JP2005068140A - Method for producing cellooligosaccharide - Google Patents

Abstract

【Task】
Cellobiose, which is expected to be used in a wide range of fields, especially cellobiose, especially in the process of producing cellobiose, it is easy and low-cost by using pulp with a specific crystallinity and water retention that is highly cellulase-reactive. Can be mass-produced on an industrial scale.
[Solution]
Pulp having a cellulase reaction substrate having a value calculated by the formula for calculating the crystallinity of cellulose I by X-ray diffraction method (formula-1) of 10% to 80% and a water retention of 200% to 1000% Is used. The pulp is obtained by performing one or more treatments of fibrillation treatment, mechanochemical treatment, and chemical treatment.
χc = ((I002C − Ia) / I002C) × 100… Formula −1
χc: Cellulose type I crystallinity (%)
I002C: Diffraction intensity of 002 plane (2θ = 22.6 °)
Ia: Diffraction intensity of amorphous part (2θ = 18.5 °)

Description

  The present invention relates to an efficient method for producing cellooligosaccharides, particularly cellobiose.

  Cellooligosaccharide, especially cellobiose, is a disaccharide with β-1,4-linked glucose, water retention, oil absorption, shape retention, heat stability, emulsion stability, low sweetness, low calorie, indigestion Food, food additives, tablet excipients, dispersants, shape retention agents, water retention agents, moisturizing agents, fillers, structures Expected to be used in a wide range of fields such as foods, pharmaceuticals, cosmetics, toiletries, feeds, liquid crystals, electronic materials, rubber / plastics, etc. Yes.

In general, as a method of obtaining cellobiose, (1) a method using an enzymatic synthesis reaction using sucrose as a raw material and (2) a method using an enzymatic decomposition reaction using cellulose as a raw material are known.
(1) As an enzymatic synthesis reaction, there is a method of obtaining cellobiose by sequentially reacting sucrose phosphorylase, glucose isomerase and cellobiose phosphorylase with sucrose as a starting material in the presence of phosphoric acid (see Patent Document 1). Are known. However, in this method, three types of expensive enzymes must be used to obtain cellobiose, and each of the three stages of reactions must be performed. Considering industrial production, the cost of the enzymes is high, and the productivity is low. there were.

On the other hand, (2) as a decomposition reaction with an enzyme, a method of decomposing a cellulose material with cellulase is known. The key to this enzymatic degradation reaction is (A) how to prevent the generated cellobiose from degrading to glucose, and (B) how to increase the reactivity of cellulase to cellulose.
(A) Regarding the point of preventing decomposition into glucose, a method of preventing the decomposition of cellobiose by selectively removing β-glucosidase that converts cellobiose into glucose from cellulase (see Patent Documents 2 and 3) In addition, a method is disclosed in which cellobiose generated by using an ultrafiltration apparatus as a reaction apparatus is extracted from the reaction system to suppress decomposition into glucose (see Patent Documents 4 and 5).
On the other hand, regarding (B) the point of increasing the reactivity of cellulase to cellulose, the use of soft cell cellulose as a raw material (see Patent Document 4), slush pulp that has not undergone a drying step or slush pulp having a specific physical property value It is disclosed that the raw material is used (see Patent Documents 5 and 6).
However, when considering the industrial production of cellobiose, since soft cell cellulose is rare, it is not suitable as a raw material.

  Patent Document 6 describes that the reactivity can be enhanced by defining the water retention and freeness of slush pulp. However, in order to industrially produce cellobiose, even the slush pulp described in Patent Document 6 has a problem of low cellulase reactivity and high cost. In addition, unlike dry pulp, slush pulp contains a large amount of water, so that there are problems that the transportation cost of raw materials increases and that slush pulp rots rarely during transportation or storage. Although this point can be solved by on-site cellobiose production at a pulp production plant, there is a problem that the production location is limited.

  For this reason, when using slush pulp, the reactivity of cellulase is further improved, and the reactivity of dry pulp, which is easy to handle, is increased to that of slush pulp or more, and cellooligosaccharides, particularly high efficiency. If cellobiose could be manufactured, it was thought that it would bring a big leap in the industrial production of cellobiose.

Japanese Patent No. 2815023 Japanese Patent Publication No. 6-83674 Japanese Patent Laid-Open No. 5-115293 JP-A-2-295492 Japanese Patent Publication No.8-2312 JP-A-9-107987

  The present invention was devised to solve the above-described problems of the prior art, and is an industrially advantageous production method capable of producing cellooligosaccharides, particularly cellobiose, in particular, in a simple operation and in large quantities. It was made to provide.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention significantly promote the enzymatic degradation reaction of cellulose by allowing cellulase to act on pulp having a specific range of crystallinity and water retention. In order to obtain the above-mentioned pulp, the degree of crystallinity of cellulose is reduced and the surface area of cellulose is increased by performing any one or more of fibrillation treatment, mechanochemical treatment, alkali treatment as a pretreatment. Based on this finding, the present invention has been made.

Regarding the details of the above reaction, since the crystal structure and fiber form of cellulose are changed and the accessibility of cellulose is improved by performing the above-described treatment on the pulp in advance, the enzymatic degradation reaction of cellulose by cellulase is dramatically improved. I think it was promoted.
That is, the present invention
(1) Pulp having a value calculated by the formula for calculating the crystallinity of cellulose type I by X-ray diffraction (Formula-1) is 10% to 80% and the water retention is 200% to 1000%. A method for producing a cellooligosaccharide characterized by the above.
χc = ((I002C − Ia) / I002C) × 100… Formula −1
χc: Cellulose type I crystallinity (%)
I002C: Diffraction intensity of 002 plane (2θ = 22.6 °)
Ia: Diffraction intensity of amorphous part (2θ = 18.5 °) (2) The pulp is subjected to one or more of fibrillation treatment, mechanochemical treatment, and chemical treatment. A method for producing cellooligosaccharides.
It is about.

  By using the method of the present invention, a pulp having a specific crystallinity and water retention is used as a substrate for cellulase in the step of producing cellooligosaccharides, particularly cellobiose, among them, by enzymatic degradation of the pulp with cellulase. In both dry pulp and cellulase, the enzymatic decomposition reaction is remarkably accelerated, and cellooligosaccharides, particularly cellobiose, can be obtained in high yield.

In the present invention, the pulp that is the substrate for cellulase has a value (hereinafter, simply referred to as crystallinity) calculated by a calculation formula for cellulose I crystallinity by X-ray diffraction method of 10% to 80%, preferably 15%. The water retention is 200% to 1000%, preferably 250% to 900%.
When the water retention is less than 200%, the swelling of the pulp and the fluffing of the fiber surface are small, that is, the surface area of the cellulose is small, which leads to a decrease in the accessibility of the cellulose. When the water retention exceeds 1000%, the cellulase reaction efficiency is compared with the pulp pretreatment cost, and the effect is remarkably reduced.

  The water retention referred to here is defined by J.TAPPI NO 26-78, and is the percentage of water held by the pulp relative to the absolutely dry pulp after a certain amount of pulp suspension is centrifuged (3000G). It is shown by. The higher the water retention, the lower the interfiber binding force and the more water contained in the fiber, that is, the cellulose surface area tends to increase.

  When the crystallinity of the pulp exceeds 80%, the structural change of the cellulose in the pulp before and after the pretreatment is small, so that accessibility is not improved, which is not preferable. Further, when the crystallinity is less than 10%, the cellulase reaction efficiency is compared with the pulp processing cost, and the effect is remarkably reduced.

  The cellulose type I referred to here is a crystalline form of natural cellulose, and the degree of crystallinity is the ratio of the amount of crystal region of cellulose to the total amount, and was measured by the X-ray diffraction method in the present invention. The degree of crystallinity is also related to the physical and chemical properties of cellulose, and the larger the value, the higher the crystallinity of cellulose and the less non-crystalline parts, so the hardness, density, etc. increase, but elongation, flexibility, Accessibility and chemical reactivity to water and solvents are reduced.

  Further, mercerized pulp is commercially available as a pulp that has been subjected to chemical treatment in advance. However, if the product satisfies the above physical properties, it can be used as it is as a raw material for producing cellooligosaccharides.

  As a raw material pulp used for obtaining the above-mentioned pulp, either a pulp that has not undergone a drying process (slush pulp) or a pulp that has undergone a drying process (dry pulp) can be used in the pulp manufacturing process.

  Slush pulp includes bleached or unbleached kraft slash wood pulp, bleached or unbleached sulfite slush wood pulp, bleached or unbleached dissolved slush wood pulp, bleached or unbleached kraft slash non-wood pulp, bleached or unbleached sulfite slush Non-wood pulp, bleached or unbleached dissolved slush non-wood pulp, coarse or refined slush linter pulp can be used. It should be noted that the removal of lignin in the pulp increases the pore volume, and the removal of hemicellulose and the like increases the pure cellulose content. Wood pulp, bleach-dissolved slush non-wood pulp, and refined slush linter pulp are preferred. Further, the moisture contained in the slush pulp depends on the pulp production conditions, that is, the conditions of the dehydration step, but is desirably 50 to 99%, more preferably 60 to 98%. When the water content is less than 50%, the pore volume in the pulp may be crushed and keratinized due to the share generated by the dehydration process, which may cause a decrease in the accessibility of cellulose. If it exceeds 1, the pulp processing efficiency decreases, and the pretreatment cost increases, which is not preferable.

  Dry pulp includes bleached or unbleached kraft wood pulp, bleached or unbleached sulfite wood pulp, bleached or unbleached dissolved wood pulp, bleached or unbleached kraft non-wood pulp, bleached or unbleached sulfite non-wood pulp, bleached Or unbleached non-wood pulp, crude or refined linter pulp, waste paper pulp, deinked pulp, cellulose produced by microorganisms such as squirt cellulose and acetic acid bacteria, etc., or the above cellulosic materials with caustic soda solution, copper ammonia solution, morpholine Regenerated cellulose dissolved in some solvent such as a derivative and spun again, mercerized pulp treated with a caustic soda solution, or the like can be used. In addition, since the cellulose pure content becomes high by removing the lignin content in the pulp and removing hemicellulose and the like, among the illustrated cellulose materials, bleach-dissolved wood pulp, bleach-dissolved non-wood pulp Finely selected linter pulp, regenerated cellulose, and mercerized pulp are preferred.

  The moisture contained in the dry pulp depends on the pulp production conditions, that is, the conditions of the drying process, but is preferably 5 to 30%. If the moisture content is less than 5%, the keratinization of the pulp, that is, hydrogen bonding between molecules and molecules may proceed, and the accessibility of cellulose may be reduced.On the other hand, if the moisture content exceeds 30%, It is not preferable because the transportation cost is high and it easily perishes.

In the present invention, in order to obtain a pulp highly reactive with cellulase, the crystallinity and water retention of cellulose type I are regulated within a specific range. In order to reduce the crystallinity, mechanochemical treatment and / or chemical treatment may be mainly performed, and in order to keep the water retention within the range of the present invention, fibrillation treatment may be mainly performed. The process will be described in detail below.
In addition, it is more efficient to reduce the crystallinity of the pulp, increase the water retention, and increase the reactivity of the cellulase by combining a plurality of treatments rather than one treatment.

  The fibrillation treatment uses a machine that grinds and shears pulp for the purpose of increasing the surface area of cellulose and facilitating the adsorption of cellulase to the cellulose surface. (1) Fine structure of cellulose fibers (2) make the cellulose fiber surface fluffy, or branch the fibers to increase the fiber surface area (external fibrillation), (3) fine fibers And consequently increase the fiber surface area.

  For the fibrillation treatment, a known model may be used as long as it can be fibrillated by grinding and shearing cellulose fibers under wet conditions. For example, beaters (Niagara Beater Kumagaya Riki Kogyo), PFI mills (PFI Mill Kumagaya Riki Kogyo), refiners (Disk Refiner Tozai Seiki) or high-pressure homogenizers (Niro)・ Soavi), ultra-high pressure homogenizer (Shinmaru Enterprises), Nanomizer (made by Special Machinery Industry), stone mill type grinder (both Supermass colloider and serendipeater are made by Masuyuki Sangyo), etc.

  The pulp concentration during the fibrillation treatment is 1.0 to 20%, more preferably 2.0 to 15%. If the pulp concentration is less than 1.0%, not only will the throughput increase significantly, but water will act as a plasticizer and the degree of fibrillation will decrease, and if the pulp concentration exceeds 20%, the pulp concentration Is too high, the fluidity is lowered, and it is difficult to perform the fibrillation treatment. The fibrillation time is preferably 3 minutes to 5 hours, more preferably 5 minutes to 3 hours, although the optimum value varies depending on the pulp concentration and the model used. When the fibrillation treatment time is less than 3 minutes, there is little change in the shape of the pulp, and there is little increase in the water retention, and when the treatment time exceeds 5 hours, the reaction efficiency of the cellulase and the pulp treatment cost are reduced. In comparison, the effect is remarkably reduced, which is not preferable. In addition, in the model which performs a continuous process like a stone mill type grinder, it can process repeatedly or repeatedly in multiple times so that it may become said process time.

  The mechanochemical treatment of the present invention is performed for the purpose of reducing the crystallinity of the cellulose fiber, and by applying strong energy to the cellulose fiber, the crystal structure of the cellulose itself is changed, that is, the crystallinity is lowered. It is to let you.

  As long as the mechanochemical treatment is a treatment capable of reducing the crystallinity of cellulose, a known method may be used, but a treatment using a medium mill is preferable. A known model may be used as the media mill. A mill (manufactured by Kotobuki Giken Kogyo), an aquamizer (manufactured by Hosokawa Micron), an ultra visco mill (manufactured by IMEX) and the like can be mentioned.

  The mechanochemical treatment is performed in a state where 100 parts of pulp is dispersed in 0 to 10,000 parts of water, more preferably 0 to 5000 parts of water. When the amount of water exceeds 10,000 parts, not only the treatment amount is remarkably increased, but also water acts as a plasticizer and the mechanochemical treatment is lowered, which is not preferable.

  As media for the media mill used for mechanochemical treatment, commercially available alumina, ceramic, titanium, zirconia, glass, etc. with φ1 mm to φ50 mm can be used, and the filling rate is 50% to 95% Is preferred. When the filling rate is less than 50%, the frequency of contact with cellulose is reduced and a mechanochemical reaction is less likely to occur, and when the filling rate exceeds 95%, the movement of the medium is hindered.

  The treatment time is preferably 5 minutes to 5 hours, more preferably 10 minutes to 3 hours. In the present invention, the time for mechanochemical treatment is important. When the treatment time is less than 5 minutes, there is almost no change in the crystal structure of the cellulose, and when it exceeds 5 hours, the cellulose I-type crystallinity decreases and the crystal structure becomes amorphous, but the cellulose fiber Is agglomerated and hardened, which is not preferable because the water retention is significantly reduced. In general, there is a correlation between the water retention and the surface area of cellulose, and when the water retention decreases, the surface area decreases. Therefore, even if the crystallinity decreases, the reactivity of cellulase decreases.

  The chemical treatment in the present invention aims at lowering the crystallinity of cellulose, and alkali treatment represented by mercerization treatment is preferable.

As the alkali treatment, it is preferable to adjust 100 parts of pulp so as to be 500 to 10,000 parts of an aqueous alkaline dispersion having a pulp concentration of 1.0 to 20% and a caustic soda concentration of 3 to 30%, and then treat for 5 minutes to 5 hours. More preferably, 100 parts of pulp is adjusted to become an alkaline water dispersion having a pulp concentration of 2.0 to 15% and a caustic soda concentration of 5 to 20%, and is treated for 10 minutes to 3 hours.
If the pulp concentration is less than 1.0%, not only will the throughput increase significantly, but the amount of caustic soda will increase to maintain the caustic soda concentration, i.e. the chemical cost will increase significantly, and the pulp concentration will increase by 20%. When exceeding, it is not preferable because the pulp concentration is too high, the fluidity is lowered, and the alkalinization treatment proceeds non-uniformly.

  When the caustic soda concentration is less than 3%, no change in the crystal structure of the cellulose is observed, and when the caustic soda concentration exceeds 30%, the cellulose I-type crystals have already completely disappeared and become amorphous. High concentration treatment is not preferable because it leads to a significant increase in chemical cost.

  When the alkali treatment time is shorter than 5 minutes, the progress of the mercerization reaction is small and the degree of decrease in the cellulose I-type crystallinity is small, and even if the alkali treatment time exceeds 5 hours, the further crystal structure of cellulose Since the change of is not seen, it is not preferable.

  The cellulase that can be used in the present invention is not particularly limited, and is derived from various sources such as Trichoderma, Aspergillus, Irpex, Aeromonas, Clostridium, Bacillus, Pseudomonas, Penicillium, Humicola, and / or the like. Those produced by gene recombination can be used alone or in admixture of two or more. In addition, as the enzyme source, a commercially available cellulase preparation, a culture of the above-mentioned bacteria, or a filtrate thereof can also be used directly.

  The cellooligosaccharides in the present invention, especially the method for producing cellobiose among them, will be described in detail.

  Pulp having a specific crystallinity and water retention is used as a substrate, usually suspended in an aqueous reaction solution within a range of 0.5 to 20% by weight, and cellulase, an enzyme, is added to 0.5 to 30% by weight per substrate for enzymatic degradation. Perform the reaction. At this time, the pH and temperature of the reaction solution are not limited as long as the enzyme is not inactivated, but the pH is usually 2 to 10 and the temperature is 20 to 70 ° C. In addition, when the operation is performed for a long time, as a countermeasure against the propagation of various bacteria or the decay of the reaction solution, a known preservative that does not affect the enzymatic decomposition reaction, for example, benzoic acid, sodium benzoate, sucrose Esters, alcohols and the like can also be added.

  Moreover, although reaction time changes with enzyme decomposition reaction conditions, about 3 to 48 hours are usually enough. In addition, as a reaction apparatus, in order to prevent cellulase reactivity inhibition by produced cellobiose and cello-oligosaccharide by β-glucosidase, in particular, to prevent cellobiose from being decomposed, only produced cellooligosaccharide containing cellobiose and glucose are continuously used. Any apparatus that can be removed outside the system can be used without any problem. If it dares to illustrate the apparatus, it is preferable to use an ultrafiltration apparatus. The ultrafiltration membrane used in the ultrafiltration device is not particularly limited as long as the cellulase used does not leak, but preferably a polyamide-based, cellulose acetate-based, It is preferable to use an ultrafiltration membrane represented by an inorganic membrane such as an organic membrane such as an ethersulfone type, a ceramic filter, and a sintered metal filter alone or in combination. After completion of the reaction, the cello-oligosaccharide, especially the enzyme degradation solution containing cellobiose is adjusted to the purity using a known purification means as necessary, and in some cases, the powder is dried by passing through a known dry grinding step. It can also be converted.

  Hereinafter, although an example explains the present invention in full detail, the present invention is not limited by this. “Parts” and “%” indicating the blending amounts all indicate “parts by weight of solids” and “% by weight of solids”.

[Raw material 1]
A commercially available dissolving pulp (NDPT, manufactured by Nippon Paper Chemicals) was used as a slush pulp collected in a wet state before the drying step. The water retention of the raw material was 210%, and the value calculated by the calculation formula of cellulose I type crystallinity by X-ray diffraction method was 83.3%.

[Raw material 2]
The raw material 1 was subjected to fibrillation treatment. Mass colloider (manufactured by Masuko Sangyo Co., Ltd.) with 10 L of water suspension adjusted to a pulp concentration of 3% and a GC-10-46 grinder (mass collider grindstone) set to a clearance of 20 μm Then, the treatment was repeated under the condition of 1800 rpm and the treatment time was 1 hour in total. The water retention of the raw material was 533%, and the value calculated by the calculation formula of cellulose I-type crystallinity by X-ray diffraction method was 68.5%.

[Raw material 3]
The raw material 1 was subjected to mechanochemical treatment. Place a ceramic ball of φ30mm in a 1L pot so that the filling rate is 65%, and then add 333ml of water suspension adjusted to a pulp concentration of 6% of raw material 1, and a vibrating ball mill (manufactured by Chuo Kako) For 30 minutes. The water retention of the raw material was 383%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 74.3%.

[Raw material 4]
The raw material 1 was subjected to alkali treatment. Immerse raw material 1 in a 15% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5%, stir at room temperature for 30 minutes (200 rpm), remove excess alkali with a centrifuge, and then adjust the pH to medium. Washed with ion-exchanged water until it became neutral. The water retention of the raw material was 267%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 40.4%.

[Raw material 5]
The raw material 1 was subjected to alkali treatment and fibrillation treatment. Immerse raw material 1 in a 15% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5%, stir at room temperature for 30 minutes (200 rpm), remove excess alkali with a centrifuge, and then adjust the pH to medium. Washed with ion-exchanged water until it became neutral. Thereafter, 300 ml of the water suspension readjusted to a pulp concentration of 10% was treated with a PFI mill (manufactured by Kumagaya Riken Kogyo) until it reached 10,000 revolutions. The water retention of the raw material was 648%, and the value calculated by the calculation formula of cellulose I type crystallinity by X-ray diffraction method was 39.9%.

[Raw material 6]
The raw material 1 was subjected to fibrillation treatment. 300 ml of an aqueous suspension prepared by adjusting the raw material 1 so as to have a pulp concentration of 10% was processed with a PFI mill (manufactured by Kumagaya Riki Kogyo) until 10,000 rotations. The water retention of the raw material was 235%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 82.3%.

[Raw material 7]
The raw material 1 was subjected to alkali treatment. Immerse raw material 1 in a 2% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5%, stir at room temperature for 10 minutes (200 rpm), remove excess alkali with a centrifuge, and then adjust the pH to medium. Washed with ion-exchanged water until it became sex. The raw material had a water retention of 233%, and a value calculated by a calculation formula for cellulose I-type crystallinity by X-ray diffraction was 81.7%.

[Raw material 8]
The raw material 1 was subjected to mechanochemical treatment. Place a ceramic ball of φ30mm into a 1L pot so that the filling rate is 65%, and then add 333ml of water suspension adjusted to a pulp concentration of 15%, and a vibrating ball mill (manufactured by Chuo Kako) For 24 hours. The water retention of the raw material was 112%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 52.2%.

[Raw material 9]
Commercially available dissolving pulp (NDPT dry product, Nippon Paper Chemicals, moisture 7.5%) was used. The water retention of the raw material was 175%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 86.6%.

[Raw material 10]
The raw material 9 was subjected to fibrillation treatment. 10L of water suspension with raw material 9 adjusted to 3% pulp concentration, with a mass colloider (made by Masuko Sangyo Co., Ltd.) with a GC-10-46 grinder set to a clearance of 20μm, rotation speed 1800rpm The treatment was repeated so that the treatment time was 1 hour in total. The water retention of the raw material was 463%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 69.5%.

[Raw material 11]
The raw material 9 was subjected to mechanochemical treatment. Place a ceramic ball of φ30mm in a 1L pot so that the filling rate is 65%, and then add 333ml of water suspension adjusted to a pulp concentration of 6%, and a vibration ball mill (manufactured by Chuo Kako) For 30 minutes. The water retention of the raw material was 327%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 74.3%.

[Raw material 12]
The raw material 9 was subjected to alkali treatment. The material 9 was immersed in a 15% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5%, stirred at room temperature for 30 minutes (200 rpm), and after removing excess alkali with a centrifuge, the pH was medium. Washed with ion-exchanged water until it became neutral. The water retention of the raw material was 257%, and the value calculated by the calculation formula of cellulose I-type crystallinity by X-ray diffraction method was 41.7%.

[Raw material 13]
The raw material 9 was subjected to alkali treatment and mechanochemical treatment. The material 9 was immersed in a 15% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5%, stirred at room temperature for 30 minutes (200 rpm), and after removing excess alkali with a centrifuge, the pH was medium. Washed with ion-exchanged water until it became neutral. Thereafter, 300 ml of the water suspension readjusted to a pulp concentration of 10% was treated with a PFI mill (manufactured by Kumagaya Riken Kogyo) until it reached 10,000 revolutions. The water retention of the raw material was 628%, and the value calculated by the calculation formula of cellulose I-type crystallinity by X-ray diffraction method was 41.1%.

[Raw material 14]
The raw material 9 was subjected to fibrillation treatment. 300 ml of an aqueous suspension prepared by adjusting the raw material 9 so as to have a pulp concentration of 10% was processed with a PFI mill (manufactured by Kumagaya Riki Kogyo) until 10,000 rotations. The water retention of the raw material was 205%, and the value calculated by the calculation formula of cellulose I type crystallinity by X-ray diffraction method was 85.3%.

[Raw material 15]
The raw material 9 was subjected to alkali treatment. The material 9 is immersed in a 2% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5%, stirred at room temperature for 10 minutes (200 rpm), and after removing excess alkali with a centrifuge, the pH is medium. Washed with ion-exchanged water until it became neutral. The water retention of the raw material was 198%, and the value calculated by the calculation formula of cellulose I type crystallinity by X-ray diffraction method was 85.7%.

[Raw material 16]
The raw material 9 was subjected to mechanochemical treatment. A 1 L pot was filled with φ30 mm ceramic balls so that the filling rate would be 85%, and 20 parts of the raw material 9 was further charged, followed by treatment with a vibrating ball mill (manufactured by Chuo Kakoki) for 10 hours. The water retention of the raw material was 83%, and the value calculated by the calculation formula for cellulose I-type crystallinity by X-ray diffraction method was 34.1%.

[Example 1]
An appropriate amount of acetate buffer (pH 5.5) was added to 10 g (solid content conversion) of raw material 2 to prepare a 1% suspension. The suspension and 0.5 g of commercially available cellulase (Cell riser, manufactured by Nagase Seikagaku) are placed in an ultrafilter with a stirrer (flat membrane type) set with a polysulfone flat membrane with a molecular weight cut off of 10000, and the stirrer is rotated. The saccharification reaction was carried out at several 200 rpm and 40 ° C. for 8 hours. During this reaction, the reaction solution (saccharified solution) in the ultrafilter is continuously permeated through the ultrafiltration membrane by pressurizing the ultrafilter to 0.2 to 0.3 MPa. Extracted as. At the same time, an acetic acid buffer solution (pH 5.5) having the same amount as the extracted permeate was continuously replenished to the ultrafilter, and the conditions were set so that the volume in the ultrafilter was almost constant. After completion of the reaction, the amount of cellobiose produced in the extracted permeate was measured, and the weight of unreacted material contained in the suspension remaining in the ultrafilter was measured.

[Examples 2 to 4, Comparative Examples 1 to 4]
Using the raw materials in Table 1, an enzymatic decomposition reaction with cellulase was performed in the same manner as in Example 1. The test results are shown in Table 2.

[Examples 5-8, Comparative Examples 5-8]
Using the raw materials in Table 1, an enzymatic decomposition reaction with cellulase was performed in the same manner as in Example 1. The test results are shown in Table 3.

<Measurement method>
<Water retention level>
The water retention was measured according to J.TAPPI NO26-78.
<Cellulose type I crystallinity>
Cellulose type I crystallinity was determined by measuring the X-ray diffraction of the cellulosic material using an X-ray diffractometer (RAD-2C system, manufactured by Rigaku Denki). Cellulose type I crystallinity was calculated by the method of Segal et al. (L. Segal, JJ Greely et al, Text. Res. J., 29, 786, 1959) and the method of Kamide et al. (K. Kamide et al, Polymer J , 17, 909, 1985), and the diffraction intensity of 2002 = 4 ° to 32 ° of the diffraction diagram obtained by X-ray diffraction measurement is used as the baseline, and the diffraction intensity of the 002 plane (2θ = 22.6 °) and amorphous It was calculated from the diffraction intensity of the part (2θ = 18.5 °) by the following formula. That is, the larger this value, the higher the crystallinity of cellulose and the less the non-crystalline portion.
χc = ((I002C − Ia) / I002C) × 100
χc: Cellulose type I crystallinity (%)
I002C: Diffraction intensity of 002 plane (2θ = 22.6 °)
Ia: Diffraction intensity of amorphous part (2θ = 18.5 °)

<Method for quantifying cellobiose>
Cellobiose was quantitatively calculated from the peak area using high performance liquid chromatography for the permeate extracted from the ultrafilter. The column was measured with a differential refractometer (Showa Denko) under the conditions of Senshu Chemical's NH2 (φ4.6mm × 250mm), eluent acetonitrile / water (65/35), and flow rate 1ml / min. It was.

<Measurement method of unreacted rate>
After completion of the enzymatic decomposition reaction, unreacted substances were collected from the suspension remaining in the ultrafilter, and the weight was measured to calculate the unreacted ratio (residual ratio).

  From the results of Table 2 and Table 3, when the bottoming raw material is either dry or slush pulp, the pulp is pretreated in advance, the crystallinity is lowered to a certain value, and the water retention is increased. Both cellobiose production and reaction rate were improved. Comparing dry pulp and slush pulp, slush pulp had higher cellobiose production and reaction rate. This is presumably because dry pulp undergoes a drying process, so that the degree of bonding between celluloses is larger than that of slush pulp, and the effect of pretreatment is less likely to occur as in slush pulp.

Claims (2)

It is characterized by using a pulp having a value calculated by the calculation formula (Formula-1) of cellulose type I crystallinity by X-ray diffraction method of 10% to 80% and water retention of 200% to 1000%. A method for producing cellooligosaccharides.
χc = ((I002C − Ia) / I002C) × 100… Formula −1
χc: Cellulose type I crystallinity (%)
I002C: Diffraction intensity of 002 plane (2θ = 22.6 °)
Ia: Diffraction intensity of amorphous part (2θ = 18.5 °) The method for producing cellooligosaccharide according to claim 1, wherein the pulp is subjected to any one or more of fibrillation treatment, mechanochemical treatment, and chemical treatment.