JP5385561B2 - Method for producing sugar - Google Patents

Description

  The present invention relates to a method for producing sugar.

Cellulose obtained by pulverizing cellulose-containing raw materials such as pulp is used as industrial raw materials such as cellulose ether raw materials, cosmetics, foods, and biomass materials. In recent years, in particular, attempts have been made to produce sugar from biomass materials and to convert it into ethanol, lactic acid, or the like by a fermentation method or the like from efforts to address environmental problems (for example, Patent Document 1). When sugar is produced from biomass using an enzyme such as cellulase, it is useful to use cellulose having an amorphous cellulose crystal structure as a pretreatment step. For example, Patent Document 1 discloses that cellulose is made amorphous by using a cellulose solvent such as lithium chloride / dimethylacetamide.
Moreover, the method of processing a pulp mechanically with a grinder and reducing the crystallinity degree of a cellulose is known (for example, refer patent documents 2-5).
Examples 1 and 4 of Patent Document 2 disclose a method of treating sheet-like pulp with a vibrating ball mill or a twin screw extruder, and Examples 1 to 3 of Patent Document 3 disclose a method of treating pulp with a ball mill. Examples 1 and 2 of Patent Document 4 disclose a method of treating cellulose powder obtained by chemical treatment such as hydrolysis of pulp with a ball mill or an airflow type pulverizer. Patent Document 5 discloses a method of treating a pulp with a medium mill such as a vibrating ball mill in a state where the pulp is dispersed in water.
However, these methods are not satisfactory in efficiency and productivity in reducing the crystallinity of cellulose.

On the other hand, as a saccharification method of cellulose, there are known a method using a specific cellulase (Patent Document 6) and a method of hydrolyzing cellulose or hemicellulose treated with hydrogen peroxide (Patent Documents 7 and 8). Yes.
However, these methods are not satisfactory in terms of saccharification efficiency and productivity.

JP 2006-223152 A JP 62-236801 A JP 2003-64184 A JP 2004-331918 A JP 2005-68140 A JP 2003-135052 A JP 2007-74992 A Japanese Patent Laid-Open No. 2007-74993

  In the present invention, sugar can be efficiently obtained by performing an enzymatic reaction such as cellulase using a cellulose-containing raw material as a substrate and a non-crystallized cellulose having a reduced degree of crystallinity of cellulose I, and has excellent productivity. It aims at providing the manufacturing method of sugar.

The present inventors can solve the above-mentioned problems by carrying out an enzyme reaction such as cellulase after performing a pretreatment (amorphization) with a vibration mill filled with a rod using a specific cellulose-containing material as a starting material. I found.
That is, the present invention is a method for saccharifying amorphous cellulose prepared from a cellulose-containing raw material having a cellulose I-type crystallinity of more than 33% represented by the following formula (1), wherein water is removed from the raw material. The cellulose content in the remaining components was 20% by mass or more, and the cellulose-containing raw material was treated with a vibration mill filled with a rod to reduce the cellulose I-type crystallinity to 33% or less. This is a method for producing sugar, in which after the crystallized cellulose is prepared, cellulase and / or hemicellulase is allowed to act on the amorphous cellulose.
Cellulose type I crystallinity (%) = [(I _22.6 -I _18.5 ) / I _22.6 ] × 100 (1)
[I _22.6 is the diffraction intensity of the grating plane (002 plane) (diffraction angle 2θ = 22.6 °) in X-ray diffraction, and I _18.5 is the diffraction intensity of the amorphous portion (diffraction angle 2θ = 18.5 °). ]

  The sugar production method of the present invention can efficiently produce non-crystallized cellulose having a reduced cellulose I-type crystallinity from the cellulose-containing raw material in the pretreatment, so that the sugar is efficiently produced by enzymatic reaction. be able to.

The present invention is a method for saccharifying non-crystalline cellulose prepared from a cellulose-containing raw material having a cellulose I-type crystallinity of more than 33% represented by the following calculation formula (1), wherein the residue obtained by removing water from the raw material The amorphous content in which the cellulose content is 20% by mass or more and the cellulose-containing raw material is treated with a vibration mill filled with a rod to reduce the cellulose I-type crystallinity to 33% or less. This is a method for producing sugar, characterized in that cellulase and / or hemicellulase is allowed to act on the non-crystalline cellulose after preparing the cellulose.
Cellulose type I crystallinity (%) = [(I _22.6 -I _18.5 ) / I _22.6 ] × 100 (1)
[I _22.6 is the diffraction intensity of the grating plane (002 plane) (diffraction angle 2θ = 22.6 °) in X-ray diffraction, and I _18.5 is the diffraction intensity of the amorphous portion (diffraction angle 2θ = 18.5 °). ]
Hereinafter, the present invention will be described in detail. In the present specification, cellulose I type crystallinity may be simply referred to as “crystallinity”.

[Cellulose-containing raw material]
The cellulose-containing raw material used in the present invention has a cellulose content in the remaining components excluding water from the raw material of 20% by mass or more, preferably 40% by mass or more, more preferably 60% by mass or more.
The cellulose content used in the present invention means the total amount of cellulose and hemicellulose.
There is no restriction | limiting in particular in the said cellulose-containing raw material, Various wood chips, pruned branch materials, thinning materials, branch wood, etc .; Wood pulp manufactured from wood, Cotton linter pulp obtained from the fiber around cotton seeds Pulps such as newspapers, corrugated cardboard, magazines, fine paper, etc .; plant stems and leaves such as rice straw and corn stalks; plant shells such as rice husks, palm husks and coconut husks. Among these, pulps, papers, plant shells, and woods are preferable, and pulps and papers are more preferable.
In the case of commercially available pulp, the cellulose content in the remaining components excluding water is generally 75 to 99% by weight, and other components include lignin and the like. Moreover, the cellulose I type crystallinity degree of a commercially available sheet-like pulp is 60% or more normally.
The water content in the cellulose-containing raw material is preferably 20% by mass or less, more preferably 15% by mass or less, and particularly preferably 10% by mass or less. If the water content in the cellulose-containing raw material is 20% by mass or less, it can be easily pulverized, and the crystallinity can be easily reduced by the pulverization process described later, and the subsequent sugar production can be efficiently performed. it can.

[Cellulose type I crystallinity]
The non-crystallized cellulose prepared in the present invention has a cellulose I-type crystallinity reduced to 33% or less. Cellulose type I crystallinity is calculated by the Segal method from the diffraction intensity value by the X-ray diffraction method, and is defined by the following calculation formula (1).
Cellulose type I crystallinity (%) = [(I _22.6 -I _18.5 ) / I _22.6 ] × 100 (1)
[I _22.6 is the diffraction intensity of the grating plane (002 plane) (diffraction angle 2θ = 22.6 °) in X-ray diffraction, and I _18.5 is the diffraction intensity of the amorphous portion (diffraction angle 2θ = 18.5 °). ]
If the degree of crystallinity is 33% or less, the chemical reactivity of cellulose is improved. For example, in the production of cellulose ether, alkali celluloseization easily proceeds when an alkali is added, resulting in cellulose etherification reaction. The reaction conversion rate can be improved. From this viewpoint, the degree of crystallinity is preferably 20% or less, more preferably 10% or less, and particularly preferably 0% in which cellulose I-type crystals are not detected by analysis. The cellulose I type crystallinity defined by the calculation formula (1) may be a negative value in calculation, but in the case of a negative value, the cellulose I type crystallinity is 0%.
Here, the cellulose I type crystallinity is the ratio of the amount of crystal region of cellulose to the total amount. Cellulose type I is a crystalline form of natural cellulose. Cellulose type I crystallinity is also related to the physical and chemical properties of cellulose, and the higher the value, the higher the crystallinity of cellulose and the less non-crystalline parts. Flexibility, solubility in water and solvents, and chemical reactivity are reduced.

[Vibration mill]
In the present invention, the cellulose-containing raw material is pulverized by a vibration mill filled with a rod. By pulverizing with a vibration mill filled with a rod, the cellulose in the raw material can be efficiently amorphized.
As a vibration mill used in the present invention, a vibration mill manufactured by Chuo Kako Co., Ltd., a small vibration rod mill 1045 type manufactured by Yoshida Seisakusho Co., Ltd., a vibration cup mill P-9 type manufactured by Fritsch, Germany, and Nichito Kagaku Co., Ltd. A small vibration mill NB-O type or the like made from can be used. The processing method may be either a batch type or a continuous type.

〔rod〕
The rod filled in the vibration mill is a rod-shaped medium, and a rod having a cross section of a polygon such as a square or a hexagon, a circle or an ellipse can be used.
There is no restriction | limiting in particular as a material of a rod, For example, iron, stainless steel, an alumina, a zirconia, silicon carbide, silicon nitride, glass etc. are mentioned.
The outer diameter of the rod is preferably in the range of 0.5 to 200 mm, more preferably 1 to 100 mm, and still more preferably 5 to 50 mm. The length of the rod is not particularly limited as long as it is shorter than the length of the crusher container. If the size of the rod is in the above range, a desired pulverizing force can be obtained, and the cellulose can be efficiently amorphized without contaminating the powdered cellulose by mixing fragments of the rod and the like.
The filling range of the rod is preferably in a range of 10 to 97%, more preferably 15 to 95%, although a suitable range varies depending on the type of vibration mill. When the filling rate is within this range, the contact frequency between the cellulose and the rod is improved, and the grinding efficiency can be improved without hindering the movement of the medium. Here, the filling rate refers to the apparent volume of the rod relative to the volume of the vibration mill.
The treatment time of the vibration mill cannot be determined unconditionally depending on the type of vibration mill, the type of rod, the size, the filling rate, etc., but from the viewpoint of reducing the crystallinity, it is preferably 0.01 to 50 hr, more preferably 0.05-20 hr, more preferably 0.1-10 hr. Although processing temperature does not have a restriction | limiting in particular, From a viewpoint of preventing deterioration by a heat | fever, Preferably it is 5-250 degreeC, More preferably, it is 10-200 degreeC.

By the above-mentioned treatment method, amorphous cellulose having a cellulose I-type crystallinity of 33% or less can be efficiently obtained from the cellulose-containing raw material, and the pulverized product is fixed inside the mill during the vibration mill treatment. Without being dry.
The average particle size of the obtained amorphous cellulose is preferably 25 to 150 μm, more preferably 30 to 100 μm, from the viewpoint of chemical reactivity and handleability when the amorphous cellulose is used as an industrial raw material. In particular, when the average particle size is 25 μm or more, it is possible to suppress the occurrence of “maco” when the non-crystalline cellulose is brought into contact with a liquid such as water.

In addition, in the vibration mill treatment in the present invention, it is preferable to use a cellulose-containing raw material having a bulk density of 100 kg / m ³ or more from the viewpoint of more efficiently performing pulverization and amorphousization in a vibration mill filled with rods. 120 kg / m ³ or more is more preferable, and 150 kg / m ³ or more is more preferable. If this bulk density is 100 kg / m ³ or more, the cellulose-containing raw material has an appropriate volume, so that handleability is improved. Moreover, since the amount of raw materials charged into the vibration mill can be increased, the processing capacity is improved. On the other hand, the upper limit of the bulk density is preferably 500 kg / m ³ or less, more preferably 400 kg / m ³ or less, and still more preferably 350 kg / m ³ or less from the viewpoint of handleability and productivity. From these viewpoints, the bulk density is preferably 100 to 500 kg / m ³ , more preferably 120 to 400 kg / m ³ , and still more preferably 150 to 350 kg / m ³ .

  The cellulose-containing raw material supplied to the vibration mill preferably has an average particle size in the range of 0.01 to 1 mm from the viewpoint of efficiently dispersing the pulverized raw material in the vibration mill. If this average particle size is 1 mm or less, when supplying into the vibration mill, the pulverized raw material can be efficiently dispersed in the vibration mill and reach a predetermined particle size without requiring a long time. Can do. On the other hand, the lower limit of the average particle diameter is preferably 0.01 mm or more from the viewpoint of productivity. From these viewpoints, the average particle diameter is more preferably 0.01 to 0.7 mm, and even more preferably 0.05 to 0.5 mm. In addition, said average particle diameter and bulk density can be measured by the method as described in an Example.

[Pretreatment of vibration mill grinding]
In this invention, it is preferable to pre-process the cellulose containing raw material supplied to a vibration mill. For example, by processing the cellulose-containing raw material with an extruder, the bulk density and the average particle diameter of the cellulose-containing raw material can be set to the above-described preferable ranges.
It is preferable to coarsely pulverize the cellulose-containing raw material before feeding it into the extruder. The size of the coarsely pulverized product is preferably 1 to 50 mm, more preferably 1 to 30 mm. By roughly pulverizing to 1 to 50 mm, the extruder treatment can be performed efficiently and easily, and the load required for pulverization can be reduced.

  Examples of the method for coarsely pulverizing the cellulose-containing raw material include a method using a shredder or a rotary cutter. When a rotary cutter is used, the size of the coarsely pulverized product obtained can be controlled by changing the opening of the screen. The opening of the screen is preferably 1 to 50 mm, more preferably 1 to 30 mm. If the opening of the screen is 1 mm or more, a coarsely pulverized product having an appropriate bulkiness is obtained, and the handleability is improved. If the opening of the screen is 50 mm or less, it has an appropriate size as a pulverized raw material in the subsequent pulverization process, so that the load can be reduced.

By treating the cellulose-containing raw material, preferably the coarsely pulverized cellulose-containing raw material, with an extruder, a compressive shear force can be applied to break the crystal structure of the cellulose, whereby the cellulose-containing raw material can be powdered.
As a method of mechanically pulverizing by applying a compressive shear force, conventionally used impact type pulverizers such as a cutter mill, a hammer mill, a pin mill, etc., make the pulverized material fluffy and bulky. And the mass-based throughput is reduced. On the other hand, by using an extruder, a pulverized raw material having a desired average particle diameter and bulk density can be obtained, and handleability can be improved.

The extruder may be either a single-screw type or a twin-screw type, but a twin-screw extruder is preferable from the viewpoint of increasing the conveyance capability.
The twin screw extruder is an extruder in which two screws are rotatably inserted into a cylinder, and conventionally known ones can be used. The rotation directions of the two screws may be the same or opposite directions, but the rotation in the same direction is preferable from the viewpoint of increasing the conveyance capability.
The screw engagement conditions may be any of full-engagement, partial meshing, and non-meshing extruders. However, from the viewpoint of improving the processing capability, the complete meshing type and the partial meshing type are preferable.

As an extruder, it is preferable to provide what is called a kneading disk part in any part of a screw from a viewpoint of applying a strong compressive shear force.
The kneading disc part is composed of a plurality of kneading discs, which are combined continuously and shifted in a constant phase, for example by 90 °, with a narrow gap as the screw rotates. An extremely strong shearing force can be imparted by forcibly passing the cellulose-containing raw material. As a configuration of the screw, it is preferable that the kneading disk portion and the plurality of screw segments are alternately arranged. In the case of a twin screw extruder, it is preferable that the two screws have the same configuration.

As a processing method, the cellulose-containing raw material, preferably the coarsely pulverized cellulose-containing raw material, is charged into an extruder and continuously processed. The shear rate is preferably 10 sec ^-1 or more, more preferably 20~30000sec ^-1, 50~3000sec ^-1 is particularly preferred. If the shear rate is 10 sec ⁻¹ or more, pulverization proceeds effectively. Other treatment conditions are not particularly limited, and preferably a treatment temperature of 5 to 200 ° C.
Also, as the number of passes by the extruder, a sufficient effect can be obtained even with one pass, but from the viewpoint of reducing the crystallinity and the degree of polymerization of cellulose, if one pass is insufficient, perform two or more passes. Is preferred. Further, from the viewpoint of productivity, 1 to 10 passes is preferable. By repeating the pass, coarse particles are pulverized and a powdery cellulose-containing raw material with little variation in particle size can be obtained. When performing two or more passes, in consideration of production capacity, a plurality of extruders may be arranged in series for processing.

[Saccharification with enzymes such as cellulase]
Since the non-crystalline cellulose obtained by the above-described treatment has a low crystallinity, it is possible to efficiently obtain a mixture of glucose or oligosaccharides such as cellobiose and cellotriose by enzymatic treatment with cellulase. Considering the case of using it for ethanol fermentation or lactic acid fermentation after saccharification treatment, it is preferable to decompose to monosaccharide. Cellulase as used herein refers to an enzyme that hydrolyzes the glycosidic bond of β-1,4-glucan of cellulose, and is a generic term for enzymes called endoglucanase, exoglucanase or cellobiohydrolase, β-glucosidase, and the like. is there. Moreover, it is possible to raise the efficiency of saccharification by making hemicellulases, such as xylanase, act simultaneously.

The cellulase and hemicellulase used for the saccharification treatment are not particularly limited, and commercially available cellulase preparations and those derived from animals, plants, and microorganisms can be used. Examples of cellulases include cellulase preparations derived from Trichoderma reesei such as Cellcrust 1.5L (Novozymes), cellulases derived from Bacillus sp. KSM-N145 (FERM P-19727), or Bacillus sp (Bacillus sp.) KSM-N252 (FERM P-17474), Bacillus sp. (Bacillus sp.) KSM-N115 (FERM P-19726), Bacillus sp. (Bacillus sp.) KSM-N440 (FERM P-19728), Bacillus sp (Bacillus sp.) KSM-N659 (FERM P-19730) cellulase from each strain, such as, furthermore, Trichoderma viride (Trichoderma viride), Aspergillus Akureatasu (Aspergillus acleatus), Clostridium thermocellum (Clostridium thermocellum), Clostridium ester Korariumu (Clostridium stercorarium), Clostridium josui (Clostridium josui) cellulose Eggplant Fimi (Cellulomonas fimi), Acremonium cell Lori caustics (Acremonium celluloriticus), Irupekkusu Rakuteusu (Irpex lacteus), Aspergillus niger (Aspergillus niger), Humicola insolens (Humicola insolens) derived cellulase mixtures and Pyrococcus horikoshii (Pyrococcus horikoshii) from And thermostable cellulase. Among these, preferably a cellulase derived from Trichoderma reesei , Trichoderma viride , or Humicola insolens , such as Cell Crust 1.5L (Novozymes), TP-60 (Meiji Seika Co., Ltd.) Company) or Ultraflo L (Novozymes) can be used to produce sugar efficiently.

Further, hemicellulase Bacillus sp Examples (Bacillus sp.) KSM-N546 (FERM P-19729) of Kishinaraze from other Aspergillus niger (Aspergillus niger), Trichoderma viride (Trichoderma viride), Humicola insolens (Humicola insolens) , Bacillus alcalophilus (Bacillus alcalophilus) derived from a xylanase, further, Thermomyces (Thermomyces), Ou Leo bus Shijiumu (Aureobasidium), Streptomyces (Streptomyces), Clostridium (Clostridium), Thermotoga (Thermotoga), Thermoascus (Thermoascus) Xylanase derived from the genus Caldocellum , Thermomonospora, and the like. An enzyme having hemicellulase activity contained in the cellulase mixture can also be used.
Although these enzymes can be used alone, it is effective to use these enzymes in combination for more efficient sugar production. Moreover, the efficiency of sugar production can be improved by further adding a specific cellulase component such as β-glucosidase to these enzymes. Examples of β-glucosidase to be added include enzymes derived from Aspergillus niger (for example, Novozymes 188 or Megazyme β-glucosidase), Trichoderma reesei , Penicillium emersonii ii. An enzyme etc. are mentioned.

  About the reaction conditions in the case of saccharifying the non-crystallized cellulose obtained by the process mentioned above by the enzyme process by a cellulase, it can select suitably by the crystallinity of the non-crystallized cellulose obtained by the pre-process, and the enzyme to be used. . For example, when 1.5 L of cell crust manufactured by Novozymes is used as cellulase and cellulose having a crystallinity of 0% derived from pulp is used as a substrate, the substrate suspension is 0.5 to 20% (w / v). Cellulose 1.5L is added to 0.001-15% (v / v) (corresponding to 0.00017-2.5% as protein) to pH 2-10 (appropriate depending on the type of enzyme used) It is preferable to select a suitable pH, and when 1.5 liters of cell crust is used, the reaction temperature is preferably 10 to 90 ° C. (preferably depending on the type of enzyme used) in a buffer solution of pH 3 to 7, particularly preferably around pH 5. The reaction is preferably performed at a temperature of 20 to 70 ° C., particularly preferably about 50 ° C. when a cell crust of 1.5 L is used, and a reaction time of 30 minutes to 5 days, preferably 0.5 to 3 days. It can be produced sugar by Rukoto.

The average particle size, bulk density, X-ray diffraction intensity, and moisture content of the non-crystalline cellulose or cellulose-containing raw material were measured by the methods described below. Moreover, the saccharification reaction of various celluloses, such as an amorphous cellulose, was performed on condition of the following.
(1) Measurement of average particle diameter The average particle diameter was measured using a laser diffraction / scattering particle size distribution measuring apparatus “LA-920” (manufactured by Horiba, Ltd.). The measurement conditions were that the sample was treated with ultrasonic waves for 1 minute before the particle size measurement, water was used as a dispersion medium at the time of measurement, and the measurement was performed at a temperature of 25 ° C.
(2) Measurement of bulk density The bulk density was measured using "Powder Tester" manufactured by Hosokawa Micron Corporation. The measurement was performed by vibrating the sieve, dropping the sample through a chute, receiving it in a specified container (capacity 100 mL), and measuring the mass of amorphous cellulose in the container.

(3) Measurement of X-ray diffraction intensity X-ray diffraction intensity was measured using the “Rigaku RINT 2500VC X-RAY diffractometer” manufactured by Rigaku Corporation under the following conditions, and based on the above formula (1), cellulose I The type crystallinity was calculated.
The measurement conditions were X-ray source: Cu / Kα-radiation, tube voltage: 40 kv, tube current: 120 mA, measurement range: 2θ = 5-45 °. The measurement sample was prepared by compressing a pellet having an area of 320 mm ² × thickness of 1 mm. The X-ray scan speed was measured at 10 ° / min.
(4) Measurement of moisture content The moisture content was measured at 150 ° C using an infrared moisture meter ("FD-610", manufactured by Kett Scientific Laboratory).
(5) Measurement of cellulose content The ground sample was subjected to Soxhlet extraction with ethanol / benzene mixed solvent (1: 1) for 6 hours, further Soxhlet extraction with ethanol for 4 hours, and the sample after extraction was vacuum-dried at 60 ° C. did. To 2.5 g of the obtained sample, 150 mL of water, 1.0 g of sodium chlorite and 0.2 mL of acetic acid were added, and the mixture was heated at 70 to 80 ° C. for 1 hour. Subsequently, an operation of adding sodium chlorite and acetic acid and heating was repeated, and the treatment was repeated 3 to 4 times until the sample was decolorized white. The white residue was filtered through a glass filter (1G-3), washed with cold water and acetone, dried at 105 ° C. until a constant weight was obtained, the residue weight was determined, and the cellulose content was calculated according to the following formula.
Cellulose content (mass%) = [residue weight (g) / sampled amount (g)] × 100

(6) Saccharification reaction The enzymatic saccharification reaction was carried out under the following conditions. 3 mL of enzyme reaction solution (100 mM citrate buffer (pH 5.0), 30 μg / mL tetracycline (added as preservative)), screw tube with lid (No. 5, The suspension was suspended in Φ27 × 55 mm (Marem Co., Ltd.), an appropriate amount of enzyme was added, and the mixture was reacted at 50 ° C. with shaking (150 rpm, constant temperature shaker “BR-15CF” manufactured by Taitec Corporation) for a predetermined time. After completion of the reaction, the precipitate and the supernatant were separated by centrifugation (17,000 × g, 5 minutes), and the amount of reducing sugar released into the supernatant was quantified by the DNS method and HPLC method shown below. As a control, the same analysis was performed for an unreacted enzyme reaction solution.
(7) DNS method (Biochemical experiment method Quantitative method for reducing sugar: Quantitative analysis of the scientific society) DNS solution DNS solution (0.5% 3,5-dinitrosalicylic acid, 30% sodium potassium tartrate tetrahydrate, 1.6 An appropriate amount of the supernatant was added to 1 mL of (% sodium hydroxide), and the mixture was heated and colored at 100 ° C. for 5 minutes. After cooling, colorimetric determination was performed at a wavelength of 535 nm. The amount of reducing sugar in the supernatant was calculated from a calibration curve using glucose as the standard sugar. In this method, since the degree of color development differs from that of glucose depending on the type of sugar produced, a value different from the following HPLC method and the like may be obtained.
(8) Quantification of sugar by HPLC method
Dionex DX500 chromatography system; column: CarboPac PA1 (Dionex 4 × 250 mm), detector: ED40 pulsed amperometric detector, eluent: solution A; 100 mM sodium hydroxide solution, solution B; 1M sodium acetate Contains 100 mM sodium hydroxide solution, solution C; ultrapure water was used. From the injection, sugars were analyzed with a linear gradient of 10% initial concentration A solution: 90% C solution, 0-15 minutes, A solution 95%: B solution 5%. 0.01% (w / v) glucose as standard (Wako Pure Chemical Industries, Ltd.), xylose (Wako Pure Chemical Industries, Ltd.), xylobiose (Wako Pure Chemical Industries, Ltd.), cellobiose (Seikagaku Corporation) Used).
(9) Protein quantification Using a DC protein assay kit (Bio Rad), the protein amount was calculated from a calibration curve using bovine serum albumin as a standard protein.

Production Example 1
[Shredder treatment]
Shredder (Meiko Shokai Co., Ltd.) sheet wood pulp ("Blue Bear Ultra Ether" manufactured by Borregard, 800 mm x 600 mm x 1.5 mm, crystallinity 81%, cellulose content 96 mass%, water content 7 mass%) Manufactured, “MSX2000-IVP440F”) to obtain a chip-like pulp of about 10 mm × 5 mm × 1.5 mm.
[Vibration mill treatment]
100 g of the obtained chip-like pulp was put into a vibration mill (manufactured by Chuo Kako Co., Ltd., “MB-1”, container total capacity 3.5 L), and as a rod, diameter 25 mm, length 218 mm, material stainless steel, cross section Sixteen rods having a circular shape were filled in a vibration mill (filling rate 49%), and the treatment was performed for 3 hours under the conditions of an amplitude of 8 mm and a circular rotation of 1200 cpm. The average particle size of the amorphous cellulose obtained after the vibration mill treatment was 80 μm. Moreover, the temperature of the obtained amorphous cellulose was 85 degreeC by the heat_generation | fever accompanying a process.
After completion of the treatment, no pulp sticking matter was found on the wall or bottom of the vibration mill. The obtained amorphous cellulose was taken out from the vibration mill, the average particle size of the obtained amorphous cellulose was measured, and the crystallinity was calculated from the X-ray diffraction intensity. The results are shown in Table 1.

Production Example 2
(Extruder processing)
Chip-like pulp obtained by subjecting the same pulverized raw material as in Production Example 1 to the same shredder treatment as in Example 1 was charged into a twin-screw extruder (“EA-20” manufactured by Suehiro EPM Co., Ltd.) at 2 kg / hr, and sheared. One-pass processing was performed while flowing cooling water from the outside at a speed of 660 sec ⁻¹ , a screw rotation speed of 300 rpm. The twin-screw extruder is a fully meshing type co-rotating twin-screw extruder, and the screws arranged in two rows are combined with a screw portion having a screw diameter of 40 mm and 12 blocks alternately (90 °). The two screws have the same configuration. The temperature of the twin screw extruder was 30 to 70 ° C. due to heat generated by the treatment.
The pulp obtained after the extruder treatment had an average particle size of 121 μm and a bulk density of 254 kg / m ³ .
[Vibration mill treatment]
Next, using the pulp obtained after the extruder treatment, vibration mill pulverization was performed in the same manner as in Production Example 1 to obtain amorphous cellulose. After completion of the pulverization, no fixed pulp was found on the wall or bottom of the vibration mill. The results are shown in Table 1.

Production Example 3
As a rod filled in the vibration mill, 13 rods with a diameter of 30 mm, a length of 218 mm, stainless steel, and a circular cross section were filled in the vibration mill (filling rate 57%), and the processing time of the vibration mill was changed to 1 hr. Amorphous cellulose was obtained by the same method and conditions as in Production Example 2 except for the above. The results are shown in Table 1.

Production Example 4
Amorphized cellulose was obtained by the same method and conditions as in Production Example 2, except that the number of rods filled in the vibration mill was changed to 14 and the vibration mill was filled (filling rate 62%). The results are shown in Table 1.

Production Example 5
Manufactured except that the vibration mill is filled with a rod with a diameter of 36 mm, a length of 218 mm, and made of stainless steel (filling rate 51%) and the processing time of the vibration mill is changed to 1 hr. Amorphized cellulose was obtained by the same method and conditions as in Example 2. The results are shown in Table 1.

Production Example 6
Manufactured except that the vibration mill is filled with a rod with a diameter of 30 mm, a length of 218 mm and made of stainless steel (filling rate 48%), and the processing time of the vibration mill is changed to 3 hr. Amorphized cellulose was obtained by the same method and conditions as in Example 2. The results are shown in Table 1.

Production Examples 7 to 10
As cellulose-containing raw materials, fine paper (Production Example 7: Cellulose content 83% by mass, moisture content 5.7% by mass), cardboard (Production Example 8: Cellulose content 84% by mass, moisture content 7.2% by mass), Production Example 1 using newspaper (Production Example 9: cellulose content 83% by mass, moisture content 7.7% by mass) and rice husk (Production Example 10: cellulose content 60% by mass, moisture content 13.6% by mass) After shredding with the method and conditions described in 1., the extruder was treated with the method and conditions of Production Example 2. The properties of the cellulose-containing raw materials obtained after the extruder treatment were as follows: Production Example 7: fine paper / average particle size 71 μm / bulk density 274 kg / m ³ , Production Example 8: cardboard / average particle size 93 μm / bulk density 216 kg / m ^3. Production Example 9: Newspaper / average particle size 61 μm / bulk density 303 kg / m ³ , Production Example 10: rice husk / average particle size 85 μm / bulk density 380 kg / m ³ .
Further, the vibration mill treatment was carried out by the method and conditions of Production Example 6 to produce amorphous cellulose (Production Example 7: fine paper / crystallinity 0% / average particle size 42 μm, Production Example 8: cardboard / crystallinity 0). % / Average particle size 48 μm, Production Example 9: newspaper / crystallinity 0% / average particle size 55 μm, Production Example 10: rice husk / crystallinity 0% / average particle size 48 μm).

Production Examples 11-12
[Coarse pulverization of pruned branches]
As cellulose-containing raw materials, pruned branches of rod-shaped street trees (Production Example 11: φ10 mm × 300 mm, moisture content 12% by mass), pruned branches of rod-shaped mandarin trees (Production Example 12: φ10 mm × 500 mm, cellulose content 64% by mass) And a moisture content of 22% by mass) was applied to a plastic pulverizer (JC-2 type manufactured by Morita Seiki Kogyo Co., Ltd.) to form a chip of about 2 mm × 3 mm × 1 mm. Then, it dried with the drier until the water content of the pruned branch of the obtained chip-shaped roadside tree and a mandarin tree became 2.3 mass% and 3.5 mass%, respectively. The bulk density of the pruned branch of the chip-shaped mandarin tree thus obtained was 224 kg / m ³ .
[Vibration milling of pruned branches]
The obtained chip-shaped pruned branches were subjected to vibration mill treatment under the method and conditions of Production Example 6 to produce amorphous cellulose (Production Example 11; roadside tree branch / crystallinity 0% / average particle size 49 μm). Production Example 12: tangerine tree branch / 0% crystallinity / average particle size 44 μm).

Comparative production example 1
A shredder process was performed in the same manner as in Production Example 1 to obtain chip-like pulp. Next, 100 g of this chip-like pulp is put into a rolling mill (manufactured by Nippon Ceramic Science Co., Ltd., “Pot Mill ANZ-51S”, container volume 1.0 L, 10 mmφ zirconia balls are filled 1.8 kg, filling rate 53%). Then, the treatment was performed for 48 hours under the condition of a rotation speed of 100 rpm. The obtained pulp did not pulverize and remained almost chip-like. By the above method, the crystallinity was calculated from the X-ray diffraction intensity of the obtained pulp. The results are shown in Table 2.

Comparative production example 2
A shredder treatment was performed in the same manner as in Production Example 1 to obtain chip-like pulp. Next, 500 g of this chip-shaped pulp was put into a cutter mill (manufactured by Dalton Co., Ltd., “Power Mill P-02S type”), and subjected to a treatment for 0.5 hr under the condition of a rotational speed of 3000 rpm. The obtained pulverized product became cottony and could not obtain amorphous cellulose. The results are shown in Table 2.

Comparative production example 3
A shredder treatment was performed in the same manner as in Production Example 1 to obtain chip-like pulp. Next, 500 g of this chip-like pulp was put into a hammer mill ("SAMPLE-MILL" manufactured by Dalton Co., Ltd.) and treated for 0.5 hr under the condition of a rotational speed of 13500 rpm. The obtained pulverized product became cottony and could not obtain amorphous cellulose. The results are shown in Table 2.

Comparative production example 4
A shredder treatment was performed in the same manner as in Production Example 1 to obtain chip-like pulp. Next, 500 g of this chip-like pulp was put into a pin mill (Hosokawa Micron Co., Ltd., “Coloplex”), and treated for 0.25 hr under the condition of a rotational speed of 13000 rpm. The obtained pulverized product became cottony and could not obtain amorphous cellulose. The results are shown in Table 2.

Comparative Production Example 5
A shredder treatment was performed in the same manner as in Production Example 1 to obtain chip-like pulp. Subsequently, a twin-screw extruder process was performed, and powder pulp was obtained without performing a pulverizer process. The average particle diameter of the obtained powder pulp was measured by the above method, and the crystallinity was calculated from the X-ray diffraction intensity. The results are shown in Table 2.

Comparative production examples 6-11
The shredder process and the extruder process are similarly performed using the cellulose-containing raw materials of Production Examples 7 to 10, or the pruned branches of the roadside trees and the pruned branches of the mandarin orange trees of Production Examples 11 to 12 are similarly roughened by a plastic grinder. Powdered or chip-like cellulose without pulverization and then vibration milling (Comparative Production Example 6: fine paper / crystallinity 71% / average particle size 71 μm, comparative production example 7: cardboard / crystallinity 71 % / Average particle size 93 μm, comparative production example 8: newspaper / crystallinity 56% / average particle size 61 μm, comparative production example 9: rice husk / crystallinity 47% / average particle size 85 μm, comparative production example 10: roadside tree Pruned branch / crystallinity 51% / size: 2 mm × 3 mm × 1 mm, Comparative Production Example 11: pruned branch of mandarin orange / crystallinity 46% / size: 2 mm × 3 mm × 1 mm.

Example 1 and Comparative Example 1
As Example 1, non-crystallized cellulose (crystallinity 0%, particle size 57 μm) prepared by extruder processing and vibration mill processing filled with rods in Production Example 6, and Comparative Example 1 also non-production Example 6 Saccharification reaction with cellulase enzyme preparation (Cell Crust 1.5 L, manufactured by Novozymes) of powder pulp (crystallinity 76%, particle size 156 μm) prepared by performing only the extruder treatment as pretreatment at the time of preparing crystallized cellulose Went. 3 mL of enzyme reaction solution (100 mM citrate buffer (pH 5.0), 3% (v / v) cell crust 1.5 L (corresponding to 0.5% protein), 30 μg) / ML tetracycline), and the enzyme reaction was performed for 18 hours, 26 hours, and 42 hours with shaking and stirring at 50 ° C. After completion of the reaction, the precipitate and the supernatant were separated by centrifugation, and the amount of reducing sugar released in the supernatant was quantified (converted to glucose) by the DNS method. The results are shown in Table 3.

Examples 2 to 4 and Comparative Examples 2 to 4
As Examples 2 to 4, amorphous cellulose was prepared under the conditions of Production Example 6 described above (Examples 2 to 4: Crystallinity 0% / average particle size 57 μm). In Comparative Examples 2 to 4, powder pulp (Comparative Examples 2 to 4: crystallinity 76% / average particle size 156 μm) prepared under the conditions of Comparative Production Example 5 described above was used. A saccharification reaction was performed with these cellulase enzyme preparations (Cell Crust 1.5 L, manufactured by Novozymes). An enzyme reaction was carried out for 3 days while shaking and agitating 0.15 g of amorphous cellulose or powdered pulp at 50 ° C. in a 3 mL enzyme reaction solution. The enzyme reaction solution was Example 2 and Comparative Example 2: 100 mM citrate buffer (pH 5.0), 3% (v / v) cell crust 1.5 L (corresponding to 0.5% protein), 30 μg / mL tetracycline, Example 3 and Comparative Example 3: 100 mM citrate buffer (pH 5.0), 1.5% (v / v) cell crust 1.5 L (corresponding to protein 0.25%), 30 μg / mL tetracycline, Example 4 And Comparative Example 4: 100 mM citrate buffer (pH 5.0), 0.6% (v / v) cell crust 1.5 L (corresponding to 0.1% protein), 30 μg / mL tetracycline. After completion of the reaction, the precipitate and the supernatant were separated by centrifugation, and the amount of reducing sugar released in the supernatant was quantified (converted to glucose) by the DNS method. The results are shown in Table 4.

Example 5
The saccharified solution supernatant (reaction 72 hours) obtained in Example 2 was analyzed by the following method using a Dionex DX500 chromatography system.
Column: CarboPac PA1 (Dionex 4 × 250 mm), detector: ED40 pulsed amperometric detector, eluent: solution A; 100 mM sodium hydroxide solution, solution B; 100 mM sodium hydroxide solution containing 1M sodium acetate, C Liquid: Ultrapure water From the injection, the initial concentration was analyzed by linear gradient of 10% of liquid A: 90% of liquid C, 0-15 minutes, 95% of liquid A: 5% of liquid B. 0.01% (w / v) glucose as standard (Wako Pure Chemical Industries, Ltd.), xylose (Wako Pure Chemical Industries, Ltd.), xylobiose (Wako Pure Chemical Industries, Ltd.), cellobiose (Seikagaku Corporation) Used). Glucose showed peaks at a retention time of about 5.5 minutes, xylose at about 6.5 minutes, xylobiose at about 14 minutes, and cellobiose at about 14.5 minutes. The saccharified solution supernatant was diluted 100 times and 10 μL was injected. The results are shown in Table 5.

Examples 6-11
Cellulase enzyme preparation (Examples 6 to 8: TP-60, manufactured by Meiji Seika Co., Ltd., protein 650 mg / ml) of amorphous cellulose (crystallinity 0% / average particle size 57 μm) prepared under the conditions of Production Example 6 above. g, Examples 9 to 11: Saccharification reaction was performed using Ultraflo L, manufactured by Novozymes, 50 mg protein / mL. The enzyme reaction was performed for 3 days while shaking and stirring 0.15 g of amorphous cellulose in a 3 mL enzyme reaction solution at 50 ° C. The enzyme reaction solution was Example 6: 100 mM citrate buffer (pH 5.0), 0.83% (w / v) TP-60 (corresponding to protein 0.54%), 30 μg / mL tetracycline, Example 7: 100 mM citrate buffer (pH 5.0), 0.42% (w / v) TP-60 (corresponding to 0.28% protein), 30 μg / mL tetracycline, Example 8: 100 mM citrate buffer (pH 5.0) ), 0.17% (w / v) TP-60 (corresponding to 0.11% protein), 30 μg / mL tetracycline, Example 9: 100 mM citrate buffer (pH 5.0), 10% (v / v) Ultraflo L (corresponding to 0.5% protein), 30 μg / mL tetracycline, Example 10: 100 mM citrate buffer (pH 5.0), 5% (v / v) Ultraflo L (tamper) 0.25% as a quality), 30 μg / mL tetracycline, Example 11: 100 mM citrate buffer (pH 5.0), 2% (v / v) Ultraflo L (corresponding to 0.1% protein), 30 μg / mL tetracycline. After completion of the reaction, the precipitate and the supernatant were separated by centrifugation, and the amount of reducing sugar released in the supernatant was quantified (converted to glucose) by the DNS method. The results are shown in Table 6.

Examples 12 and 13
As Examples 12 and 13, a cellulase enzyme preparation of amorphous cellulose (crystallinity 0% / average particle size 57 μm) prepared under the conditions of Production Example 6 described above (Example 12: Cellcrust 1.5 L, Novozymes) Manufactured, Example 13: Cellulose 1.5L and saccharification reaction with β-glucosidase as Novozyme 188, manufactured by Novozymes) were performed. The enzyme reaction was performed for 3 days while shaking and stirring 0.15 g of amorphous cellulose in a 3 mL enzyme reaction solution at 50 ° C. The enzyme reaction solution was Example 12: 100 mM citrate buffer (pH 5.0), 0.6% (v / v) cell crust (corresponding to 0.1% protein), 30 μg / mL tetracycline, Example 13: 100 mM Citrate buffer (pH 5.0), 0.6% (v / v) cell crust 1.5 L (corresponding to 0.1% as protein), 0.03% (v / v) Novozyme 188 (protein 0.0075) % Equivalent), 30 μg / mL tetracycline. After completion of the reaction, the precipitate and the supernatant were separated by centrifugation, and the amount of reducing sugar released in the supernatant was quantified (converted to glucose) by the DNS method. The results are shown in Table 7.

Examples 14 and 15
The saccharified solution supernatant (reaction 75 hours) obtained in Examples 12 and 13 was analyzed with a DX500 chromatography system manufactured by Dionex as in Example 5 described above. The results are shown in Table 8.

Examples 16-18 and Comparative Examples 5-7
As Examples 16 to 18, the non-crystallized cellulose obtained in the above Production Examples 7 to 9 (Example 16: fine paper / crystallinity 0% / average particle size 42 μm, Example 17: cardboard / crystallinity) 0% / average particle size 48 μm, Example 18: newspaper / crystallinity 0% / average particle size 55 μm), and Comparative Examples 5-7 as powdered cellulose obtained in Comparative Production Examples 6-8 (Comparison) Example 5: fine paper / crystallinity 71% / average particle size 71 μm, comparative example 6: cardboard / crystallinity 71% / average particle size 93 μm, comparative example 7: newspaper / crystallinity 56% / average particle size 61 μm ) Was used to carry out a saccharification reaction using a cellulase enzyme preparation (Cell Crust 1.5 L, manufactured by Novozymes). The enzyme reaction was carried out for 3 days with shaking and stirring at 50 ° C. in 0.1 mL of amorphous cellulose or powdered cellulose in 3 mL of the enzyme reaction solution. The enzyme reaction solutions were Examples 16 to 17 and Comparative Examples 5 to 6: 100 mM citrate buffer (pH 5.0), 1.5% (v / v) cell crust 1.5 L (corresponding to 0.25% protein) 30 μg / mL tetracycline, Example 18 and Comparative Example 7: 100 mM citrate buffer (pH 5.0), 12% (v / v) cell crust 1.5 L (corresponding to 2% protein), 30 μg / mL tetracycline . After completion of the reaction, the precipitate and the supernatant were separated by centrifugation, and the amount of reducing sugar released in the supernatant was quantified (converted to glucose) by the DNS method. The results are shown in Table 9.

Examples 19-21 and Comparative Examples 8-10
As Examples 19 to 21, the non-crystalline cellulose obtained in the above Production Examples 10 to 12 (Example 19: rice husk / 0% crystallinity / average particle size 48 μm, Example 20: pruned branch of street tree / Example 21: tangerine tree branch / crystallinity 0% / average particle size 44 μm), and Comparative Examples 8 to 10 as obtained in the above Comparative Production Examples 9 to 11 Powdered or chip-like cellulose (Comparative Example 8: rice husk / crystallinity 47% / average particle size 85 μm, comparative example 9: pruned branch of street tree / crystallinity 51% / size: 2 mm × 3 mm × 1 mm Comparative Example 10: Pruned branch of mandarin orange tree / crystallinity 46% / size: 2 mm × 3 mm × 1 mm) and saccharification reaction with cellulase enzyme preparation (Cell Crust 1.5 L, manufactured by Novozymes) It was. The enzyme reaction was carried out for 3 days with shaking and stirring at 50 ° C. in 0.1 mL of amorphous cellulose or powdered cellulose in 3 mL of the enzyme reaction solution. The enzyme reaction solution was 100 mM citrate buffer (pH 5.0), 3% (v / v) cell crust 1.5 L (corresponding to 0.5% protein), 30 μg / mL tetracycline. After completion of the reaction, the precipitate and the supernatant were separated by centrifugation, and the amount of reducing sugar released in the supernatant was quantified (converted to glucose) by the DNS method. The results are shown in Table 10.

From Table 1 and Table 2, the manufacturing method of the non-crystalline cellulose of Production Examples 1 to 6 efficiently obtains non-crystalline cellulose having a reduced crystallinity of cellulose as compared with Comparative Production Examples 1 to 5. It can be seen that it is excellent in productivity. Further, comparing Production Example 1 and Comparative Production Example 1, when a rod is used as the medium of the vibration mill, it is possible to efficiently obtain amorphous cellulose having a cellulose crystallinity reduced to 0%. I understand that I can do it. Furthermore, the non-crystalline cellulose obtained in Production Examples 1 to 6 had an appropriate average particle size as compared with Comparative Production Examples 2 to 5.
From the results of Table 3 and Table 4, the production methods of the sugars of the present invention shown in Examples 1 to 4 have higher productivity than Comparative Examples 1 to 4, and from the results of Table 5, the production sugars are large. It turns out that a part is glucose and is useful as a manufacturing method of the raw material for production of ethanol and lactic acid by fermentation. Furthermore, from the results in Table 6, it is possible to produce sugar efficiently regardless of the type of enzyme preparation. From the results in Tables 7 and 8, it is more efficient by adding β-glucosidase to the cellulase preparation. It turns out that sugar production is possible. Furthermore, from the results of Tables 9 and 10, the sugar production method of the present invention is not limited to pulp, but also high-quality paper, paper such as cardboard and newspaper, plant shells such as rice husks, various woods, and various celluloses. It turns out that it is effective with respect to a containing raw material.

  The sugar production method of the present invention is excellent in productivity and can efficiently obtain sugar. The obtained sugar is useful for production of fermentation such as ethanol and lactic acid.

Claims (7)

A method for saccharifying non-crystalline cellulose prepared from a cellulose-containing raw material having a cellulose I-type crystallinity of more than 33% represented by the following calculation formula (1), wherein water is removed from the raw material The cellulose content is 20% by mass or more , the moisture content of the cellulose-containing raw material is 20% by mass or less, and the cellulose-containing raw material is a raw material pretreated with an extruder , and the cellulose-containing raw material is After processing with a vibration mill filled with a rod to prepare an amorphous cellulose having a cellulose I crystallinity reduced to 33% or less, cellulase and / or hemicellulase is allowed to act on the amorphous cellulose for saccharification. , Production method of sugar.
Cellulose type I crystallinity (%) = [(I _22.6 -I _18.5 ) / I _22.6 ] × 100 (1)
[I _22.6 is the diffraction intensity of the lattice plane (002 plane) (diffraction angle 2θ = 22.6 °) in X-ray diffraction, and I _18.5 is the diffraction intensity of the amorphous portion (diffraction angle 2θ = 18.5 °). Show   The method for producing sugar according to claim 1, wherein the treatment time of the vibration mill filled with the rod is 0.01 to 50 hr. The manufacturing method of the saccharide | sugar of Claim 1 or 2 whose bulk density of a cellulose containing raw material is 100-500 kg / m < ³ >.   The sugar manufacturing method according to any one of claims 1 to 3, wherein the cellulose-containing raw material has an average particle diameter of 0.01 to 1 mm. The manufacturing method of the saccharide | sugar in any one of Claims 1-4 whose extruder is a twin-screw extruder. The method for producing sugar according to any one of claims 1 to 5 , wherein the cellulose-containing raw material is at least one selected from the group consisting of pulps, papers, plant shells, and woods. The method for producing a saccharide according to any one of claims 1 to 6, wherein the average particle size of non-crystalline cellulose having a cellulose I type crystallinity reduced to 33% or less is 25 to 150 µm.