As a result, the glucose rate per buttock dry weight was 55% (particle size 250 μm or more) and 54% (particle size 250 μm or less) (w / w) regardless of the particle size. Thus, in the pulverization process, loss of easily degradable carbohydrates, particularly starch, did not occur.

Example 1 [Composition data of Koshihikari raw material and ingredient data of cocoon and other (leaf sheath / leaf blade) when separated by hand]
(1) Preparation of crushed material from buttocks, leaf sheath and leaf blades 10 g of rice straw [Cultivar name: Koshihikari, harvested rice straw harvested and sun-dried and stored at room temperature (water content 10% Below)] is cut at 1 cm intervals and weighed in increments of 5 g, and repeated using New Power Mill PM-2002 (Osaka Chemical Co., Ltd.) until the pulverized product passes through the 500 μm sieve. went. This was used to acquire component data for the whole rice straw.

  In addition, 3 mm above and 3 mm below were cut from each node of 30 g of the above-mentioned rice straw [variety name: Koshihikari], and the stems and other stems and leaves (leaf sheath and leaf blades) were separated manually. The separated straws, leaf sheaths and leaf blades were weighed, and the weight ratio relative to the total weight of the rice straw was calculated ("dry weight" in Table 1). Thereafter, only the cocoon, leaf sheath, and leaf blade were cut at 1 cm intervals, and the above-mentioned pulverization was performed using a new power mill. This was used for component data acquisition in each fraction of the hip and leaf sheath / leaf blade.

  Easily degradable carbohydrates (glucose, fructose, sucrose, starch) per dry weight of rice straw powder and each fraction sample (particle size 500 μm or less, water content 10% or less) obtained by grinding as described above And β- (1 → 3), (1 → 4) -glucan) were measured by the following method. Here, saccharification of easily degradable carbohydrates is performed by water extraction for glucose and fructose, sucrose for invertase reaction after water extraction, starch for α-amylase and amyloglucosidase reaction, β- (1 → 3) , (1 → 4) -glucan was obtained by the reaction of lichenase and β-glucosidase.

(2) Calculation of starch rate per dry weight of rice straw and each fraction sample Starch rate per dry weight of rice straw and each fraction sample (particle size 500 μm or less, water content 10% or less) It was performed with a starch kit (Megazyme).

In other words, 10 mg each of rice straw and each fraction sample were weighed and prepared in a 1.5 ml plastic tube. One of them was added with 0.5 ml of water (containing 0.02% NaN ₃ ) and stirred vigorously for 10 minutes. After stirring, the sample was immediately cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), and a part of the supernatant was sampled. After diluting this with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.), and the free glucose value per dry weight was calculated as G (Table 1). "Glc.")

The other is a thermostable α-amylase (50 mM MOPS buffer, 0.02% NaN ₃ , 5 mM CaCl ₂ , pH 7.0) with 300 μl (30 U) of enzyme solution and heated in a 100 ° C heat block. Treated for 10 minutes (violent stirring every 2 minutes). The sample is then cooled to 50 ° C., 400 μl sodium acetate buffer (200 mM, 0.02% NaN ₃ , pH 4.5) and 10 μl (2 U) amyloglucosidase are added and rotated in a 50 ° C. heat block. The saccharification reaction was performed for 30 minutes. After the reaction, the sample was immediately cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), and a part of the supernatant was sampled. After diluting this with water, the amount of glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose value after the enzyme reaction per dry weight was calculated to be StaG.

  The starch ratio per dry weight was calculated by subtracting the G value from the StaG value and converting it to the amount of starch (“Starch” in Table 1).

(3) Calculation of β- (1 → 3), (1 → 4) -glucan ratio per dry weight of rice straw and each fraction sample Rice straw and each fraction sample (particle size 500 μm or less, water content 10 %-Or less) was calculated using the Mixed-linkage Beta-glucan kit (Megazyme). The β- (1 → 3), (1 → 4) -glucan ratio per dry weight was calculated.

In other words, 10 mg each of rice straw and each fraction sample were weighed and prepared in a 1.5 ml plastic tube. One of them was added with 0.5 ml of water (containing 0.02% NaN ₃ ) and stirred vigorously for 10 minutes. After stirring, the sample was quickly cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), and a part of the supernatant was sampled. After diluting this with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.), and the free glucose value per dry weight was calculated as G.

  The other was added with 480 μl of sodium acetate buffer (20 mM, pH 5.0) and treated for 10 minutes in a heat block at 100 ° C. (violent stirring every 2 minutes). Thereafter, the sample was cooled to 40 ° C., 20 μl (1 U) of lichenase was added, and the saccharification reaction was performed for 60 minutes while rotating in a 40 ° C. heat block. After the reaction, the sample was immediately cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), and 100 μl of the supernatant was sampled. Β-Glucosidase (0.2 U, 50 mM sodium acetate buffer, pH 4.0) enzyme solution (100 ul) was added thereto, and the saccharification reaction was performed for 30 minutes while rotating in a 40 ° C. heat block. After the reaction, the sample was immediately cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), and a part of the supernatant was sampled. After diluting this with water, the amount of glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.), and the glucose value after the enzyme reaction per dry weight was calculated as BetaG.

  Β- (1 → 3), (1 → 4) -glucan ratio per dry weight is calculated by subtracting G value from BetaG value and converting to β- (1 → 3), (1 → 4) -glucan amount ("B-1,3-1,4-Glucan" in Table 1).

(4) Calculation of sucrose ratio per dry weight of rice straw and each fraction sample Calculation of sucrose ratio per dry weight of rice straw and each fraction sample (particle size 500 μm or less, moisture content 10% or less) Was performed with Sucrose, D-fructose and D-glucose kit (Megazyme).

Specifically, 20 mg each of rice straw and each fraction sample was weighed, placed in a 1.5 ml plastic tube, 1 ml of water (containing 0.02% NaN ₃ ) was added, and the mixture was vigorously stirred for 10 minutes. After stirring, the sample was immediately cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), 10 μl of the supernatant was sampled, and placed in two wells of 96 plates. Of these, 1 well measured the amount of free glucose using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.) and calculated the free glucose value per dry weight as G.

  In another well, 20 μl (4 U) of invertase enzyme solution (sodium citrate buffer, pH 4.6) was added, reacted at 30 ° C. for 10 minutes, and a part was sampled. After diluting this with water, the amount of glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.), and the free glucose value per dry weight was calculated as SucG.

  The sucrose ratio per dry weight was calculated by subtracting the G value from the SucG value and converting it to the amount of sucrose ("Suc." In Table 1).

(5) Calculation of fructose rate per dry weight of rice straw and each fraction sample Calculate the fructose rate per dry weight of rice straw and each fraction sample (particle size 500 μm or less, water content 10% or less). D-fructose and D-glucose kit (Megazyme) was used.

Specifically, 20 mg each of rice straw and each fraction sample was weighed, placed in a 1.5 ml plastic tube, 1 ml of water (containing 0.02% NaN ₃ ) was added, and the mixture was vigorously stirred for 10 minutes. After stirring, the sample was immediately cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), 10 μl of supernatant was sampled, and placed in a well of 96 plates. To this well, 200 μl of water, 10 μl of imidazole buffer (2 M, pH 7.6) and 10 μl of NADP ⁺ • ATP (12.5 mg / ml, 36.7 mg / ml) aqueous solution were added and reacted at 30 ° C. for 3 minutes. . Thereafter, the absorbance at 340 nm was measured and designated as A1. After measuring A1, add 10 μl of mixed enzyme solution of hexokinase (0.85 U) and Glucose-6-phosphate dehydrogenase (0.42 U), react at 30 ° C for 10 minutes, and measure the absorbance at 340 nm to obtain A2. (The following reaction was carried out after confirming stable absorbance while measuring absorbance at 2-minute intervals). After A2 measurement, 10 μl (2 U) of Phosphoglucose isomerase was added and reacted at 30 ° C. for 10 minutes, and the absorbance at 340 nm was measured to be A3. A value obtained by subtracting A2 from A3 and a fructose calibration curve for each concentration were prepared, and the fructose rate per dry weight was calculated ("Fru." In Table 1).

(6) Results Table 1 shows the above measurement results. Koshihikari had a starch ratio of 2.18% per dry weight based on the total powder. In addition, there is a marked difference in starch content between the buttocks and the leaf sheaths and leaf blades, and the total easily degradable carbohydrate content is 1.78% in the buttocks, which is higher than the leaf sheath and leaf blades (0.54%). showed that.

Glc.：Glucose、Fru.：Fructose、Suc.：Sucrose
全含有量^１：各試料30 g当たりの全易分解性糖質量（g）
量率^２：易分解性糖質量率（％）＝［各試料中の全易分解性糖質量（g）／稈と葉鞘・葉身の全易分解性糖質量の合計（g）］×100

Glc .: Glucose, Fru .: Fructose, Suc .: Sucrose
Total content ¹ : Total easily degradable sugar mass (g) per 30 g of each sample
Quantity ratio ² : Mass ratio of readily degradable sugar (%) = [total mass of easily degradable sugar in each sample (g) / total of all easily degradable sugar masses of cocoons, leaf sheaths and leaf blades (g)] × 100

Example 2 [Glucidation data using cellulase and xylanase preparations of koji and other (leaf sheath / leaf blade) when Koshihikari was hand-sorted]
In addition to easily degradable sugars, rice straw contains cellulose with glucose as a unit, hemicellulose with units of xylose and the like. These celluloses and hemicelluloses are less susceptible to enzymatic degradation by cellulase preparations and hemicellulase preparations due to their crystal structures, chemical modifications, and positional relationships. Therefore, saccharification experiments of cellulose and hemicellulose were carried out, including unseparated rice straw, hand-separated cocoons and leaf sheath / leaf blade parts, and mechanically degradable carbohydrates of each part.

Rice straw and each fraction sample [variety name: Koshihikari, crushed in Example 1, whole (unseparated rice straw) and straw, leaf sheath, leaf blades] 50 mg each, 1.5 ml plastic tube 958 μl of sodium citrate buffer solution (50 mM, pH 4.8, NaN ₃ 0.01%) was added to the mixture and treated in a heat block at 100 ° C. for 10 minutes (violent stirring every 2 minutes). The sample was then cooled to 50 ° C., and cellulase preparation (12 μl, Celluclast 1.5 L, Novozymes Japan), hemicellulase preparation (6 μl, Viscozyme L, Novozymes Japan), β-glucosidase preparation (4 μl, Novozyme 188 (Sigma)), amyloglucosidase (10 μl (2 U), Megazyme) and invertase enzyme solution (10 μl (4 U), TOYOBO) were added to make up to 1 ml to prepare a reaction solution. Thereafter, the saccharification reaction was carried out for 24 hours while rotating in a heat block at 50 ° C. After the reaction, a part was sampled and diluted with water, and then the amount of glucose after saccharification was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

The saccharification rate of the glycan after saccharification was calculated by the following formula from the glucose amount after saccharification and the total glucose amount of each sample [measured after two-step sulfuric acid treatment according to Test Example 1 (2)] (Table 2 "glycan saccharification rate"). The total glucose rate (%) was calculated from the total glucose amount per dry weight of each sample ("Total Glc. Rate" in Table 2).
Glycan saccharification rate (%) = (free glucose amount after enzyme saccharification / total glucose amount of each sample) × 100

The saccharification rate of cellulose is determined from the amount of glucose obtained by subtracting the amount of glucose derived from easily degradable carbohydrates obtained in Example 1 from the amount of free glucose after enzymatic saccharification and the total amount of glucose after the above two-stage sulfuric acid treatment. From the amount of glucose obtained by subtracting the amount of glucose derived from the easily degradable carbohydrate determined in Example 1, the calculation was performed according to the following formula (“cellulose saccharification rate” in Table 2).
Cellulose saccharification rate (%) = [(free glucose amount after enzymatic saccharification−glucose amount derived from easily degradable carbohydrate) / (total glucose amount−glucose amount derived from easily degradable carbohydrate)] × 100

  In addition, the cellulose ratio per dry weight of rice straw and each fraction sample is the amount of glucose obtained by subtracting the amount of glucose derived from the easily degradable carbohydrates obtained in Example 1 from the total amount of glucose after the above two-stage sulfuric acid treatment. Was calculated in terms of cellulose content ("cellulose ratio" in Table 2).

The saccharification rate of hemicellulose after saccharification is calculated by measuring the amount of xylose after saccharification (D-xylose kit (Megazyme)), the amount of xylan in rice straw and each fraction sample (two-stage sulfuric acid). The amount of xylose after the treatment was measured with a D-xylose kit (Megazyme Co., Ltd. and converted as xylan) and calculated according to the following formula (“hemicellulose saccharification rate” in Table 2). The “total xylan ratio” in Table 2 is a value per dry weight of rice straw and each fraction sample of the above total xylan amount.
Hemicellulose saccharification rate (%) = (decomposed xylan amount after enzymatic saccharification / total xylan amount of each sample) × 100

  The results are shown in Table 2. The overall glycan saccharification rate of Koshihikari rice straw was 27.91%, the cellulose saccharification rate was 22.82%, and the hemicellulose saccharification rate was 3.65%. The glycan saccharification rate, the saccharification rate of cellulose, and the saccharification rate of hemicellulose were all high in the buttocks, showing 30.16, 30.25, and 4.54%, respectively. In addition, the glycan saccharification rate in the buttocks and the saccharification rate of cellulose both showed saccharification more than twice that of the leaf sheath and leaf blades.

Glc.：Glucose
総Glc.率¹、総キシラン率¹：2段階硫酸処理で測定（各試料の乾重あたり対する率）

Glc .: Glucose
Total Glc. Rate ¹ , Total xylan rate ¹ : Measured by two-step sulfuric acid treatment (rate per dry weight of each sample)

Example 3 [Saccharification data using a cellulase / xylanase preparation after alkali treatment of leaves (leaf sheath / leaf blade) other than cocoon when Koshihikari was hand-sorted]
Koshihikari's leaf sheath and leaf blades [Examples 1 and 2] have the lowest easily degradable carbohydrate rate (0.54%) and total saccharification rate (13.16%), and chemical or physical pretreatment is required for the saccharification reaction Is done. Therefore, in order to increase the total saccharification rate of the leaf sheath / leaf blade part of Koshihikari, which has a low rate of easily degradable carbohydrates and a low total saccharification rate, the following examination was conducted.

Weigh 50 mg of the fraction sample [variety name: Koshihikari, leaf sheath / leaf body part crushed in Example 1], put each in two 1.5 ml plastic tubes, one of which is sodium citrate buffered 0.25 ml of a liquid (200 mM, pH 4.8, NaN ₃ 0.01%), an aqueous NaOH solution (50 mM, 0.5 ml), and an aqueous HCl solution (100 mM, 0.25 ml) were added. In the other one, only NaOH aqueous solution (50 mM, 0.5 ml) was added, and after alkali treatment at 121 ° C for 15 minutes, sodium citrate buffer (200 mM, pH 4.8, NaN ₃ 0.01%) 0.25 ml And HCl aqueous solution (100 mM, 0.25 ml) were added.

  Next, a total saccharification experiment was performed on each tube by enzymatic reaction. Cellulase preparation (12 μl, Celluclast 1.5 L, Novozymes Japan), hemicellulase preparation (6 μl, Viscozyme L, Novozymes Japan) and β-glucosidase preparation (4 μl, Novozyme188, Sigma) Was added, and saccharification reaction was carried out for 12, 24 and 91 hours while rotating in a heat block at 50 ° C., and a part was sampled at each time. After diluting this with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.). The glycan saccharification rate and the hemicellulose saccharification rate were calculated according to Example 2.

  The results are shown in Table 3. The glycan saccharification rate and the saccharification rate of hemicellulose were all high in the alkali-treated samples, and showed 44.5 and 18.97% in the 91-hour reaction.

Example 4 [Composition data of milky queen raw material, sulfuric acid two-stage saccharification data, enzymatic saccharification data, and comparison of cocoon and other (leaf sheath / leaf blade) data when separated by hand]
Example of rice straw 10 g [variety name: milky queen, harvested rice straw (moisture content of 10% or less) cut and stored in a cold room immediately at 4 ° C] cut at 1 cm intervals According to 1, the crushing / saccharification / degradable carbohydrates were quantified. In addition, rice bran and other stems and leaves (leaf sheath and leaf blade) were separated from the rice straw 30 g [variety name: Milky Queen] according to Example 1. The separated cocoons and leaf sheaths / leaf blades were weighed, and pulverized / saccharified / degradable carbohydrates were quantified according to Example 1.

  The results are shown in Table 4. The whole rice straw [variety name: Milky Queen] had the highest sucrose ratio (4.63%) as an easily degradable carbohydrate, and the total easily degradable sugar ratio was 12.61%. The rice straw of this example is different from Koshihikari of Example 1 (total easily degradable carbohydrate rate 2.49%), but the preservation method after harvest (Koshihikari of Example 1 is More easily degradable carbohydrates (3.78 g) could be easily saccharified and recovered by sun drying. In addition, the total easily degradable sugar ratio of cocoon, leaf sheath and leaf blades is 28.16% in the buttocks, and the readily degradable sugar mass ratio is 91.8%, which is more than 90% of the total easily degradable sugar mass of rice straw. I was concentrating on my niece.

Glc.：Glucose、Fru.：Fructose、Suc.：Sucrose
全含有量^１：各試料30 g当たりの全易分解性糖質量（g）
量率^２：易分解性糖質量率（％）＝［各試料中の全易分解性糖質量（g）／稈と葉鞘・葉身の全易分解性糖質量の合計（g）］×100

Glc .: Glucose, Fru .: Fructose, Suc .: Sucrose
Total content ¹ : Total easily degradable sugar mass (g) per 30 g of each sample
Quantity ratio ² : Mass ratio of readily degradable sugar (%) = [total mass of easily degradable sugar in each sample (g) / total of all easily degradable sugar masses of cocoons, leaf sheaths and leaf blades (g)] × 100

  Thereafter, rice straw and each fraction sample (variety name: milky queen, pulverized as above, whole, straw, leaf sheath and leaf blades) were weighed 50 mg each, and total saccharification experiment and 2 steps according to Example 2 Sulfuric acid treatment was performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 5. In the whole rice straw (variety name: Milky Queen), the glycan saccharification rate was 40.01%, the saccharification rate of cellulose was 29.89%, and the saccharification rate of hemicellulose was 7.60%.

  On the other hand, the glycan saccharification rate, the saccharification rate of cellulose and the saccharification rate of hemicellulose were all high in the buttocks and showed glycan saccharification rate and saccharification rate of hemicellulose in leaf sheath and leaf. The saccharification was about twice that of the body.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)

Example 5 [Without mechanical separation roller, 3 cm cut, grinder mill, 1 mm cut With milky queen raw material crushed with a grinder mill and separated using a cylinder, component data of low specific gravity part and high specific gravity part, sulfuric acid 2 Staged saccharification data, enzymatic saccharification data]
30 g of rice straw [variety name: milky queen] used in Example 4 was cut at intervals of 3 cm in length and put into a grinder mill (RDI-15, Glow Engineering), and a rotating grinder (diameter 15 cm). And grinding with a grinding grinder of 500 μm and a rotation speed of 1000 rpm. Grind the crushed material through a sieve with a mesh size of 1 mm and finely pulverize with a particle size of 1 mm or less. The rice straw was collected and weighed. Grained rice straw (20.5 g) with a particle size of 1 mm or more is placed in a 1 m long x 7 cm inner diameter open top cylindrical tube, and 1 L / sec of air is vented from the bottom to separate the specific gravity difference. It was. The crushed material blown from the upper part of the cylindrical tube due to the difference in specific gravity was collected as a low specific gravity part, and the crushed rice straw remaining in the cylindrical tube was collected as a high specific gravity part and weighed. The recovered pulverized rice straw (1 mm sieve passing part, high specific gravity part, low specific gravity part) was further crushed to a particle size of 0.5 mm or less using a Were mill.

  Then, the rice straw and each fraction sample were subjected to saccharification reaction and quantitative experiment of easily degradable carbohydrates according to Example 1, and each easily degradable sugar content per dry weight and total easily degradable sugar mass were calculated. did. Dry weight recovery rate, sugar concentration rate, and sugar recovery rate were measured according to Example 4. Rice straw [variety name: milky queen] dry weight (35.2%: 30 g of rice straw yielded 10.6 g) ), Easy-degradable carbohydrate rate (91.8%) and total easily-degradable sugar mass (2.97 g) as ideal values (100%), dry weight of each fraction sample, easily-degradable carbohydrate rate and total easily-degradable It calculated by comparing with the amount of sex sugar.

  The results are shown in Table 6. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate of the 1 mm sieve passing part, high specific gravity part, and low specific gravity part obtained by mechanical pulverization / separation were (60, 43, 26), (27, 68), respectively. 19), (152, 33, 50), compared to the ideal value (100, 100, 100) of the buttocks separated by hand, in this embodiment, the low specific gravity part (dry weight recovery rate 152) that is blown by the wind %), The high specific gravity part (sugar concentration rate of 68%) showed the highest sugar concentration rate, which is characteristic of the buttocks.

^１ :各試料の乾重あたりの全易分解性糖質含有率
^２ :各試料30 gから機械的分離したサンプル稲わらの全易分解性糖質量
^３ :実施例４によって分離された稈部位（乾重10.6 g）に対する各試料の乾重比
^４ :実施例４によって分離された稈部位（易分解性糖質率91.8％）に対する各試料の易分解性糖質率比
^５ :実施例４によって分離された稈部位（全易分解性糖質量2.97 g）に対する各試料の全易分解性糖質量比
^６ :実施例４によって分離された稈部位の乾重・易分解性糖質含有率

¹ : Total easily degradable carbohydrate content per dry weight of each sample
² : Total degradable sugar mass of sample rice straw mechanically separated from 30 g of each sample
³ : Dry weight ratio of each sample to the heel part (dry weight 10.6 g) separated in Example 4
⁴ : Ratio of easily degradable carbohydrate ratio of each sample to the sputum site separated according to Example 4 (easily degradable carbohydrate ratio 91.8%)
⁵ : Total easily degradable sugar mass ratio of each sample to the sputum site (total easily degradable sugar mass 2.97 g) separated in Example 4
⁶ : Dry weight / easily degradable carbohydrate content of the sputum part separated in Example 4

  Thereafter, 50 mg of each fraction sample [variety name: milky queen, pulverized and separated as above, 1 mm sieve passing part, high specific gravity part, low specific gravity part] was weighed and total saccharified according to Example 2. The experiment and two-stage sulfuric acid treatment were performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 7. The glycan saccharification rate and the saccharification rate of cellulose were high in the high specific gravity pulverized product, showing 46.52 and 31.43% respectively.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定
^２：実施例４によって分離された稈部位のグリカン糖化率、セルロース糖化率及びヘミセルロース糖化率

Glc .: Glucose
Total Glc. Rate ¹ , Total xylan rate ¹ : Measured by two-step sulfuric acid treatment
² : Glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the heel part separated according to Example 4

Example 6 [With mechanical separation roller, 3 cm cut, grinder mill, without 1 mm cut Milky queen raw material was passed through a roller, cut to 3 cm, crushed with a grinder mill, and separated using a cylinder, low specific gravity part, Component data of high specific gravity part, sulfuric acid two-stage saccharification data, enzymatic saccharification data]
30 g of rice straw [variety name: milky queen] used in Example 4 was crushed while passing through a roller with a gap of 0 mm (iron, diameter: 14 cm) loaded with 100 kg in a vertical direction. Then, it cut | judged at intervals of 3 cm in length, thrown into the grinder mill (RDI-15, glow engineering company), and grind | pulverized according to Example 5. FIG. However, separation using a sieve with a mesh size of 1 mm, which was performed in Example 5 after pulverization, was not performed, and the following experiment was performed using the entire recovered amount after pulverization. That is, after the pulverization, the low specific gravity part and the high specific gravity part were separated and crushed according to Example 5 from the total amount of 28.1 g of the pulverized product. Thereafter, a saccharification reaction of a readily degradable sugar and a quantitative experiment were performed according to Example 1, and the content per dry weight and the total easily degradable sugar mass were calculated. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate were calculated according to Example 5.

  The results are shown in Table 8. The dry weight recovery rate, sugar concentration rate and sugar recovery rate of the high specific gravity part and low specific gravity part were (96, 78, 75) and (153, 23, 35), respectively. Compared with the ideal value (100, 100, 100) of the heel part separated by hand, rice straw is first passed through a roller, and in this example, rice straw of 1 mm or less after grinding with a grinder mill is not collected. The sugar concentration rate, which is the characteristic of the high specific gravity part to the buttocks, is the highest (78%), and the dry weight recovery rate (96%) was also equivalent to the theoretical value. By this method, the straw of the rice straw was efficiently separated.

^１ :各試料の乾重あたりの全易分解性糖質含有率
^２ :各試料30 gから機械的分離したサンプル稲わらの全易分解性糖質量
^３ :実施例４によって分離された稈部位（乾重10.6 g）に対する各試料の乾重比
^４ :実施例４によって分離された稈部位（易分解性糖質率91.8％）に対する各試料の易分解性糖質率比
^５ :実施例４によって分離された稈部位（全易分解性糖質量2.97 g）に対する各試料の全易分解性糖質量比
^６ :実施例４によって分離された稈部位の乾重・易分解性糖質含有率

¹ : Total easily degradable carbohydrate content per dry weight of each sample
² : Total degradable sugar mass of sample rice straw mechanically separated from 30 g of each sample
³ : Dry weight ratio of each sample to the heel part (dry weight 10.6 g) separated in Example 4
⁴ : Ratio of easily degradable carbohydrate ratio of each sample to the sputum site separated according to Example 4 (easily degradable carbohydrate ratio 91.8%)
⁵ : Total easily degradable sugar mass ratio of each sample to the sputum site (total easily degradable sugar mass 2.97 g) separated in Example 4
⁶ : Dry weight / easily degradable carbohydrate content of the sputum part separated in Example 4

  Thereafter, 50 mg of each sample [variety name: milky queen, pulverized and separated as above, high specific gravity part, low specific gravity part] was weighed and subjected to total saccharification experiment and two-stage sulfuric acid treatment according to Example 2. The total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate and hemicellulose saccharification rate were calculated.

  The results are shown in Table 9. The glycan saccharification rate and the hemicellulose saccharification rate were high in the high specific gravity pulverized product, showing 47.94 and 6.36%, respectively, values similar to the glycan saccharification rate and hemicellulose saccharification rate (55.29, 7.00%) in Example 4 It was found that the high specific gravity part obtained by this pulverization / separation method has a high total saccharification (Examples 2 and 4), and most of the straw of rice straw occupies. By this method, the straw of the rice straw was efficiently separated.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）
^２：実施例４によって分離された稈部位のグリカン糖化率、セルロース糖化率及びヘミセルロース糖化率

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)
² : Glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the heel part separated according to Example 4

Example 7 [With mechanical separation roller, 1 cm cut, grinder mill, 1 mm cut With milky queen raw material passed through a roller, cut into 1 cm, crushed with a grinder mill, separated using a cylinder, low specific gravity part, Component data of high specific gravity part, sulfuric acid two-stage saccharification data, enzymatic saccharification data]
30 g of rice straw [variety name: milky queen] used in Example 4 was crushed while passing through a roller with a gap of 0 mm (iron, diameter: 14 cm) loaded with 100 kg in a vertical direction. Then, it cut | judged at intervals of 1 cm in length, thrown into the grinder mill (RDI-15, glow engineering company), and grind | pulverized according to Example 5. FIG. After pulverization, the crushed rice straw having a particle size of 1 mm or less was collected through a sieve having a mesh size of 1 mm and weighed. The remaining 25.2 g of the pulverized product was subjected to separation and crushing of a low specific gravity part and a high specific gravity part according to Example 5. Then, the saccharification reaction and quantitative experiment of the easily degradable carbohydrates in the 1 mm sieve passing part, high specific gravity part, and low specific gravity part were performed according to Example 1, and the content per dry weight and the total easily degradable sugar mass were calculated. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate were calculated according to Example 5.

  The results are shown in Table 10. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate of the 1 mm sieve passing part, high specific gravity part, and low specific gravity part obtained by mechanical pulverization / separation were (35, 19, 7), (62 76, 47), (170, 23, 39). Compared to the ideal value (100, 100, 100) of the buttocks separated by hand, in this example there are more low specific gravity parts (dry weight recovery rate 170%) that are blown by the wind, but high specific gravity parts (sugar concentration rate 76) %) Showed the highest sugar concentration rate characteristic of the buttocks.

^１ :各試料の乾重あたりの全易分解性糖質含有率
^２ :各試料30 gから機械的分離したサンプル稲わらの全易分解性糖質量
^３ :実施例４によって分離された稈部位（乾重10.6 g）に対する各試料の乾重比
^４ :実施例４によって分離された稈部位（易分解性糖質率91.8％）に対する各試料の易分解性糖質率比
^５ :実施例４によって分離された稈部位（全易分解性糖質量2.97 g）に対する各試料の全易分解性糖質量比
^６ :実施例４によって分離された稈部位の乾重・易分解性糖質含有率

¹ : Total easily degradable carbohydrate content per dry weight of each sample
² : Total degradable sugar mass of sample rice straw mechanically separated from 30 g of each sample
³ : Dry weight ratio of each sample to the heel part (dry weight 10.6 g) separated in Example 4
⁴ : Ratio of easily degradable carbohydrate ratio of each sample to the sputum site separated according to Example 4 (easily degradable carbohydrate ratio 91.8%)
⁵ : Total easily degradable sugar mass ratio of each sample to the sputum site (total easily degradable sugar mass 2.97 g) separated in Example 4
⁶ : Dry weight / easily degradable carbohydrate content of the sputum part separated in Example 4

  Thereafter, 50 mg of each sample [variety name: milky queen, pulverized / separated above, 1 mm sieve passing part, high specific gravity part, low specific gravity part] was weighed and subjected to total saccharification experiments and 2 according to Example 2. Stepped sulfuric acid treatment was performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 11. The glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate were all high in the high specific gravity pulverized product, showing 52.81, 27.53, and 7.23%, respectively.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）
^２：実施例４によって分離された稈部位のグリカン糖化率、セルロース糖化率及びヘミセルロース糖化率

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)
² : Glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the heel part separated according to Example 4

Example 8 [With mechanical separation roller, 2 cm cut, grinder mill, 1 mm cut With milky queen raw material passed through a roller, cut to 2 cm, crushed with a grinder mill, separated using a cylinder, low specific gravity part, Component data of high specific gravity part, sulfuric acid two-stage saccharification data, enzymatic saccharification data]
30 g of rice straw [variety name: milky queen] used in Example 4 was crushed while passing through a roller with a gap of 0 mm (iron, diameter: 14 cm) loaded with 100 kg in a vertical direction. Then, it cut | judged at intervals of 2 cm in length, thrown into the grinder mill (RDI-15, glow engineering company), and grind | pulverized according to Example 5. FIG. After pulverization, the crushed rice straw having a particle size of 1 mm or less was collected through a sieve having a mesh size of 1 mm and weighed. The remaining 25.7 g of the pulverized product was subjected to separation and crushing of the low specific gravity part and the high specific gravity part according to Example 5. Thereafter, the saccharification reaction and quantitative experiment of the easily degradable carbohydrates in the 1 mm sieve passage part, high specific gravity part, and low specific gravity part were carried out according to Example 1, and the content per dry weight and the total easily degradable sugar mass were calculated. . The dry weight recovery rate, sugar concentration rate, and sugar recovery rate were calculated according to Example 5.

  The results are shown in Table 12. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate of the 1 mm sieve passing part, high specific gravity part, and low specific gravity part obtained by mechanical pulverization / separation were (29, 23, 7), (121 66, 80), (114, 11, 12). Compared with the ideal value of the buttocks separated by hand (100, 100, 100), in this example, the sugar concentration rate that is the characteristic of the buttocks is the highest (66%) in the high specific gravity part that is not easily blown by the wind (66%), Although the dry weight recovery rate (121%) was higher than the theoretical value, a high sugar recovery rate (80%) was shown. By this method, the straw of the rice straw was efficiently separated.

^１ :各試料の乾重あたりの全易分解性糖質含有率
^２ :各試料30 gから機械的分離したサンプル稲わらの全易分解性糖質量
^３ :実施例４によって分離された稈部位（乾重10.6 g）に対する各試料の乾重比
^４ :実施例４によって分離された稈部位（易分解性糖質率91.8％）に対する各試料の易分解性糖質率比
^５ :実施例4によって分離された稈部位（全易分解性糖質量2.97 g）に対する各試料の全易分解性糖質量比
^６ ：実施例４によって分離された稈部位の乾重・易分解性糖質含有率

¹ : Total easily degradable carbohydrate content per dry weight of each sample
² : Total degradable sugar mass of sample rice straw mechanically separated from 30 g of each sample
³ : Dry weight ratio of each sample to the heel part (dry weight 10.6 g) separated in Example 4
⁴ : Ratio of easily degradable carbohydrate ratio of each sample to the sputum site separated according to Example 4 (easily degradable carbohydrate ratio 91.8%)
⁵ : Ratio of total readily degradable sugar mass of each sample to the sputum site (total easily degradable sugar mass 2.97 g) separated in Example 4
⁶ : Dry weight / easily degradable carbohydrate content of the sputum part separated in Example 4

  Thereafter, 50 mg of each sample [variety name: milky queen, pulverized / separated above, 1 mm sieve passing part, high specific gravity part, low specific gravity part] was weighed, respectively. Stepped sulfuric acid treatment was performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 13. The glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate were all high in the high-specific gravity pulverized product, showing 55.92, 36.48, and 7.21%, respectively. This result was the closest value to 55.29, 36.09 and 7.00% of the buttocks of Example 4. By this method, the straw of the rice straw was efficiently separated.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）
^２：実施例４によって分離された稈部位のグリカン糖化率、セルロース糖化率及びヘミセルロース糖化率

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)
² : Glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the heel part separated according to Example 4

Example 9 [with mechanical separation roller, 3 cm cut, grinder mill, 1 mm cut with Milky Queen raw material passed through a roller, cut to 3 cm, crushed with a grinder mill, separated using a cylinder, low specific gravity part, Component data of high specific gravity part, sulfuric acid two-stage saccharification data, enzymatic saccharification data]
30 g of rice straw [variety name: milky queen] used in Example 4 was crushed while passing through a roller with a gap of 0 mm (iron, diameter: 14 cm) loaded with 100 kg in a vertical direction. Then, it cut | judged at intervals of 3 cm in length, thrown into the grinder mill (RDI-15, glow engineering company), and grind | pulverized according to Example 5. FIG. After pulverization, the crushed rice straw having a particle size of 1 mm or less was collected through a sieve having a mesh size of 1 mm and weighed. The remaining 23.8 g of the pulverized product was subjected to separation and crushing of the low specific gravity part and the high specific gravity part according to Example 5. Then, the saccharification reaction and quantitative experiment of the easily degradable carbohydrates in the 1 mm sieve passing part, high specific gravity part, and low specific gravity part were performed according to Example 1, and the content per dry weight and the total easily degradable sugar mass were calculated. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate were calculated according to Example 5.

  The results are shown in Table 14. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate of each 1mm sieve passing part, high specific gravity part, and low specific gravity part obtained by mechanical grinding / separation are (42, 43, 18) (94, 76 72), (121, 21, 25). Compared with the ideal value (100, 100, 100) of the buttocks separated by hand, in this example, the sugar concentration rate that is the characteristic of the buttocks is the highest (76%) in the high specific gravity part that is not easily blown by the wind, The dry weight recovery rate (94%) was also high. By this method, the straw of the rice straw was efficiently separated.

^１ :各試料の乾重あたりの全易分解性糖質含有率
^２ :各試料30 gから機械的分離したサンプル稲わらの全易分解性糖質量
^３ :実施例４によって分離された稈部位（乾重10.6 g）に対する各試料の乾重比
^４ :実施例４によって分離された稈部位（易分解性糖質率91.8％）に対する各試料の易分解性糖質率比
^５ :実施例４によって分離された稈部位（全易分解性糖質量2.97 g）に対する各試料の全易分解性糖質量比
^６ :実施例４によって分離された稈部位の乾重・易分解性糖質含有率

¹ : Total easily degradable carbohydrate content per dry weight of each sample
² : Total degradable sugar mass of sample rice straw mechanically separated from 30 g of each sample
³ : Dry weight ratio of each sample to the heel part (dry weight 10.6 g) separated in Example 4
⁴ : Ratio of easily degradable carbohydrate ratio of each sample to the sputum site separated according to Example 4 (easily degradable carbohydrate ratio 91.8%)
⁵ : Total easily degradable sugar mass ratio of each sample to the sputum site (total easily degradable sugar mass 2.97 g) separated in Example 4
⁶ : Dry weight / easily degradable carbohydrate content of the sputum part separated in Example 4

  Thereafter, 50 mg of each sample [variety name: milky queen, pulverized / separated above, 1 mm sieve passing part, high specific gravity part, low specific gravity part] was weighed, respectively. Stepped sulfuric acid treatment was performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 15. The glycan saccharification rate was 47.57%, which was high in the high specific gravity part, and was higher than that of the pulverized product with the 1 mm sieve and the low specific gravity part. By this method, the straw of the rice straw was efficiently separated.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）
^２：実施例４によって分離された稈部位のグリカン糖化率、セルロース糖化率及びヘミセルロース糖化率

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)
² : Glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the heel part separated according to Example 4

Example 10 [with mechanical separation roller, 4 cm cut, grinder mill, 1 mm cut milky queen raw material is passed through a roller, cut to 4 cm, crushed with a grinder mill, separated using a cylinder, low specific gravity part, Component data of high specific gravity part, sulfuric acid two-stage saccharification data, enzymatic saccharification data]
30 g of rice straw [variety name: milky queen] used in Example 4 was crushed while passing through a roller with a gap of 0 mm (iron, diameter: 14 cm) loaded with 100 kg in a vertical direction. Then, it cut | judged at intervals of 4 cm in length, thrown into the grinder mill (RDI-15, glow engineering company), and grind | pulverized according to Example 5. FIG. After pulverization, the pulverized rice straw having a particle size of 1 mm or less was collected through a sieve having a mesh size of 1 mm and weighed. The remaining 24.0 g of the pulverized product was subjected to separation and crushing of the low specific gravity part and the high specific gravity part according to Example 5. Thereafter, the saccharification reaction and quantitative experiment of the easily degradable carbohydrates in the 1 mm sieve passage part, high specific gravity part, and low specific gravity part were carried out according to Example 1, and the content per dry weight and the total easily degradable sugar mass were calculated. . The dry weight recovery rate, sugar concentration rate, and sugar recovery rate were calculated according to Example 5.

  The results are shown in Table 16. The dry weight recovery rate, sugar concentration rate, and sugar recovery rate of each part 1 mm sieve passing part, high specific gravity part, and low specific gravity part obtained by mechanical pulverization / separation were (39, 26, 10), (61 71, 43), (157, 16, 25). Compared with the ideal value (100, 100, 100) of the buttocks separated by hand, in this example, there are many low specific gravity parts (dry weight recovery rate 157%) blown by the wind, but high specific gravity parts (sugar concentration rate) 71%) showed the highest sugar concentration rate characteristic of the buttocks.

  From the results of Examples 5, 6, 7, 8, 9, and 10, when the rice straw was mechanically pulverized, the high specific gravity part was the straw part of the rice straw because the sugar concentration rate was high in the high specific gravity part in any pulverization method. Is considered to be reflected. In addition, the effect of separating the ridges was improved by passing the roller and cutting the rice straw from 2 cm to 3 cm in length and crushing.

^１ :各試料の乾重あたりの全易分解性糖質含有率
^２ :各試料30 gから機械的分離したサンプル稲わらの全易分解性糖質量
^３ :実施例４によって分離された稈部位（乾重10.6 g）に対する各試料の乾重比
^４ :実施例４によって分離された稈部位（易分解性糖質率91.8％）に対する各試料の易分解性糖質率比
^５ :実施例４によって分離された稈部位（全易分解性糖質量2.97 g）に対する各試料の全易分解性糖質量比
^６ :実施例４によって分離された稈部位の乾重・易分解性糖質含有率

¹ : Total easily degradable carbohydrate content per dry weight of each sample
² : Total degradable sugar mass of sample rice straw mechanically separated from 30 g of each sample
³ : Dry weight ratio of each sample to the heel part (dry weight 10.6 g) separated in Example 4
⁴ : Ratio of easily degradable carbohydrate ratio of each sample to the sputum site separated according to Example 4 (easily degradable carbohydrate ratio 91.8%)
⁵ : Total easily degradable sugar mass ratio of each sample to the sputum site (total easily degradable sugar mass 2.97 g) separated in Example 4
⁶ : Dry weight / easily degradable carbohydrate content of the sputum part separated in Example 4

  Thereafter, 50 mg of each sample [variety name: milky queen, pulverized / separated above, 1 mm sieve passing part, high specific gravity part, low specific gravity part] was weighed, respectively, and total saccharification experiments were conducted according to Example 2. Two-stage sulfuric acid treatment was performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 17. The glycan saccharification rate was 48.50%, which was high in the high specific gravity pulverized product.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）
^２：実施例４によって分離された稈部位のグリカン糖化率、セルロース糖化率及びヘミセルロース糖化率

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)
² : Glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the heel part separated according to Example 4

Example 11 [Composition data of cocoon and other (leaf sheath / leaf blade) separated from the whole component data for each sample of Yume Aoba]
Rice straw [Cultivar name: Dream Aoba harvested at each season (heading, milk ripening, paste ripening, yellow ripening and full ripening) and dried at 70 ° C for 48 hours or more (water content 10% or less)] The sample was cut at intervals of 1 cm in length and divided into whole or buttocks and other parts (leaf sheath / leaf blade) according to Example 1, and crushed / saccharified / degradable carbohydrates were quantified according to Example 1.

  The results are shown in Table 18. The content of the readily degradable sugars in the whole rice straw changed from the heading to the full maturity (harvest). The total content of heading, milk ripening, paste ripening, yellow ripening and full ripening was 15.73, 18.61, 10.25, 7.18, and 12.69%, respectively. In the period, the content rate showed a tendency to increase again. The most important factor in the change in the rate was the starch rate, which showed 8.76, 8.57, 2.45, 0.39 and 4.25% for each period. Moreover, the sucrose rate showed the same change pattern as the starch rate, and changes with time of 2.36, 4.57, 2.89, 1.05, and 2.96% were observed for each period. In both cases, the rate increased again during the harvest period. In addition, the total easily degradable sugar content of the buttocks shows a high value regardless of the season, and more than 70% of the total easily degradable sugar mass is concentrated in the buttocks after the milk ripening period. It was.

Glc.：Glucose、Fru.：Fructose、Suc.：Sucrose
全含有量^１：各試料30 g当たりの全易分解性糖質量（g）
量率^２：易分解性糖質量率（％）＝［各試料中の全易分解性糖質量（g）／稈と葉鞘・葉身の全易分解性糖質量の合計（g）］×100

Glc .: Glucose, Fru .: Fructose, Suc .: Sucrose
Total content ¹ : Total easily degradable sugar mass (g) per 30 g of each sample
Quantity ratio ² : Mass ratio of readily degradable sugar (%) = [total mass of easily degradable sugar in each sample (g) / total of all easily degradable sugar masses of cocoons, leaf sheaths and leaf blades (g)] × 100

Example 12 [Comparison data of total sulfuric acid two-stage saccharification data and enzymatic saccharification (cellulase / xylanase preparation, etc.) data and hand-separated cocoon and other (leaf sheath / leaf blade) data for samples of heading stage of Yume Aoba]
Rice straw and each fraction sample [variety name: Yume Aoba, heading stage, whole pulverized in Example 11, cocoon and leaf sheath / leaf blade] were weighed 50 mg each, and total saccharification experiment was conducted according to Example 2. Two-stage sulfuric acid treatment was performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 19. The glycan saccharification rate of whole rice straw was 64.46%, the cellulose saccharification rate was 51.01%, and the hemicellulose saccharification rate was 9.74%. The glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the buttocks and others (leaf sheath / leaf blade) were all high in the buttocks, showing 74.55, 42.90, and 9.27%.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)

Example 13 [Comparison data of total sulfuric acid two-stage saccharification data and enzymatic saccharification (cellulase / xylanase preparation, etc.) data, and hand-separated cocoon and other (leaf sheath / leaf blade)]
Rice straw and each fraction sample [variety name: Yume Aoba, fully matured, whole pulverized in Example 11, cocoon and leaf sheath / leaf blade] were weighed 50 mg each, and total saccharification experiment was conducted according to Example 2. Two-stage sulfuric acid treatment was performed, and the total glucose rate, glycan saccharification rate, cellulose rate, cellulose saccharification rate, total xylan rate, and hemicellulose saccharification rate were calculated.

  The results are shown in Table 20. The glycan saccharification rate of whole rice straw was 51.78%, the cellulose saccharification rate was 38.41%, and the hemicellulose saccharification rate was 6.69%. The glycan saccharification rate, cellulose saccharification rate, and hemicellulose saccharification rate of the buttocks and others (leaf sheath / leaf blade) were all high in the buttocks, showing 63.55, 47.47, and 7.72%.

Glc.：Glucose
総Glc.率^１、総キシラン率^１：2段階硫酸処理で測定（各試料の乾重あたり対する率）

Glc .: Glucose
Total Glc. Rate ¹ , total xylan rate ¹ : measured by two-stage sulfuric acid treatment (rate per dry weight of each sample)

Example 14 [Ethanol fermentation of rice straw by parallel double fermentation]
The parallel multi-fermentation of each sample [variety name: milky queen, heel part and leaf sheath / leaf body part separated in Example 4 and high specific gravity part and low specific gravity part mechanically separated in Example 9] The enzyme system and Saccharomyces cerevisiae NBRC0224 were used.

  That is, weigh out 200 mg of each sample [saddle part and high specific gravity part] and prepare two in 10 ml vials. Add 2 ml of sodium citrate buffer (50 mM, pH 4.8). Then, high-temperature and high-pressure sterilization (121 ° C., 15 minutes) was performed. After that, one of them was filtered (0.2 μm) cellulase preparation (24 μl, Celluclast 1.5 L, Novozymes Japan), hemicellulase preparation (12 μl, Viscozyme L, Novozymes Japan) and β-glucosidase preparation (8 μl, Novozyme188, Sigma) was added and shilled with a sterile rubber stopper and aluminum cap. Thereafter, the enzyme reaction was carried out at 30 ° C. for 48 hours. After the reaction, the sample was immediately cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), and a part of the supernatant was sampled. After diluting this with water, the amount of glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

For the other one, after adding the above enzyme solution, the yeast solution (the culture solution previously cultured in YPD medium at 30 ° C. for 16 hours was centrifuged twice (5,000 g, 5 minutes) and washed (saline) ) and was Schilling with sterile rubber stoppers and aluminum caps simultaneous saccharification that fermentation during the initial OD _600nm was adjusted to 1) was added 0.1 ml. Thereafter, parallel double fermentation was performed at 30 ° C. for 48 hours. After fermentation, the sample was quickly cooled to 4 ° C., centrifuged (15,000 g, 3 minutes), a portion of the supernatant was sampled, and filtered (0.2 μm). After diluting with water, Shimadzu HPLC (LC-20AD, SIL-20AC, CTO-20AC, RID-10A) and Aminex ^R HPX-87H column (300 mm x 7.8 mm, Bio-RAD) were used. Ethanol quantification was performed.

  100% of the theoretical amount of ethanol obtained from the amount of glucose after enzymatic saccharification and the fructose ratio determined in Examples 4 and 9 (two molecules of ethanol are produced from one molecule of glucose or one molecule of fructose). The ethanol fermentation yield (%) obtained by parallel double fermentation was calculated.

Since the saccharification rate is low in the leaf sheath / leaf blade part and the low specific gravity part, the rice straw alkali treatment performed in Example 3 was performed on these parts in order to perform an efficient saccharification reaction and ethanol fermentation. That is, weighed 200 mg of each sample [leaf sheath / leaf blade part and low specific gravity part], prepared two in 10 ml vials, added 1 ml NaOH aqueous solution (50 mM), 121 After alkali treatment at 15 ° C. for 15 minutes, 0.5 ml of sodium citrate buffer (200 mM, pH 4.8, NaN ₃ 0.01%) and 0.5 ml of aqueous HCl (100 mM) were added. One of them was added with only the above enzyme solution, and the other was added with the enzyme solution and the above yeast solution to carry out an enzyme reaction and parallel double fermentation, respectively. Thereafter, glucose quantification and ethanol quantification were performed in the same manner as described above, and the ethanol fermentation yield was calculated.

  The results are shown in Table 21. In all samples, the fermentation yield of ethanol exceeded 75%. The amount of each ethanol exceeded the amount of easily degradable carbohydrates determined in Examples 4 and 9, and ethanol production from cellulose was confirmed. In particular, ethanol production was also high in the buttocks and high specific gravity areas containing a large amount of easily degradable carbohydrates. In addition, it succeeded in obtaining a high ethanol fermentation yield after alkali treatment also in the leaf sheath / leaf blade part and low specific gravity part with low saccharification.

  The present invention relates to the development of efficient rice saccharification technology, and is expected to lead to the development of bioethanol production technology and biorefinery technology. In particular, it is extremely important as a new innovation for the development of domestic bioethanol production technology, which is an urgent issue in Japan. In addition, it will lead to the development of rice stalk and leaf utilization technology in rice growing areas mainly in Asia and the western United States.

It is a graph which shows the crushing particle size distribution by the physical crushing of a cocoon, a leaf sheath, and a leaf blade. ● shows the results for the leaf sheath / leaf blade, and ○ shows the result for the buttocks.

Claims (10)

  After cutting the rice shoots and leaves so that more than 70% of the whole fragments are 10cm in length by weight ratio, the bond between the buds in the cut and the other shoots and leaves is cut off, A method for saccharification of rice characterized by enzymatic saccharification of the fraction enriched with straw after separating the other leaves and stems.   After crushing and / or heat treatment at 80 ° C. or higher and 130 ° C. or lower for “the fraction enriched with koji”, cellulose degrading enzyme, starch degrading enzyme, β- (1 → 3), (1 → 4 2. The method for saccharification of rice according to claim 1, wherein enzymatic saccharification is performed under conditions containing at least one enzyme selected from the group consisting of glucan-degrading enzymes and hemicellulose-degrading enzymes.   From the group consisting of acid treatment, alkali treatment and hydrothermal treatment after pulverizing the fraction other than the “fraction enriched with straw” obtained by separating the straw and other stems and leaves The method for saccharification of rice according to claim 1 or 2, wherein the saccharification is performed by performing at least one selected pretreatment.   The method for saccharification of rice according to any one of claims 1 to 3, wherein the rice shoots and leaves are the above-ground part of the plant body after cutting the above-ground part of the rice and separating the straw or a cut product thereof.   The enzymatic saccharification is carried out after storing the "fraction enriched with koji" in a state where the water content is lowered to 20% (w / w) or less. Saccharification method of rice.   The method for saccharification of rice according to any one of claims 1 to 5, wherein the method of dividing the binding portion between the straw and the other stem and leaves is a method by grinding.   The saccharification of rice according to any one of claims 1 to 6, wherein the method of separating the cocoon and the other shoots and leaves is a method of separating the cocoon and the other shoots and leaves by a specific gravity difference. Law.   The paddy rice according to any one of claims 1 to 7, wherein, before the step of separating the cocoon and the other stem and leaf part, the size of the cocoon gap is reduced by pressing and crushing the cocoon. Saccharification method.   The saccharification of rice according to any one of claims 1 to 8, characterized in that the method of separating the straw from the other stems and leaves is a method of separating both from the difference in behavior when blown by wind. Law.   A method for producing ethanol, characterized by using the rice saccharification method according to claim 1.

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