JPH082312B2 - Method for producing cellooligosaccharide - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing cellooligosaccharides such as cellobiose, cellotriose and cellotetraose, which are used as sweeteners for dietary diets or health drinks.

[0002]

2. Description of the Related Art Generally, the above-mentioned cellooligosaccharide is obtained by hydrolyzing cellulose by a hydrolysis reaction by a cellulase which is one of enzymes. By the way, conventionally, the following proposals have been made in order to create an environment in which cellulase easily works in the above-mentioned hydrolysis reaction.

(1) By swelling the cellulose in a mixed solution of concentrated hydrochloric acid and concentrated sulfuric acid, the cellulose is non-crystallized and processed into cellulose that is easily hydrolyzed by cellulase (see Japanese Patent Publication No. 57-53801). . (2) A flexible cell, which is a highly reactive cellulose-containing fiber, is selected from the cellulose and hydrolyzed by cellulase (see JP-A-2-295492).

[0004]

However, in the above-mentioned ones, in the former case, the cellulose must be processed, and in the latter case, the soft cell which is a cellulose-containing fiber highly reactive to cellulase. Therefore, it is difficult to mass-produce cellooligosaccharides at low cost, because it has to be selectively separated.

[0005] The present invention aims to solve the above problems and provides a method for producing cellooligosaccharides capable of inexpensively mass-producing cellooligosaccharides.

[0006]

By the way, as a result of various studies on the method of overcoming the above-mentioned problems, the present inventors have found that (a) 130 as a raw material containing natural lignocellulose.
Cellulose fibers obtained by subjecting pulping reaction by cooking at a high temperature ranging from ℃ to 180 ° C. are in a state in which the amorphous state of the cellulose fibers is maintained in a wet state which has not been dried after cooking. (B) In the process of the non-crystalline cellulose losing water by drying, hydrogen bonds between cellulose molecular chains become strong and crystallinity increases, and (c) hydrogen bonds between cellulose molecular chains are once formed. Since it is not cut even if water is added again, it is necessary to perform the swelling treatment or the selective separation of highly reactive cellulose-containing parenchymal fibers as described above. I focused on that point. Thus, an attempt was made to cause a raw material containing natural lignocellulose to undergo a pulping reaction by digestion, and to partially decompose wet pulp obtained after the digestion without being dried by the action of cellulase. As a result, surprisingly, wet pulp in a wet state that did not undergo drying after digestion was found to have a significantly higher hydrolysis reaction for cellulase than cellulose that has undergone drying, and the present invention has been completed. there were.

The method for producing cellooligosaccharide according to the present invention comprises:
A raw material containing natural lignocellulose, for example, a wet pulp obtained by cooking by sulfite cooking or kraft cooking and not undergoing drying after the cooking is partially decomposed by the action of cellulase to form at least cellobiose among cellooligosaccharides. It is to collect.

The cellulase used in the present invention is Tr
Other than the genus ichoderma viride, Asper
gillus, Irpex, Aeromonas
Genus, Clostridium, Bacillus,
Those of various origins such as Pseudomonas genus and Penicillium genus can be used.

[0009]

EFFECTS OF THE INVENTION According to the method for producing cellooligosaccharides according to the present invention, a raw material containing natural lignocellulose, which is in a state in which decrystallization of cellulose fibers has progressed, is obtained by cooking and without drying after the cooking. Due to the use of wet pulp that is used, swelling treatment with an acid or the like to achieve non-crystallization of cellulose, physical treatment such as grinding, etc.
Furthermore, cellulase is highly efficiently partially decomposed without performing pretreatment such as selective separation of soft cells, which are highly reactive cellulose-containing fibers. Therefore, cellooligosaccharides are produced in high yield, and cellooligosaccharides can be inexpensively mass-produced.

A low molecular weight compound produced during partial decomposition,
If cellulase is allowed to act while continuously removing cellooligosaccharides containing cellobiose and glucose from the reaction system by an ultrafiltration membrane: ・ Inhibition of cellulase action by cellobiose ・ Degradation of cellooligosaccharide by β-glucosidase Since the two factors can be suppressed, the cellooligosaccharide can be obtained in a higher yield.

[0011]

EXAMPLES Next, examples of the method for producing cellooligosaccharides according to the present invention will be described in detail in correspondence with comparative examples. -Example 1-The pH of the raw material containing natural lignocellulosic sulphite pulp which has not been dried after cooking and which has not been dried is adjusted to pH 5.
A 2 wt% slurry solution was prepared by adding 5 of the acetate buffer solution. Next, while stirring the prepared slurry liquid, the slurry liquid was heated to 45 ° C.
While maintaining the temperature of 5 ° C., 0.1 to the slurry liquid
Cellulase from the genus Trichoderma viride was added at wt% and the cellulose was hydrolyzed by stirring for 24 hours. Thus, when the hydrolysis reaction solution was filtered, a sugar solution containing 21% cellobiose was obtained in a yield of 31% based on the original unbleached sulfite pulp.

COMPARATIVE EXAMPLE In the same manner as in Example 1, acetic acid having a pH of 5.5 was obtained after freeze-drying the wet unfiltered sulfite pulp that had not been dried after cooking in Example 1 for 24 hours. A 2 wt% slurry solution was prepared by adding a buffer solution. Further, while stirring the prepared slurry liquid, the slurry liquid was heated to 45 ° C., and while maintaining the temperature of 45 ° C., 0.1 wt% of Tr was added to the slurry liquid.
Cellulase of the genus ichoderma viride was added and the cellulose was hydrolyzed by stirring for 24 hours. Thus, when the hydrolysis reaction solution was filtered, a sugar solution containing 11% cellobiose was obtained in a yield of 14% based on the original unbleached sulfite pulp.

Example 2 As in Example 1, 2 wt% was obtained by adding an acetic acid buffer solution having a pH of 5.5 to a non-dried, non-dried raw sulphite pulp after cooking a raw material containing natural lignocellulose.
% Slurry solution was prepared. Next, the prepared slurry liquid was charged into a bioreactor equipped with a polysulfone membrane having a molecular weight cutoff of 10,000, and the slurry liquid was heated to 45 ° C. while being circulated by a pump, and the temperature of this 45 ° C. 0.1 wt% to the slurry liquid while holding
Cellulase of the genus Trichoderma viride was added and reacted for 4 hours to hydrolyze the cellulose. Thus, when the hydrolysis reaction liquid passed through the polysulfane membrane which is the ultrafiltration membrane was concentrated, the sugar liquid containing 71% cellobiose was collected at 87% of the original unsulfated sulfite pulp. Obtained at a rate.

-Comparative Example-Acid having a pH of 5.5 was lyophilized in the same manner as in Example 2 after freeze-drying the wet unfiltered sulfite pulp which had not been dried after cooking in Example 2 for 24 hours. A 2 wt% slurry solution was prepared by adding a buffer solution. In addition, the prepared slurry liquid was charged into a bioreactor equipped with a polysulfone membrane having a molecular weight cutoff of 10,000, and the slurry liquid was heated to 45 ° C. while being circulated by a pump, and the temperature of 45 ° C. Similarly, 0.1 wt% Trichoderm with respect to the slurry liquid is retained.
Cellulase of the genus a viride was added and reacted for 4 hours to hydrolyze the cellulose. In this way, the hydrolysis reaction liquid that passed through this polysulfone membrane was concentrated. As a result, the sugar liquid containing 31% cellobiose was mixed with the original unfiltered sulfite pulp at 3%.
Obtained in a yield of 4%.

Example 3 As in Example 1, 2 wt% was obtained by adding an acetic acid buffer solution having a pH of 5.5 to a sulphite pulp in a wet state which had not been dried after cooking of a raw material containing natural lignocellulose.
A slurry liquid of was prepared. Next, the prepared slurry liquid was charged into a bioreactor equipped with a polysulfone membrane having a molecular weight cutoff of 10,000, and the slurry liquid was heated to 45 ° C. while being circulated by a pump, and the temperature of this 45 ° C. Cellulase derived from the genus Trichoderma viride of 0.1 wt% was added to the slurry liquid while maintaining the above, and the reaction was carried out for 4 hours to hydrolyze the cellulose. Thus, when the hydrolysis reaction liquid passing through this police fan membrane was concentrated, 68
A sugar solution containing 100% cellobiose was obtained in a yield of 84% based on the original sulphite pulp.

Comparative Example In the same manner as in Example 3, an acetic acid buffer having a pH of 5.5 was prepared by freeze-drying the sulphite pulp in a wet state which had not been dried after cooking in Example 3 for 24 hours. A 2 wt% slurry liquid was prepared by adding the liquid. In addition, the prepared slurry liquid was charged into a bioreactor equipped with a polysulfone membrane having a molecular weight cutoff of 10,000, and the slurry liquid was heated to 45 ° C. while being circulated by a pump, and the temperature of 45 ° C. Similarly, 0.1 wt% Trichoderm with respect to the slurry liquid is retained.
Cellulase of the genus a viride was added and reacted for 4 hours to hydrolyze the cellulose. In this way, when the hydrolysis reaction liquid passing through the polysulfone membrane was concentrated, a sugar liquid containing 28% cellobiose was obtained in a yield of 29% based on the original sulfite pulp.

Example 4 As in Example 1, the raw lignocellulosic raw material was poured into the unrefined kraft pulp in a wet state which had not been dried after cooking.
A 2 wt% slurry solution was prepared by adding H5.5 acetate buffer. Next, the prepared slurry liquid was charged into a bioreactor equipped with a polysulfone membrane having a molecular weight cutoff of 10,000, and the slurry liquid was heated to 45 ° C. while being circulated by a pump, and the temperature of this 45 ° C. Of 0.1% by weight relative to the slurry liquid while holding
Cellulase derived from the genus hoderma viride was added and reacted for 4 hours to hydrolyze the cellulose. In this way, when the hydrolysis reaction liquid passing through this police fan membrane was concentrated, a sugar liquid containing 41% cellobiose was obtained in a yield of 44% based on the original unpurified kraft pulp.

-Comparative Example 1-Acid having a pH of 5.5 was lyophilized in the same manner as in Example 4 after lyophilizing the unfiltered kraft pulp in a wet state which had not been dried after cooking in Example 4 for 24 hours. A 2 wt% slurry solution was prepared by adding a buffer solution. Also,
The prepared slurry liquid was charged into a bioreactor equipped with a polysulfone membrane having a molecular weight cutoff of 10,000, and the slurry liquid was heated to 45 ° C. while being circulated by a pump, and the temperature of 45 ° C. was maintained. Meanwhile, 0.1 wt% of Trichoderma was similarly added to the slurry liquid.
Cellulase of the genus viride was added and reacted for 4 hours to hydrolyze the cellulose. In this way, when the hydrolysis reaction liquid passing through the polysulfone membrane was concentrated, a sugar liquid containing 21% cellobiose was obtained in a yield of 27% based on the original unpurified kraft pulp.

-Comparative Example 2-Dried KC Flock W-50 (cellulosic raw material: manufactured by Nippon Paper Industries Co., Ltd.) was prepared in the same manner as in Example 4 to have a pH of 5.5.
A 2 wt% slurry solution was prepared by adding the acetic acid buffer solution of 1. In addition, the prepared slurry liquid was charged into a bioreactor equipped with a polysulfone membrane having a molecular weight cutoff of 10,000, and the slurry liquid was circulated by a pump at 45 ° C.
While maintaining the temperature of 45 ° C., the temperature of the slurry liquid is adjusted to 0.1 wt% of Trich.
Cellulase derived from the genus errama viride was added and reacted for 4 hours to hydrolyze the cellulose. In this way, when the hydrolysis reaction liquid passing through the polysulfone membrane was concentrated, a sugar liquid containing 31% cellobiose was obtained in a yield of 23% with respect to the original KC Flock W-50.

In the present Example, the cellulase originated from Trichoderma viride was used, but Aspergillus genus, Irpex genus,
Aeromonas, Clostridium, B
acillus genus, Pseudomonas genus, Pen
Those of origin such as genus icillum can also be used.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takahiko Tsunomiya 4-6-23 Kashima, Yodogawa-ku, Osaka City, Osaka Prefecture Japan Chemical Machinery Manufacturing Co., Ltd. (72) Inventor Masami Ueda Kashima, Yodogawa-ku, Osaka-shi, Osaka 4-6-23 Japan Chemical Machinery Manufacturing Co., Ltd.

Claims (2)

[Claims] 1. A method of partially decomposing a wet pulp obtained by digesting a raw material containing natural lignocellulose, which is obtained after the digestion without drying after the digestion, to collect at least cellobiose among cellooligosaccharides. A process for producing a characteristic cellooligosaccharide. 2. The partial decomposition is progressed while continuously removing the cellooligosaccharide containing cellobiose and glucose generated during the partial decomposition by the action of the cellulase from the reaction system by an ultrafiltration membrane. The method for producing a cellooligosaccharide according to claim 1.