TWI429748B - Cellulase and fiber oligosaccharide production method - Google Patents

Description

Method for producing cellulase and cellooligosaccharide

The present invention relates to a method for producing an enzyme solution which selectively produces a cellooligosaccharide in a high yield, which has high cellulase activity and high cellulose saccharide selectivity; and provides a fiber using the enzyme solution A method of producing oligosaccharides.

The general term for cellooligosaccharide cellobiose, cellotriose, cellotetraose, celloose, and cellulose, which are 1 to 6 grape melanose units, and various sugars bonded by β-1,4. In recent years, the cellooligosaccharide is similar to other oligosaccharides, and its physiological function is gradually becoming clear, and it is expected to be used as a new material for functional foods (see Non-Patent Document 1). In general, as a method for obtaining cellobiose, (1) a method of synthesizing a reaction using an enzyme using sucrose as a raw material, and (2) a method of using a decomposition reaction of an enzyme using cellulose as a raw material.

As a synthesis reaction using the enzyme of the above (1), there is known a method in which sucrose phosphorylase, glucose isomerase, and cellobiose phosphorylase are sequentially used in the presence of phosphoric acid using sucrose as a raw material. A method of obtaining a cellobiose by a reaction (refer to Patent Document 1). However, in this method, three kinds of expensive enzymes are used in order to obtain cellobiose, and it is necessary to carry out a three-stage reaction separately. If industrial production is considered, there is a problem that the cost of the enzyme is low and the productivity is low.

On the other hand, as a decomposition reaction using the enzyme in the above (2), a method of decomposing a cellulose-based material by cellulase is known. About this The key to the enzyme decomposition reaction is how to improve the cellulose oligosaccharide production of the enzyme solution containing cellulase when producing cellooligosaccharide from cellulose, and how to prevent the formation of cellobiose into glucose. Refer to Non-Patent Document 2).

In view of the above problems, more attempts have been made previously to increase the yield of cellooligosaccharides in the decomposition of cellulase.

As a method of decomposing cellulase by using cellulase to selectively obtain cellooligosaccharide, the following may be mentioned.

Patent Document 2 discloses a method for producing a cellooligosaccharide by using a cellulose raw material containing a large amount of amorphous cellulose, performing a hydrolysis reaction using a cellulase in the presence of lignin, and optionally in a reaction liquid. At least the cellobiose in the cellooligosaccharide produced by the hydrolysis reaction is collected; and in Patent Document 3, a method for producing cellooligosaccharide is disclosed: the raw material containing natural lignocellulose is distilled, and is not dried after being evaporated. The resulting wet slurry is partially hydrolyzed by cellulase to collect at least the cellobiose in the cellooligosaccharide.

These production methods are such that β-glucosidase, which is a cellosome degrading enzyme contained in the cellulase, is adsorbed to lignin to inhibit the action of β-glucosidase, thereby inhibiting the decomposition of cellooligosaccharide into glucose. Thereby increasing the reaction selectivity of cellooligosaccharides. However, in these production methods, the obtained saccharified solution contains a large amount of lignin, and as a result, the yield of cellooligosaccharide becomes low. Further, there is a problem in that in order to obtain a high-purity cellooligosaccharide, a delignification treatment from a saccharification solution is required, which complicates the purification step.

Patent Document 4 discloses a manufacturing method as follows: The lignocellulose of 1 to 20% by mass of lignin reacts with lignin-degrading bacteria such as cellulase and white decay fungi to produce cellobiose which is one of cellooligosaccharides. In the production method, the effect of the cellulase on the substrate can be improved without the treatment of the delignification of the cellulose, but in the decomposition product, the lignin decomposition product is mixed in addition to the cellobiose, so the same as above The yield of cellooligosaccharide is low. Further, there is a problem in that in order to obtain a high-purity cellobiose, a removal step of the lignin decomposition product is required, and the purification step is complicated.

Further, as a method of decomposing the cellulase using a specific cellulase to increase the yield of cellooligosaccharide, the following may be mentioned.

Patent Document 5 discloses a method for producing cellooligosaccharide by a method for producing cellooligosaccharide from a cellulosic material in an aqueous reaction solution by the action of a cellulase produced by a microorganism belonging to the genus Cellovibrio. : The product inhibition is released by combining the ultrafiltration reactor to generate accumulated cellooligosaccharides. According to this method, cellooligosaccharide containing only cellobiose or cellotriose can be obtained as a decomposition product of decomposition of an enzyme of a cellulose-based substance. However, there is a problem that an enzyme produced by a microorganism belonging to the genus Cellovibrio is difficult to act on crystalline cellulose, so in order to shorten the reaction time and increase the yield, it is necessary to use amorphous cellulose as a substrate, and the step is changed. It’s complicated.

In Patent Document 6, in the method of decomposing cellulose by cellulase to produce cellooligosaccharide, a method for producing cellooligosaccharide is disclosed in which cellulase is previously equilibrated to a weak acidity of pH 3.5 to 5.0. Contacting the cation exchange resin to selectively remove the β-glucosidase in the cellulase, The cellulase from which the β-glucosidase has been removed is then contacted with cellulose. According to this production method, it is possible to decompose the cellulose enzyme and reduce the glucose to obtain a decomposition product of 60% or more of the cellooligosaccharide. However, in this production method, there is a problem in that the step of removing the β-glucosidase in cellulose is complicated, and the cellooligosaccharide production step is complicated. Further, in the cellulase purification step, there is a problem that 75 to 1000 times of the cation exchange resin is required with respect to the untreated cellulase, so that the amount of cellulase treatment is limited, and the productivity of cellooligosaccharide is Insufficient, the cost of cellulase purification, and the cost of separating and purifying the cation exchange resin become high.

Patent Document 7 discloses a cellulase purification method in which any one or both of a cellulose ester or a cellulose ether ester is dissolved with a cellulase, and the pH is changed after a fixed period of time. The insoluble solid fraction is separated from the solution to selectively remove the β-glucosidase contained in the cellulase; and the cellobiose is produced by removing the cellulose of the β-glucosidase The enzyme and the cellulose are added to an aqueous medium to prepare a suspension, and the suspension is kept for a fixed time to form a cellobiose in the suspension, and the cellobiose is collected.

Patent Document 8 discloses a method for purifying a cellulase by dissolving the polyglucamine and cellulase in an aqueous medium in which pH is adjusted to dissolve polyglucosamine, and after allowing for a fixed period of time, a pH value change, separating the insoluble solid fraction from the solution, thereby selectively removing the β-glucosidase contained in the cellulase; and, in addition, the method for producing cellobiose, which has removed the β-glucose The cellulase and cellulose of the glycosidase are added to the aqueous medium to prepare a suspension, and the suspension is kept for a fixed time, and the suspension is suspended. Cellobiose is formed in the liquid, and the cellobiose is collected.

According to the production method, the cellulase is adsorbed and separated by a cellulose derivative or polyglucamine, and is brought into contact with cellulose while being adsorbed on the cellulose derivative or the polyglucamine. Increase the yield of cellobiose. However, there is a problem in that the production method requires purification of a cellulase, which complicates the production process, and the cellulose derivative or polyglucamine used in the purification of cellulase is expensive. The cost is getting higher. Further, there is a problem that the cellulase is used together with the cellulose derivative or the polyglucamine for the decomposition of the cellulose enzyme, and therefore, it is necessary to carry out the step of removing the same from the self-decomposing reaction liquid.

Patent Document 9 discloses that an insoluble cellulose or a cellulose-containing material and a cellulase derived from Trichoderma are incubated in an aqueous medium to separate a solid fraction, and a water-soluble solution is added to the solid fraction. And a method of obtaining cellobiose. According to this production method, the β-glucosidase mixed in the cellobiose production step can be reduced, and the cellobiose can be selectively obtained. However, there is a problem that the step of adsorbing the cellulase on the solid fraction and the step of separating the solid fraction must be performed, making the process very complicated.

[Non-Patent Document 1] Cellulose Communications, 5, No., 91-97 (1998)

[Non-Patent Document 2] "Cellulase", Kodansha Scientific Release, 97-104 (1987)

[Patent Document 1] Japanese Patent Laid-Open Publication No. 03-130086

[Patent Document 2] Japanese Patent Laid-Open No. Hei 05-317073

[Patent Document 3] Japanese Patent Laid-Open No. Hei 07-184678

[Patent Document 4] Japanese Patent Laid-Open No. Hei 08-089274

[Patent Document 5] Japanese Patent Laid-Open No. Hei 01-256394

[Patent Document 6] Japanese Patent Laid-Open No. Hei 05-115293

[Patent Document 7] Japanese Patent Laid-Open No. Hei 05-227957

[Patent Document 8] Japanese Patent Laid-Open No. Hei 05-227958

[Patent Document 9] Japanese Patent Laid-Open Publication No. SHO 63-226294

The problem to be solved by the present invention is to provide a cellulosic material as a raw material, and in the presence of cellulase, when cellobiose is selectively produced by decomposition of an enzyme, a large amount of fiber can be ensured without complicated steps. A method for producing an enzyme solution for culturing a supernatant enzyme solution having high activity of a protease and having a high rate of cellulose saccharide, and a method for producing a cellooligosaccharide using the enzyme solution.

The present inventors have found that a culture medium having a high cellulase activity can be obtained by culturing a microorganism belonging to the genus Trichoderma in a medium containing 0.1 to 10% by weight of cottonseed meal. Further, in a medium containing 90% or more of ammonium sulfate in addition to cottonseed meal, a microorganism belonging to the genus Trichoderma is inoculated, and cultured under appropriate conditions, whereby the β-glucosidase activity is successfully reduced.

That is, the present invention is as follows.

(1) A method for producing a cellulase-containing enzyme solution, comprising: cultivating a microorganism belonging to the genus Trichoderma in a liquid medium containing a component derived from a plant seed belonging to the family Malvaceae.

(2) The method for producing an enzyme solution according to (1), wherein: the plant seed of the Malvaceae is a cotton seed.

(3) The method for producing an enzyme solution according to (2), wherein the microorganism belonging to the genus Trichoderma is cultured in a liquid medium containing 0.1 to 10% by mass of cottonseed meal as a component derived from cottonseed.

(4) The method for producing an enzyme solution according to any one of (1) to (3), wherein the liquid medium contains at least one selected from the group consisting of a sulfate ion, a hydrogen sulfate ion, and an ammonium ion.

(5) The method for producing an enzyme solution according to any one of (1) to (3) wherein the microorganism is a strain of Trichoderma reesei.

(6) The method for producing an enzyme solution according to (4), wherein the microorganism is a strain of Trichoderma reesei.

(7) The method for producing an enzyme solution according to (5), wherein: a strain belonging to Trichoderma reesei, a strain of Trichoderma reesei GL-1 (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Deposit Center, registration number) : FERM BP-10323), and/or Trichoderma reesei AKC-015 (Independent Administrative Corporation Industrial Technology Research Institute, Patent Bio-Hosting Center, Deposit No.: FERM BP-10839), and/or GL-1 strain Or a mutant strain obtained as a mother strain of the AKC-015 strain.

(8) A method for producing an enzyme solution according to (6), wherein: a strain belonging to Trichoderma reesei, a strain of Trichoderma reesei GL-1 (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Deposit Center, registration number) :FERM BP-10323), and/or Trichoderma reesei AKC-015 (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Deposit Center, registration number: FERM BP-10839), and/or a mutant obtained by using the GL-1 strain or the AKC-015 strain as a parent strain.

(9) A method for producing a cellulase-containing enzyme solution, comprising: cultivating a strain of Trichoderma reesei AKC-015 (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Depository Center, registration number: FERM BP-10839) The steps.

(10) A method for producing a cellooligosaccharide, comprising: using any one of the claims (1), (2), (3), (6), (7), (8), and (9) The enzyme solution produced by the manufacturing method described above is a step of decomposing a cellulose enzyme.

(11) A method for producing a cellooligosaccharide, comprising: decomposing a cellulolytic enzyme using the enzyme solution produced by the production method according to (4).

(12) A method for producing a cellooligosaccharide, comprising: decomposing a cellulolytic enzyme using the enzyme solution produced by the production method according to (5).

According to the present invention, an enzyme solution having high cellulase activity and high cellulose saccharide selectivity can be produced, and by using the enzyme solution in the production of cellooligosaccharide, cellooligosaccharide can be produced in high yield without complicated steps. .

The cellulase in the present invention is a general term for a cellulose-decomposing enzyme, and if it has cellulolytic activity, it is included in the cellulase referred to in the present invention. Further, the cellulase activity in the present invention can be expressed as decomposition of carboxymethylcellulose (CMC) enzymatic activity (CMC-ase), decomposition of crystalline cellulose (MCC) enzymatic activity (MCC-ase), and The measurement was carried out by the method described below. The higher the cellulase activity, the decomposition rate of cellulose, The higher the decomposition rate, the better.

(1) CMC-ase activity

480 μl of a 1% sodium carboxymethylcellulose sodium salt solution dissolved in 20 mM acetic acid-sodium acetate buffer (pH 5) was prepared. 20 μl of an appropriately diluted enzyme solution was added thereto, and the mixture was reacted at 40 ° C for 30 minutes. The reducing sugar was subjected to colorimetric quantification by a 3,5-dinitro water field acid method after heating at 95 ° C for 15 minutes to stop the reaction. The cellobiose standard solution was used as a standard curve, and the amount of the enzyme which released 1 μmol of the reducing sugar in 1 minute in terms of cellobiose was defined as 1 enzyme unit (1 U).

(2) MCC-ase activity

1.25 mass% of crystalline cellulose was suspended in 50 mM acetic acid-sodium acetate buffer (pH 5) (manufactured by Asahi Kasei Chemical Co., Ltd., trade name: Ceolus PH-101, 60% moisture, manufactured by Sanying Co., Ltd. , the product name: Universal Mixing Mixer, 0.1 ml of the appropriately diluted enzyme solution was added to 0.4 ml of the receiving solution by kneading with a hook piece at 90 minutes and 126 rpm, and reacted at 40 ° C for 30 minutes. After heating at 95 ° C for 15 minutes to stop the reaction, the reducing sugar was subjected to colorimetric quantification by the 3,5-dinitrosalicylic acid method. The cellobiose standard solution was used as a standard curve, and the amount of the enzyme which released 1 μmol of the reducing sugar in 1 minute in terms of cellobiose was defined as 1 enzyme unit (1 U).

Further, the β-glucosidase in the present invention refers to an enzyme which decomposes cellooligosaccharide and produces glucose, and can be quantified by β-glucosidase activity. When the cellulose enzyme is decomposed to obtain glucose, the higher the β-glucosidase activity of the enzyme solution, the rate of formation of glucose and the fraction of glucose. The higher the solution rate, the more effective it is. On the contrary, in order to obtain cellooligosaccharide in a high yield, it is effective to use an enzyme solution having a high cellulase activity and a low β-glucosidase activity. The β-glucosidase activity can be measured by the following method.

(3) β-glucosidase activity

In a 200 mM acetic acid-sodium acetate buffer (pH 5), 2.0% by mass of cellobiose (made by Aldrich; special) was added to 0.3 ml of the nucleating solution, and 0.3 ml of the enzyme solution was added thereto, and the mixture was reacted at 40 ° C for 30 minutes. After that, the mixture was heated at 95 ° C for 15 minutes, and after the reaction was stopped, a glucose metering kit (glucose CII-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.), which is a combination of a mutase enzyme and a glucose oxidase, was used for quantification. The concentration of glucose in the reaction solution. The amount of enzyme that will release 1 μmol of glucose in 1 minute is defined as 1 enzyme unit (1 U).

The cellulase producing strain used in the present invention belongs to the microorganism of the genus Trichoderma. The enzyme having high cellulase activity of the present invention can be produced by inoculating and cultivating a microorganism belonging to the genus Trichoderma.

When the enzyme solution of the present invention is used for the production of cellooligosaccharide, it is necessary to obtain an enzyme solution having high cellulase activity and high cellulose saccharide selectivity, and among the microorganisms belonging to the genus Trichoderma, it is preferred to use For the strain of Trichoderma reesei, it is particularly preferable to use Trichoderma reesei GL-1 strain (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Depository Center, deposit number: FERM BP-10323), or Trichoderma reesei AKC - 015 (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Deposit Center, registration number: FERM BP-10839). In the above method, further preferably, The production of cellooligosaccharide in a high yield, using the above-mentioned T. reesei GL-1 strain or AKC-015 strain as a mother strain, can obtain cellooligosaccharide in a high yield and high selectivity, and perform a known mutation treatment. Mutant strain.

Furthermore, the GL-1 strain was deposited in the Institute of Industrial Technology and Technology, the National Institute of Advanced Industrial Science and Technology, and the Patent Bio-Reservation Center (1st, 1st, 1st, 1st, 1st, Tsukuba, Ibaraki Prefecture, Japan) 305-8566)), the registration number: FERM BP-10323; the AKC-015 strain was deposited on June 13, 2007, and is deposited in the Institute of Industrial Technology, the National Institute of Industrial Technology, and the Patent Biological Depository Center (Tsukuba City, Ibaraki Prefecture, Japan) 1 Chome 1 Place 6 (postal number 305-8566), registration number: FERM BP-10839. In addition, the GL-1 strain was deposited on October 7, 2005 in the Republic of China (Taiwan) Food Industry Development Institute (FIRDI) under the registration number BCRC930082.

The term "known mutation treatment" as used herein refers to a treatment in which a microorganism having cellulase-producing ability is subjected to mutation induction treatment using ultraviolet irradiation or a mutation inducing agent such as nitrosoguanidine, if necessary. A strain having a high production efficiency of cellooligosaccharide is selected among the strains. As a microorganism for the mutation-inducing treatment, it is preferred to use the Trichoderma reesei GL-1 strain (Independent Administrative Corporation Industrial Technology Research Institute, Patent Bio-Hosting Center, deposit number: FERM BP-10323), or Richter Wood Mold strain AKC-015 (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Depository Center, deposit number: FERM BP-10839) is used as the parent strain.

As an example of the mutation treatment, for example, the following methods can be mentioned.

Trichoderma reesei GL-1 strain or AKC-015 strain was cultured on potato dextrose agar slant medium at 28 ° C for 3 to 10 days. Spores The seeds are suspended in physiological saline to be 100,000 to 100,000,000/ml, and EMS (ethyl methane sulfonate, ethyl methanesulfonate) is subjected to mutation treatment (100 to 500 μg/ml, pH 7.0, 28 ° C, 5). ~24 hours). The selection of a strain for secreting cellulase having high cellulase production can be achieved by inducing a suspension for treating spores from the mutation, collecting spores by centrifugation, and performing thorough washing, using glucose or the like as carbon. The source is cultured, and the enzyme activity of the culture is measured by a known method. For example, a culture of each mutant treatment strain can be used, and cellobiose or crystalline cellulose can be used as a substrate for enzymatic decomposition, and the resulting reducing sugar can be quantified; and a known coloring substrate can also be used to make it The culture is subjected to an enzyme reaction to qualitatively select a target strain.

The main culture medium used in the method for producing an enzyme solution of the present invention must contain a component derived from a plant seed belonging to the Malvaceae family. Examples of the seed belonging to the plant of the Malvaceae include cotton, okra, chrysanthemum, hibiscus, hibiscus, hollyhock, etc., and cottonseed or the like can be preferably used. As a component derived from a plant seed belonging to the Malvaceae family, for example, cottonseed meal can be used. The term "cotton seed" as used herein refers to the seed of the plant belonging to the genus Malvaceae and the outer skin thereof; the so-called cottonseed meal is the residue after the cottonseed oil is extracted from the cotton seed. The cottonseed meal of the present invention, in addition to the above cottonseed, also includes seeds, cottonseed hulls, cotton linters, cotton, petals, and the like. The amount of cottonseed added to the culture medium of the microorganism belonging to the genus Trichoderma is closely related to the cellulase activity of the enzyme solution. In order to obtain a cellulase having a strong valence, the amount of cottonseed meal in the medium must be added. It is 0.1 to 10% by mass. The cottonseed meal in the medium is preferably added in an amount of 0.2 to 7% by mass, and more preferably 2 to 5% by mass.

In order to obtain an enzyme solution in which the β-glucosidase activity of the cellooligosaccharide of the present invention can be selectively produced, in addition to the component derived from the plant seed belonging to the Malvaceae, it is preferred to use the sulfate ion, At least one selected from the group consisting of hydrogen sulfate ions and ammonium ions is added to the liquid medium, and it is particularly preferable to add ammonium sulfate to the liquid medium. The amount of ammonium sulfate added to the medium is preferably from 0.01 to 10% by mass, more preferably from 0.02 to 2% by mass, in order to reduce the β-glucosidase activity. A particularly preferred range is from 0.1 to 0.5% by mass.

In the medium used in the main culture, in addition to the above, as the carbon source, cellulose powder, cellobiose, filter paper, general paper, sawdust, wheat bran, rice husk, bagasse, large Cardamom, coffee, starch, lactose, glucose, glycerin, alcohol, organic acids, etc. Further, as the nitrogen source, an inorganic ammonium salt such as ammonium nitrate, an organic nitrogen-containing compound such as urea, an amino acid, a gravy, a yeast extract, a polypeptone or a protein decomposition product may be added. As the inorganic salt, KH ₂ PO ₄ , MgSO ₄ ‧7H ₂ O, CaCl ₂ ‧2H ₂ O, FeCl ₃ ‧6H ₂ O, MnCl ₃ ‧4H ₂ O, ZnSO ₄ ‧7H ₂ O or the like can be used. A medium containing organic micronutrients can be used if necessary.

For the culture, if a normal aeration stirring culture device or a solid culture device is used, and the above medium is used, the temperature is 20 to 40 ° C, preferably 26 to 30 ° C, and the culture pH is 2 to 8, preferably 2.5 to 2. When cultured at 5.5, the cellulase activity reached the highest at 3 to 10 days. Then, the supernatant is removed from the culture solution by a known method such as centrifugation or filtration. The supernatant can be used directly as a crude enzyme solution.

The method for producing the cellooligosaccharide will be described below.

The method for decomposing the enzyme can be a known method, and is not particularly limited. As an example, a method in which the cellulosic material as a substrate is suspended in an aqueous medium and the cellulase of the present invention is added is used. Stirring or shaking is carried out while heating to carry out the saccharification reaction.

In the above method, the suspension conditions, the stirring method, the cellulase ‧ means of addition, the order of addition, and the reaction conditions such as the concentration can be appropriately adjusted so that the cellooligosaccharide can be obtained in a higher yield. The pH and temperature of the reaction solution at this time may be within a range in which the enzyme is not deactivated. Usually, when the reaction is carried out under normal pressure, the temperature is in the range of 5 to 95 ° C and the pH is in the range of 1 to 11. Just inside. Further, the pressure, temperature, and pH are the same as described above, and can be appropriately adjusted so that the cellooligosaccharide can be obtained in a higher yield, but the above-mentioned Trichoderma reesei GL-1 strain and/or AKC- are used. When 015 strains or these mutant strains are used as cellulase-producing cellulase, the cellulose enzyme is decomposed, preferably under normal pressure, in acetic acid or phosphate buffer at a temperature of 50~ The temperature is in the range of 3.0 to 5.5 at 60 ° C.

The cellulose-based material used in the present invention is preferably a water-insoluble fibrous substance containing cellulose. The source may be plant or animal, as the animal or plant which produces it, for example, wood, bamboo, wheat straw, straw, corncob powder, cotton, ramie, bagasse, kenaf, sugar beet, sea squirt, bacteria Cellulose, etc. Further, in the present invention, in addition to the natural cellulose-based material produced by the above-mentioned animals and plants, a regenerated cellulose system obtained by temporarily chemically dissolving or pulverizing the natural cellulose-based material may be used. Substance, as well as chemical modification of cellulose-based materials A cellulose derivative is a substance. These cellulose-based materials may be industrially used as raw materials such as paper, cellulose powder, and crystalline cellulose, and various regenerated fibers such as regenerated cellulose, alkali cellulose, and phosphoric acid swellable cellulose. A raw material, a cellulose derivative such as sodium carboxymethyl cellulose, is used as a raw material. Among them, when the obtained cellooligosaccharide is used for pharmaceuticals, foods, and cosmetics, it is more preferable to use a natural cellulose-based raw material. As the raw material, one type of the cellulose-based material may be used, or two or more types may be used.

The enzyme reaction can be carried out in batches or continuously. In the enzyme decomposition reaction, in order to avoid product inhibition caused by cellobiose, it is important to maintain the cellobiose concentration in the reaction system in a specific range in order to improve the productivity of cellooligosaccharide. As a method of maintaining the cellobiose concentration in the reaction system within a specific range, the method may be a method of extracting the produced cellobiose from the reaction system by membrane filtration such as ultrafiltration or reverse osmosis filtration; An organic porous substrate such as dried plant powder such as bamboo or wood, an inorganic porous substrate such as ceria, or the like, which is introduced into a reaction system to adsorb cellobiose or the like; and the cellulose substrate is fixed to the column. In the method, a reaction solution containing a cellulase is circulated, and a cellulase is fixed in a polymer or the like to circulate a reaction solution containing cellulose.

The aqueous solution containing the cellooligosaccharide obtained by decomposing the above-mentioned enzyme as a main component may be subjected to purification treatment such as decolorization, desalting, or removal of an enzyme, as needed. The purification method is not particularly limited as long as it is a known method, and for example, activated carbon treatment, ion exchange resin treatment, chromatography treatment, microfiltration, ultrafiltration, and reverse osmosis can be used. Filtration treatment such as filtration, crystallization treatment, etc. may be used alone or in combination of two or more.

The aqueous solution containing the cellooligosaccharide as a main component purified by the above method can be used as it is, and can be cured by drying as needed. The drying method is not particularly limited as long as it is a known method, and for example, spray drying, freeze drying, drum drying, film drying, layer drying, air flow drying, vacuum drying, etc. may be used, and these may be used singly or in combination of two kinds. the above.

As the medium of the cellooligosaccharide in the above-mentioned purification and drying treatment, an organic solvent or the like may be used as needed in addition to water. The organic solvent to be used herein is also not particularly limited. For example, it is preferably used in the steps of producing pharmaceuticals, foods, and the like, and can be exemplified in the "Pharmaceutical Additives Code" (Pharmacy News Agency ( It is classified as a solvent in the "issuance of the stock", "Japanese Pharmacopoeia", and "Food Additives" (issued by the Hirokawa Bookstore). The water or the organic solvent may be used singly or in combination of two or more kinds, and may be used as a single material. After dispersing a medium, the medium may be removed and dispersed in a different medium.

The form of the cellooligosaccharide which has undergone the above steps is not particularly limited. For example, it can be used in the form of a solid, a suspension, an emulsion, a syrup or a solution at normal temperature. Examples of the solid cellooligosaccharide include powders, granules, agglomerates, molded bodies, laminates, solid dispersions, and the like.

The use of the cellooligosaccharide obtained by the present invention is not particularly limited, and can be used, for example, in foods, cosmetics, pharmaceuticals, general industrial products, and the like as food ingredients, cosmetic ingredients, coloring ingredients, perfume ingredients, and pharmaceutical drugs. Effective ingredients, pesticide ingredients, feed ingredients, fertilizer ingredients, medium The reagent components for analysis and analysis, as well as additives, intermediate materials, and yeast raw materials.

The cellooligosaccharide obtained by the present invention can be used for foods, for example, jelly, pudding, yogurt, gelatinous food, mayonnaise, salad dressing, sauce, sauce, soup, processed vegetables, etc. Raw materials, curry, diced meat, meat sauce, stew (meat), canned food such as soup, frozen food, hamburger, enzyme meat, sausage, Italian sausage, ham and other livestock processed products, fish cake, fish rolls, fish Aquatic products such as ham, sausage, fried fish cake, bread, raw noodles, dried noodles, macaroni, Italian pasta, steamed buns, cake mix powder, bread premixed powder, white sauce, dumplings, spring rolls, etc. Wheat processed foods, curries, sauces, soups, sweet-cooked seafood, jams, canned, bottled, confectionery, sugar, hard candy, chocolate, biscuits, cookies, rice cakes, Japanese snacks, Western-style snacks, small Dim sum, candied fruit, pudding and other snacks, fried foods, fried meatloaf, dumplings, buns and other cooking and processing products, vegetable paste, minced meat, fruit paste, fish and shellfish paste and other paste. In addition, it can also be used in dairy products such as cream, ice milk, milk ice, foamed emulsified concentrate, condensed milk, butter, yogurt, cheese, white sauce, and processed fats such as margarine, fat spread food, and ghee. Further, it can also be used in carbonated drinks such as cola, fruit drinks and beverages mixed with carbonic acid, alcohol and dairy products, juices or beverages containing fruit drinks, milk drinks, coffee, milk, soy milk, cocoa milk, and fruit milk. Lactic acid/milk drink such as yogurt, tea drink such as sencha, oolong tea, matcha tea, black tea, etc.

The cellooligosaccharide obtained by the present invention can be expected to have a useful bacterial community such as lactic acid bacteria, lactobacilli, etc. Concentration, blood insulin concentration, lowering blood cholesterol, lowering body fat percentage, promoting fat metabolism, saccharide metabolism, improving various physiological activities such as defecation, odor, anti-caries, etc. Therefore, in addition to the above-mentioned general food use, It can also be used as a physiologically active substance for use in functional foods, health foods, health foods, and the like.

Further, the cellooligosaccharide obtained by the present invention is of high purity, and thus can be used as a chemical conversion raw material of various cellooligosaccharide derivatives.

[Examples]

The present invention will be more specifically described based on the examples and the like, but the present invention is not limited by the examples and the like.

[Example 1]

Trichoderma reesei GL-1 strain (Independent Administrative Corporation Industrial Technology Research Institute, Patent Bio-Hosting Center, Deposit No.: FERM BP-10323) was cultured on potato dextrose agar slant medium at 28 ° C for 7 days. Spores are fully formed. 1 platinum ear amount, cottonseed meal (Pharmamedia, manufactured by Trader's): 0.15 g, KH ₂ PO ₄ : 0.06 g, (NH ₄ ) ₂ SO ₄ : 0.045 g, MgSO ₄ ‧7 H ₂ O: 0.009 g, CaCl ₂ ‧2H ₂ O: 0.009 g, ADEKA NOL LG109: 0.03 ml, trace element solution (H ₃ BO ₃ 6 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄ ‧4H ₂ O 26 mg, FeCl ₃ ‧6H ₂ O 100 mg, CuSO ₄ ‧5H ₂ O 40 mg, MnSO ₄ ‧5H ₂ O 8 mg, ZnSO ₄ ‧7H ₂ O 200 mg solution dissolved in 100 ml of water): 0.03 ml dissolved and suspended in water: 30 ml, pH The value was adjusted to 4.0, 30 ml was dispensed into an Erlenmeyer flask having a volume of 150 ml, and 0.3 g of crystal cellulose (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name PH-101) was added, and then inoculated into autoclaved sterilized preculture. The medium was cultured at 28 ° C for 3 days.

Then, the culture solution and cottonseed meal: 2 g, KH ₂ PO ₄ : 0.2 g, MgSO ₄ ‧7 H ₂ O: 0.03 g, CaCl ₂ ‧2 H ₂ O: 0.03 g, ADEKA NOL LG109: 0.1 ml, trace element solution: Dissolve and suspend 0.1 ml in water: 100 ml, adjust the pH to 4.0, dispense 100 ml into an Erlenmeyer flask having a volume of 500 ml, and add 2 g of crystalline cellulose (manufactured by Asahi Kasei Chemical Co., Ltd., trade name PH-101). This was inoculated on an autoclave-sterilized main culture medium, and cultured at 28 ° C for 6 days. The culture solution was centrifuged on the 6th day, and 1 ml of the cellulase activity (CMCase) of the supernatant was measured. The results are shown in Figure 1 as pharmamedia-2%.

[Embodiment 2]

The Trichoderma reesei GL-1 strain was cultured on potato dextrose agar slant medium at 28 ° C for 7 days to fully form spores. 1 platinum ear amount, cottonseed meal (Pharmamedia, manufactured by Trader's): 0.15 g, KH ₂ PO ₄ : 0.06 g, (NH ₄ ) ₂ SO ₄ : 0.045 g, MgSO ₄ ‧7 H ₂ O: 0.009 g, CaCl ₂ ‧2H ₂ O: 0.009g, ADEKA NOL LG109: 0.03ml, trace element solution (H ₃ BO ₃ 6mg, (NH ₄ ) ₆ MO ₇ O ₂₄ ‧4H ₂ O 26mg, FeCl ₃ ‧6H2O 100mg, CuSO ₄ ‧5H ₂ O 40mg, MnSO ₄ ‧5H ₂ O 8mg, ZnSO ₄ ‧7H ₂ O 200mg dissolved and suspended in water 100ml solution): 0.03ml dissolved and suspended in water: 30ml, the pH value The mixture was adjusted to 4.0, and 30 ml was dispensed into an Erlenmeyer flask having a volume of 150 ml, and 0.3 g of crystal cellulose (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name: PH-101) was added, and then inoculated into an autoclave-sterilized preculture medium. The culture was carried out at 28 ° C for 3 days.

Then, the culture solution and cottonseed meal: 1 g, KH ₂ PO ₄ : 0.2 g, MgSO ₄ ‧7 H ₂ O: 0.03 g, CaCl ₂ ‧2 H ₂ O: 0.03 g, ADEKA NOL LG 109: 0.1 ml, trace element solution: Dissolve and suspend 0.1 ml of water in 100 ml, adjust the pH to 4.0, dispense 100 ml into an Erlenmeyer flask having a volume of 500 ml, and add 2 g of crystalline cellulose (manufactured by Asahi Kasei Chemical Co., Ltd., trade name PH-101). This was inoculated on an autoclave-sterilized main culture medium, and cultured at 28 ° C for 6 days. The culture solution was centrifuged on the 6th day, and 1 ml of the cellulase activity (CMCase) of the supernatant was measured. The results are shown in Figure 1 as pharmamedia-2%.

[Comparative Example 1]

In the same method as in the first embodiment or the second embodiment, 2 g or 1 g of wheat bran (Japanese flour), 2 g (manufactured as wheat bran - 2%, 1%), corn syrup (manufactured by Wako Pure Chemical Industries, Ltd.), 2 g or 1 g ( The main culture medium was designated as corn syrup - 2%, 1%), instead of the main culture medium using cottonseed meal (1 g in Example 1 and 2 g in Example 2), except for the examples and examples. 1 The same method was used to culture the Trichoderma reesei GL-1 strain for 6 days. The culture solution was centrifuged on the sixth day, and the cellulase activity (CMCase) of 1 ml of the supernatant was measured, and the results are shown in Fig. 1 .

[Example 3]

The Trichoderma reesei GL-1 strain was cultured on potato dextrose agar slant medium at 28 ° C for 7 days to fully form spores. 1 platinum ear amount, cottonseed meal (Pharmamedia, manufactured by Trader's): 0.15 g, KH ₂ PO ₄ : 0.06 g, (NH ₄ ) ₂ SO ₄ : 0.045 g, MgSO ₄ ‧7 H ₂ O: 0.009 g, CaCl ₂ ‧2H ₂ O: 0.009 g, ADEKA NOL LG109: 0.03 ml, trace element solution (H ₃ BO ₃ 6 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄ ‧4H ₂ O 26 mg, FeCl ₃ ‧6H ₂ O 100 mg, CuSO ₄ ‧5H ₂ O 40mg, MnSO ₄ ‧5H ₂ O 8mg, ZnSO ₄ ‧7H ₂ O 200mg dissolved and suspended in 100ml of water): 0.03ml dissolved and suspended in water: 30ml, will The pH was adjusted to 4.0, 30 ml was dispensed into an Erlenmeyer flask having a volume of 150 ml, and 0.3 g of crystal cellulose (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name PH-101) was added, and then inoculated into an autoclave-sterilized preculture. The culture was carried out for 3 days at 28 ° C on the medium.

Then, the culture solution and cottonseed meal: 2 g, (NH ₄ ) ₂ SO ₄ : 0.15 g, KH ₂ PO ₄ : 0.2 g, MgSO ₄ ‧7 H ₂ O: 0.03 g, CaCl ₂ ‧2 H ₂ O: 0.03 g, ADEKA NOL LG109: 0.1ml, trace element solution: 0.1ml dissolved and suspended in water: 100ml, adjust the pH to 4.0, dispense 100ml in a 500ml conical flask, add crystalline cellulose (Asahi Kasei Chemicals) After 2 g of the product manufactured by the company, trade name PH-101), it was inoculated on an autoclave-sterilized main culture medium, and cultured at 28 ° C for 6 days. The culture solution was centrifuged on the 6th day, and the cellulase activity (CMCase), cellulase activity (MCCase), and β-glucosidase activity of 1 ml of the supernatant were measured, and the results are shown in Tables 1 and 2. And Table 3.

[Example 4]

The Trichoderma reesei GL-1 strain was cultured on potato dextrose agar slant medium at 28 ° C for 7 days to fully form spores. 1 platinum ear amount, cottonseed meal (Pharmamedia, manufactured by Trader's): 0.15 g, KH ₂ PO ₄ : 0.06 g, (NH ₄ ) ₂ SO ₄ : 0.045 g, MgSO ₄ ‧7 H ₂ O: 0.009 g, CaCl ₂ ‧2H ₂ O: 0.009 g, ADEKA NOL LG109: 0.03 ml, trace element solution (H ₃ BO ₃ 6 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄ ‧4H ₂ O 26 mg, FeCl ₃ ‧6H ₂ O 100 mg, CuSO ₄ ‧5H ₂ O 40mg, MnSO ₄ ‧5H ₂ O 8mg, ZnSO ₄ ‧7H ₂ O 200mg dissolved and suspended in 100ml of water): 0.03ml dissolved and suspended in water: 30ml, will The pH was adjusted to 4.0, 30 ml was dispensed into an Erlenmeyer flask having a volume of 150 ml, and 0.3 g of crystal cellulose (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name: PH-101) was added, and then inoculated into an autoclave-sterilized preculture. The culture was carried out for 3 days at 28 ° C on the medium.

Then, the culture solution and cottonseed meal: 1 g, (NH ₄ ) ₂ SO ₄ : 0.15 g, KH ₂ PO ₄ : 0.2 g, MgSO ₄ ‧7 H ₂ O: 0.03 g, CaCl ₂ ‧ ₂ H ₂ O: 0.03 g, ADEKA NOL LG109: 0.1ml, trace element solution: 0.1ml dissolved and suspended in 100ml of water, adjust the pH to 4.0, dispense 100ml in an Erlenmeyer flask with a volume of 500ml, add crystal cellulose (Asahi Kasei Chemical Co., Ltd.) After producing 2 g of the product name: PH-101), it was inoculated on the autoclave-sterilized main culture medium, and cultured at 28 ° C for 6 days. The culture solution was centrifuged on the 6th day, and the cellulase activity (CMCase), cellulase activity (MCCase), and β-glucosidase activity of 1 ml of the supernatant were measured, and the results are shown in Tables 1 and 2. And Table 3.

[Example 5]

Cellulase activity (CMCase), cellulase activity (MCCase), and β-glucosidase activity of 1 mL of the supernatant of the culture solution obtained by culturing in the same manner as in Example 1 were measured, and the respective results were obtained. Shown in Tables 1, 2 and 3.

[Embodiment 6]

The cellulase activity (CMCase), cellulase activity (MCCase), and β-glucosidase activity of 1 mL of the supernatant of the culture solution obtained by culturing in the same manner as in Example 2 were measured, and the respective results were obtained. Shown in Tables 1, 2 and 3.

Table 1 is the CMCase activity (relative value) of the culture medium for each of the added amounts of cottonseed meal (hereinafter referred to as pharmamedia) and ammonium sulfate (referred to as ammonium sulfate in the table) as a component of the main culture medium. , the unit is U) for comparison. It can be seen that when ammonium sulfate is added to the medium, the CMCase can be increased even if the cottonseed meal is added in a low amount.

Table 2 is the MCCase activity (relative value) of the culture medium for each of the addition amounts of cottonseed meal (hereinafter referred to as pharmamedia) as a component of the main culture medium and ammonium sulfate (referred to as ammonium sulfate in the table). , the unit is U) for comparison. It can be seen that if ammonium sulfate is added to the medium, even cottonseed meal MCCase can also be added for low additions.

Table 3 is a β-glucosidase against the culture solution of cottonseed meal (hereinafter referred to as pharmamedia) and ammonium sulfate (referred to as ammonium sulfate in the table) as components of the main culture medium. The activity (relative value, in U) was compared. It is understood that when ammonium sulfate is added to the medium, the cellulase activities (CMCase, MCCase) shown in Tables 1 and 2 can be increased, and the β-glucosidase activity can be lowered.

Therefore, by adding cottonseed meal and ammonium sulfate to the culture medium, the cellulase activity can be increased and the β-glucosidase activity can be lowered. Therefore, if the enzyme solution of the present invention is used in the decomposition of the enzyme of the cellulose-based substance, it can be expected Cellulose oligosaccharides are obtained in high yield.

[Embodiment 7]

The cellulase activity was carried out in the same manner as in Example 4 except that cottonseed meal: 5 g was placed in 100 ml of the main culture medium. After centrifugation of the culture solution on the 7th day, 1 ml of the cellulase activity of the supernatant was carried out. (CMCase) and cellulase activity (MCCase) were 58.4 and 19.4, respectively.

[Embodiment 8]

The culture medium was cultured in the same manner as in Example 4 except that cottonseed meal: 7 g was placed in 100 ml of the main culture medium. After centrifugation, the cellulase activity (CMCase) and cellulase activity (MCCase) of 1 mL of the supernatant were 17.9 and 8.0, respectively.

[Embodiment 9]

The Trichoderma reesei GL-1 strain was cultured on potato dextrose agar slant medium at 28 ° C for 7 days to fully form spores. 1 platinum ear amount, cottonseed meal (Pharmamedia, manufactured by Trader's): 0.15 g, KH ₂ PO ₄ : 0.06 g, (NH ₄ ) ₂ SO ₄ : 0.045 g, MgSO ₄ ‧7 H ₂ O: 0.009 g, CaCl ₂ ‧2H ₂ O: 0.009 g, ADEKA NOL LG109: 0.03 ml, trace element solution (H ₃ BO ₃ 6 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄ ‧4H ₂ O 26 mg, FeCl ₃ ‧6H ₂ O 100 mg, CuSO ₄ ‧5H ₂ O 40mg, MnSO ₄ ‧5H ₂ O 8mg, ZnSO ₄ ‧7H ₂ O 200mg dissolved and suspended in 100ml of water): 0.03ml dissolved and suspended in water: 30ml, will The pH was adjusted to 4.0, 30 ml was dispensed into an Erlenmeyer flask having a volume of 150 ml, and 0.15 g of crystalline cellulose (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name: PH-101) was added, and then inoculated into an autoclave-sterilized preculture. The cells were cultured at 28 ° C for 6 days on the medium. On the 6th day after the culture, the culture solution of the GL-1 strain was centrifuged, and 1 ml of the cellulase activity (CMCase) and β-glucosidase activity of the supernatant were measured, and the CMCase activity was 29.4 U, β-glucose. The glucosidase activity was 0.145 U.

In the same operation as above, the strain used was changed from GL-1 strain to AKC-015 strain, cultured, and the culture solution was centrifuged, and the cellulase activity (CMCase) of 1 mL of the supernatant was measured. And β-glucosidase activity, CMCase activity was 37.0 U, and β-glucosidase activity was 0.046 U.

[Industrial availability]

The cellooligosaccharide obtained by the production method of the present invention can be used as a chemical conversion raw material such as a functional food material, a pharmaceutical product, and an intermediate synthetic material of other chemicals, and a raw material for fermentation, in addition to the usual food materials. It is well used in the fields of food, pharmaceuticals and general industrial products.

Figure 1 compares the cottonseed meal with the CMCase activity (relative ratio) of other main culture medium components. The culture medium cultured with cottonseed meal showed higher CMCase activity (relative ratio) under the same culture conditions as compared with other medium components.

Claims (4)

A method for producing a cellulase-containing enzyme solution, comprising: cultivating a microorganism belonging to Trichoderma reesei by directly adding 0.1 to 10% by mass of cottonseed meal as a liquid medium derived from cottonseed. The method for producing an enzyme solution according to claim 1, wherein the liquid medium contains at least one selected from the group consisting of a sulfate ion, a hydrogen sulfate ion, and an ammonium ion. The method for producing the enzyme solution of claim 2, wherein the microorganism belonging to Trichoderma reesei is the GL-1 strain of Trichoderma reesei (Independent Administrative Corporation Industrial Technology Research Institute, Patent Biological Depository Center, registration number: FERM BP-10323 ; registered number BCRC 930082). A method for producing a cellooligosaccharide, which comprises decomposing a cellulase enzyme using an enzyme solution produced by the production method according to any one of claims 1, 2 and 3.