JP5317828B2 - Method for solubilizing plant tissue and mixed enzyme preparation - Google Patents

Description

  The present invention relates to a plant tissue solubilization method and a mixed enzyme preparation.

Improvement of plant taste (for example, reduction of astringency and bitterness) and effective utilization of plant fibers have been attempted by solubilizing plant tissues by causing enzymes such as cellulase to act on plants (for example, Patent Document 1). , See Patent Document 2).
When solubilizing a plant tissue with a cellulase preparation, the difficulty of solubilizing the plant tissue varies depending on the type of plant tissue, the growth state, etc., but it is often difficult in practice. The main factor is attributed to the components of the plant.

  When disintegrating or solubilizing plant tissues with cellulase preparations, first, protopectin, hemicellulose, etc., which adhere cells to each other, are degraded by cell separation enzymes (pectinase, hemicellulase, etc.) to become single cells. The basic solubilization method is to expose the entire surface of the cell wall, then decompose the cell wall composed of cellulose or the like with a cellulase preparation and elute the contents. Only soft plant fibers can be solubilized relatively easily by this solubilization method. Many plant fibers are difficult to solubilize. Hard plant tissue is resistant to cellulase preparations. The main reason is that cell walls and intercellular substances composed of cellulose and hemicellulose are embedded with an aromatic compound such as protolignin to become a hardly decomposable compound, and inhibit the enzyme action.

  Moreover, when saccharifying a cellulosic raw material such as wood with a cellulase preparation, pretreatment is performed in order to remove the factor that inhibits the enzyme action by lignin or the like (see, for example, Non-Patent Document 1 and Non-Patent Document 2). . As a pretreatment method, a chemical method, a physical method, or a microbial method is used. However, each method has advantages and disadvantages, and an effective method has not been established yet. Another problem is that the pre-processing costs are high.

JP 2001-161258 A Japanese Patent No. 2948471 Journal of the Wood Society of Japan 28 (2) 122 (1982) Journal of the Wood Society 35 521-529 (1989)

  An object of the present invention is to provide a method for efficiently solubilizing plant tissue by increasing the degradability of cellulase without costing pretreatment in a method for solubilizing plant tissue using cellulase. is there. In addition, it is a mixed enzyme preparation for solubilizing plant tissue by acting on the plant tissue, which can efficiently solubilize the plant tissue by increasing the degradability of cellulase without costing pretreatment It is to provide an enzyme preparation.

In order to achieve the above-mentioned object, the present inventor understood various structures of plant tissues and first examined enzyme preparations having specificity for natural aromatic compounds.
The basic part of the plant tissue is composed of cellulose, hemicellulose, and lignin, to which proteins, starch, lipids, and the like are bound. Lignin is deposited between cells and cell walls, and chemically binds to cellulose and hemicellulose to give physical strength.
Hemicellulose is a heteropolymer in which various sugar residues have various bonds. There is no regularity like cellulose, and it has a complicated bonding mode. Lignin is bonded to cellulose or hemicellulose to form a basic plant structure.

Lignin is a non-enzymatic polymerization of a phenoxy radical obtained by enzymatically dehydrogenating cinnamon alcohol, which is a precursor (monolignol). The binding mode is a complex structure with no regularity, and is a huge biopolymer that forms a three-dimensional network structure.
However, not all of the basic molecular bonds are C6-C3 units, and the bonding mode is not limited to the β-O-4 type, but there are various other bonding methods. From this diversity of substrates, it will be very important to study enzyme preparations with various substrate specificities in the future.
Among natural aromatic compounds other than lignin that are widely distributed in plant tissues, many low-molecular phenolic acids exist in ester bonds with cellulose, hemicellulose, pectin, and the like. The details of these modes of existence are unknown, but the present inventors hydrolyzed this with esterases such as cinnamic acid esterase and ferulic acid esterase, thereby breaking down one corner of the hardly decomposable compound and reacting the reactivity of cellulase and hemicellulase. As a result, the present invention was completed.

That is, method of solubilizing a plant tissue according to the present invention, cellulase, full Erura acid Estella over zero, and to a method of solubilizing a plant tissue to act sequentially or simultaneously plant tissue cinnamic acid esterase in any order, In this method, 0.126 to 126 units of cellulase, 0.00006 to 0.06 unit of ferulic acid esterase, and 0.000075 to 0.075 unit of cinnamic acid esterase are allowed to act on 1 g of plant tissue.

The mixing enzyme preparation according to the present invention is a mixed enzyme preparation to solubilize plant tissue to act on plant tissue, cellulase, full Erura acid Estella over zero, and contains a cinnamic acid esterase It is a mixed enzyme preparation , and the blending ratio of cellulase, ferulic acid esterase and cinnamic acid esterase is 20 to 30 parts by mass of ferulic acid esterase and 20 to 30 mass of cinnamic acid esterase with respect to 100 parts by mass of cellulase. part der is, and cellulase activity from 100 to 10,000 units, ferulic acid esterase activity 1-100 units and, cinnamic acid esterase activity is from 1.2 to 120 units.

  According to the plant tissue solubilization method of the present invention, plant tissue can be efficiently solubilized without costing pretreatment. Moreover, according to the mixed enzyme preparation of this invention, a plant tissue can be efficiently solubilized, without costing a pretreatment.

6 is a graph showing the glucose concentration in a sample of each test section of Example 4. FIG.

The cellulase used in the present invention is not particularly limited as long as it acts on cellulose. Microbial origins include Bacillus subtilis, Aspergillus niger, Irpex lacteus
), Humicura insorens, Trichoderma viride, Trichoderm reesei, and the like. Of these, those derived from the genus Trichoderma are particularly suitable.
Normally, cellulase is produced by the solid culture method or liquid culture method using the above-mentioned producing bacteria. Cellulase can be used in all forms such as sputum, culture solution, concentrated solution or powder obtained by purifying it. Is possible. Commercially available cellulase preparations such as cellulase preparation “TP2-Kyowa” manufactured by Kyowa Kasei Co., Ltd. can also be used.

The ferulic acid esterase used in the present invention is a known enzyme that belongs to a hydrolase (hydrolase) and acts on an ester bond in the EC classification based on the reaction form of the enzyme. Substrate specificity is shown for ethyl ferulate and the like.
Cinnamic acid esterase used in the present invention is a known enzyme that belongs to a hydrolase in the EC classification and acts on an ester bond, like ferulic acid esterase. Shows substrate specificity to ethyl cinnamate and the like. Decomposing ethyl cinnamate becomes cinnamic acid and ethanol.

  The ferulic acid esterase or cinnamic acid esterase may be any substance that has esterase activity with respect to a substrate having an ester bond. For example, a microorganism belonging to the genus Aspergillus or Botrysis, etc. And a culture product obtained by solid culture or liquid culture according to a conventional method or a treated product thereof. The treated product includes (1) an aqueous extract of the culture, (2) a liquid culture filtrate, (3) a salting out method using ammonium sulfate or the like from the aqueous extract or the liquid culture solution, or a precipitation method using alcohol or the like. (4) A purified enzyme prepared from this crude enzyme powder if necessary, commercially available ferulic acid esterase, cinnamic acid esterase, and the like.

Examples of tannase used in the reference examples of the present invention include ordinary tannase, for example, tannase having an activity of hydrolyzing gallate. Specifically, a microorganism belonging to the genus Aspergillus, Penicillium, Rhizopus, Mucor, etc. and capable of producing tannase is obtained by solid culture or liquid culture according to a conventional method using a medium used for culturing these microorganisms. In addition, cultures or processed products thereof can be mentioned. The treated product includes (1) an aqueous extract of the culture, (2) a liquid culture filtrate, (3) a salting-out method using ammonium sulfate or the like from the aqueous extract or the liquid culture filtrate, or a precipitation method using alcohol or the like. And the like, and crude enzyme powder obtained by drying the precipitate, and (4) a commercially available tannase preparation, such as tannase manufactured by Kikkoman Corporation.

The plant treated with the enzyme may be a plant mainly composed of cellulose, such as tea leaves, eucalyptus, poplar leaves, roots and branches, vines such as sweet potato, corn, rice, wheat and beans. And fruits such as apples, yuzu, and grapes.
These plants may be used as they are, and may be subjected to pretreatment such as drying, pulverization, and heating as necessary.

It will be described an embodiment of the present onset Ming implementation. The cellulase, ferulic acid esterase, and cinnamic acid esterase are added to the plant, and these three types of enzymes are allowed to act on the plant tissue.
These three types of enzymes may be separately added to the plant tissue and allowed to act, but it is preferable to add and act on the plant tissue simultaneously. When adding sequentially, the order of addition of the three types of enzymes may be in any order, and is not particularly limited, but it is preferable to add cellulase, ferulic acid esterase, and cinnamic acid esterase in this order.

  When the above three types of enzymes are added and acted simultaneously, the amount of each enzyme added and the enzyme reaction conditions vary depending on the type of plant to be treated, the form of the enzyme, etc., but usually the following is recommended. .

The amount of cellulase added is 0.126 to 126 units, preferably 7.2 to 18 units per 1 g of plant tissue (one unit is 1 micromole of glucose per minute when reacted at 40 ° C. for 30 minutes in a CMC solution). The amount of the enzyme that produces
The amount of ferulic acid esterase added is 0.00006 to 0.06 units, preferably 0.006 to 0.0015 units (1 unit is the amount of enzyme that produces 1 micromole of ferulic acid per minute in water at 37 ° C.) per 1 g of plant tissue. It is preferable that
The amount of cinnamic acid esterase added is 0.000075 to 0.075 units, preferably 0.0075 to 0.018 units per 1 g of plant tissue (when 1 unit is reacted at 30 ° C. for 20 minutes, 1 micromole of cinnamic acid is added per minute. The amount of enzyme produced) is preferred.

The enzyme reaction in the case of adding and acting on the above three types of enzymes at the same time can be carried out according to a conventional method. For example, water or an aqueous solution is added to a plant, and the above three types are added thereto. These enzymes can be added simultaneously, or three kinds of enzymes can be mixed in advance, and the mixed enzyme and plant can be added to water or an aqueous solution.
The enzyme reaction conditions in this case are preferably a reaction temperature of 30 to 50 ° C., particularly 40 to 43 ° C., and a reaction time of 2 to 50 hours, particularly 3 to 6 hours. The pH of water or an aqueous solution may or may not be adjusted, but is preferably adjusted to 3.0 to 7.0, particularly 4.0 to 6.0. Moreover, it is not necessary to stir during the reaction.

  In addition, when the above three types of enzymes are separately added and allowed to act separately, water or an aqueous solution may be added to the plant, and the above three types of enzymes may be added and reacted sequentially. The addition amount of each enzyme and each enzyme reaction condition may be as follows.

The amount of cellulase added is 0.126 to 126 units, preferably 7.2 to 18 g per 1 g of plant tissue.
Preferably it is a unit.
In the cellulase treatment, the reaction temperature is preferably adjusted to 30 to 50 ° C, particularly 40 to 50 ° C. The reaction time is 2 to 50 hours, preferably 3 to 6 hours. The pH of the water or aqueous solution may be adjusted or not adjusted, but is preferably adjusted to 3.0 to 7.0, particularly 4.0 to 6.0. Moreover, it is not necessary to stir during the reaction.

  The amount of ferulic acid esterase added is 0.00006 to 0.06 unit, preferably 0.006 to 0.015 unit per 1 g of plant tissue, for example, an amount sufficient to completely or completely release ferulic acid contained in the plant tissue. Preferably there is. In the ferulic acid esterase treatment, the reaction temperature is preferably adjusted to 25 to 60 ° C, particularly 50 to 55 ° C. The reaction time is 1 to 10 hours, preferably 2 to 6 hours. The pH of the water or aqueous solution may be adjusted or not, but is preferably adjusted to 5.0 to 8.0, particularly 6.0 to 7.0. Moreover, it is not necessary to stir during the reaction.

  The amount of cinnamic acid esterase added is 0.000075 to 0.075 units, preferably 0.0075 to 0.018 units, per 1 g of plant tissue. In the treatment with cinnamic acid esterase, the reaction temperature is preferably adjusted to 30 to 50 ° C, particularly 35 to 40 ° C. The reaction time is 1 to 10 hours, preferably 2 to 6 hours. The pH may or may not be adjusted, but is preferably adjusted to 4.5 to 6.0, particularly 5.0 to 5.5. Moreover, it is not necessary to stir or not during the reaction.

  When the above three types of enzymes are separately added and acted on separately, the previously added enzyme may be deactivated and then the next enzyme may be added, or the previously added enzyme may not be deactivated. The enzyme may be added.

Next, Reference Example 1 that is closely related to the present invention will be described. Cellulase and ferulic acid esterase are allowed to act on plant tissues sequentially or simultaneously in any order. Other configurations are the same as those of the present invention .

Next, Reference Example 2 that is closely related to the present invention will be described. Cellulase and cinnamic esterase are allowed to act on plant tissues sequentially or simultaneously in any order. Other configurations are the same as those of the present invention .

Next, Reference Example 3 that is closely related to the present invention will be described. Cellulase, ferulic acid esterase, and tannase are allowed to act on plant tissues sequentially or simultaneously in any order.
The amount of tannase added is 0.002 to 2 units, preferably 0.2 to 0.5 units, per 1 g of plant. In the tannase treatment, the reaction temperature is preferably adjusted to 30 to 46 ° C, particularly 40 to 42 ° C. The reaction time is 1 to 10 hours, preferably 2 to 6 hours. The pH may or may not be adjusted, but is preferably adjusted to 4.5 to 6.0, particularly 5.0 to 5.5. Moreover, it is not necessary to stir during the reaction.
Other configurations are the same as those of the present invention .

Next, Reference Example 4 that is closely related to the present invention will be described. Cellulase, cinnamic esterase, and tannase are allowed to act on plant tissues sequentially or simultaneously in any order. Other configurations are the same as those in Reference Example 3 .

Next, a description will be given mixed enzyme preparation used et is the solubilization method of the plant tissue of the present invention.
That is, mixed enzyme preparation used et is the solubilization method of the plant tissue of the present invention, three types of enzymes mentioned above (cellulase, ferulic acid esterase, cinnamic acid esterase) in the manufacture of this type of enzyme preparation and, optionally Various carriers, excipients, and other additives that have been used in the past are formulated and formulated. The dosage forms are tablets, powders, granules, liquids, etc., and can be formulated by conventional methods.

In the mixed enzyme preparation used in the plant tissue solubilization method of the present invention, the blending ratio of cellulase, ferulic acid esterase and cinnamic acid esterase is 20 to 30 parts by mass, particularly 25 to 25 parts by mass of ferulic acid esterase with respect to 100 parts by mass of cellulase. It is preferable to set it to -27 mass parts and cinnamate esterase to 20-30 mass parts, especially 25-27 mass parts. The mixed enzyme preparation of the present invention has a cellulase activity of 100 to 10,000 units, particularly 500 to 5000 units, a ferulic acid esterase activity of 1 to 100 units, particularly 5 to 50 units, and a cinnamic acid esterase activity of 1.2 to 120. It is preferable that it is a unit, especially 6-60 units. In the mixed preparation of the present invention, for example, amylase, protease and the like may be appropriately mixed.

Next, the mixed enzyme preparation used in Reference Example 1 will be described. A mixed enzyme preparation for acting on a plant tissue to solubilize the plant tissue, which contains cellulase and ferulic acid esterase. Other configurations are the same as those of the mixed enzyme preparation used in the method for solubilizing plant tissue of the present invention .

Next, the mixed enzyme preparation used in Reference Example 2 will be described. A mixed enzyme preparation for solubilizing a plant tissue by acting on the plant tissue, comprising cellulase and cinnamic acid esterase. Other configurations are the same as those of the mixed enzyme preparation used in the method for solubilizing plant tissue of the present invention .

Next, the mixed enzyme preparation used in Reference Example 3 will be described. A mixed enzyme preparation for acting on a plant tissue to solubilize the plant tissue, which contains cellulase, ferulic acid esterase and tannase.
In the mixed enzyme preparation used in Reference Example 3 , the blending ratio of tannase is preferably 20 to 30 parts by mass, particularly 25 to 27 parts by mass with respect to 100 parts by mass of cellulase.
Other configurations are the same as those of the mixed enzyme preparation used in the method for solubilizing plant tissue of the present invention .

Next, the mixed enzyme preparation used in Reference Example 4 will be described. A mixed enzyme preparation for solubilizing a plant tissue by acting on the plant tissue, comprising cellulase, cinnamic acid esterase, and tannase. Other configurations are the same as those of the mixed enzyme preparation used in Reference Example 3 .

  Hereinafter, the present invention will be specifically described with reference to examples.

Example 1
(1) Tea leaf decomposition-1
30 ml of pure water was added to 1 g of tea leaves (raw). The following preparations 1) to 4) were added thereto.
1) Cellulase preparation (TP2-Kyowa, Kyowa Kasei) 10 mg (60 units) alone 2) Cellulase preparation (TP2-Kyowa, Kyowa Kasei) 10 mg (60 units) cinnamic acid esterase (Kyowa Kasei) 3 mg 3) Cellulase preparation (TP2-Kyowa, manufactured by Kyowa Kasei Co., Ltd.) 10 mg (60 units) and Ferulic acid esterase (Kyowa Kasei Co., Ltd.) 3 mg (0.009 units) combined 4) Cellulase preparation (TP2-Kyowa) , Kyowa Kasei Co., Ltd.) 10 mg (60 units) combined with cinnamic acid esterase (Kyowa Kasei Co., Ltd.) 3 mg (0.011 units) and ferulic acid esterase (Kyowa Kasei Co., Ltd.) 3 mg (0.009 units) went. The enzymatic reaction was carried out at 40 ° C. for 43 hours. After the reaction was completed, the reducing sugar produced in the decomposition solution was quantified. The results are shown below.
1) Cellulase preparation alone: 138 mg
2) Cinnamate esterase preparation in combination with cellulase preparation: 149mg
3) Ferulic acid esterase preparation in combination with cellulase preparation: 190mg
4) Combined use of cinnamic acid esterase and ferulic acid esterase in cellulase preparation: 248mg
From this result, it is understood that the cellulase reactivity is increased by using esterase in combination with the cellulase preparation. In particular, in the 4) section of the present invention in which three kinds of enzymes are used in combination, the amount of produced reducing sugar is about 1.79 times that in the 1) section of the cellulase preparation alone treatment. In addition, in the section 4), the overall decomposition of the decomposition liquid progressed, and the filtration speed at the time of filter paper filtration was high, and a complete transparent liquid was obtained.

Example 2
(2) Tea leaf decomposition-2
Three types of tea leaves (raw) (tea leaves-1, tea leaves-2, tea leaves-3) were used. 30 ml of pure water was added to 1 g of this tea leaf, and 10 mg of a mixed preparation containing 6 mg (36 units) of cellulase, 2 mg of ferulic acid esterase (0.006 units) and 2 mg of cinnamic acid esterase (0.0075 units) was added and reacted. As a control, 10 mg (60 units) of cellulase preparation (TP2-Kyowa) was used alone. Incubate at 40 ° C for reaction, sample after 24 hours, pore size
It filtered with the 0.2 micrometer membrane filter, and measured the total polyphenol amount of the filtrate. The results are shown in Table 1.

Mixed enzyme preparation used Ward that need use in the present invention, the total polyphenol content is significantly higher than compared to the control (singly Ward). Although the enzyme action varies depending on the type of tea leaf, the combined effect of the three types of enzymes was observed in each leaf. If over time See total polyphenol amount, the tea leaves -1, in any mixing enzyme preparations used Ward and controls are needed use in the present invention (singly-ku) also reached almost peak 6 hours after the start of the enzymatic reaction, After that it remained flat. In tea leaves -2 and tea -3, even after 6 hours, the mixture an enzyme preparation used Ward that need use in the present invention but the total polyphenol content is increased gradually, the control (singly-ku) in flat or a slightly declining Indicated.

Reference Example 1
(3) Tea leaf decomposition-3
30 ml of pure water was added to 1 g of tea leaves (raw). The following preparations 1) to 4) were added thereto.
1) Cellulase preparation (TP2-Kyowa, Kyowa Kasei) 10 mg (60 units) alone 2) Cellulase preparation (TP2-Kyowa, Kyowa Kasei) 10 mg (60 units) ferulic acid esterase (Kyowa Kasei) 3 mg 3) Cellulase preparation (TP2-Kyowa, manufactured by Kyowa Kasei Co., Ltd.) 10 mg (60 units) and tannase preparation (Kikkoman), 3 mg (0.3 units) combined 4) Cellulase preparation (TP2-Kyowa, Kyowa) 10 mg (60 units) of Kasei Co., Ltd. and 3 mg (0.009 units) of ferulic acid esterase (manufactured by Kyowa Kasei Co., Ltd.) and 3 mg (0.3 units) of tannase preparation (manufactured by Kikkoman Corp.) were used. The enzymatic reaction was carried out at 40 ° C. for 43 hours. After the reaction was completed, the reducing sugar produced in the decomposition solution was quantified. The results are shown below.
1) Cellulase preparation alone: 125mg
2) Ferulic acid esterase preparation in combination with cellulase preparation: 185mg
3) Cellulase preparation combined with tannase preparation: 160mg
4) Ferulic acid esterase preparation and tannase preparation in combination with cellulase preparation: 251mg
From this result, it is understood that the cellulase reactivity is increased by using esterase in combination with the cellulase preparation. In particular, in the 4) section of Reference Example 3 in which three kinds of enzymes are used in combination, the amount of produced reducing sugar is about twice that of the 1) section of the cellulase preparation alone treatment. In addition, in the section 4), the overall decomposition of the decomposition liquid progressed, and the filtration speed at the time of filter paper filtration was high, and a complete transparent liquid was obtained.

Reference Example 2
(4) Tea leaf decomposition-4
Three types of tea leaves (raw) (tea leaves-1, tea leaves-2, tea leaves-3) were used. 30 ml of pure water was added to 1 g of this tea leaf, and 10 mg of a mixed preparation containing 6 mg (36 units) of cellulase, 2 mg (0.006 units) of ferulic acid esterase and 2 mg (0.2 units) of tannase was added thereto and reacted. As a control, 10 mg (60 units) of cellulase preparation (TP2-Kyowa) was used alone. The mixture was incubated at 40 ° C. for reaction, sampled after 24 hours, filtered through a membrane filter having a pore size of 0.2 μm, and the total amount of polyphenols in the filtrate was measured. The results are shown in Table 2.

In the mixed enzyme preparation use group used in Reference Example 3 of the present invention, the total amount of polyphenols greatly exceeds the control (single use group). Although the enzyme action varies depending on the type of tea leaf, the combined effect of the three types of enzymes was observed in each leaf.

Reference Example 3
(5) Tea leaf decomposition-5
30 ml of pure water was added to 1 g of tea leaves (raw). The following preparations 1) to 4) were added thereto.
1) Cellulase preparation (TP2-Kyowa, Kyowa Kasei) 10 mg (60 units) alone 2) Cellulase preparation (TP2-Kyowa, Kyowa Kasei) 10 mg (60 units) cinnamic acid esterase (Kyowa Kasei) 2 mg 3) Cellulase preparation (TP2-Kyowa, manufactured by Kyowa Kasei Co., Ltd.) 10 mg (60 units) and tannase preparation (Kikkoman), 3 mg (0.3 units) combined 4) Cellulase preparation (TP2-Kyowa, Kyowa) 10 mg (60 units) of Kasei Co., Ltd. 2 mg (0.0075 units) of cinnamic acid esterase (manufactured by Kyowa Kasei Co., Ltd.) and 3 mg (0.3 units) of tannase preparation (Kikkoman Co., Ltd.) were used. The enzymatic reaction was carried out at 40 ° C. for 43 hours. After the reaction was completed, the reducing sugar produced in the decomposition solution was quantified. The results are shown below.
1) Cellulase preparation alone: 130mg
2) Cinnamate esterase preparation in combination with cellulase preparation: 168mg
3) Cellulase preparation combined with tannase preparation: 156mg
4) Cinnamate esterase and tannase preparation in combination with cellulase preparation: 220mg
From this result, it is understood that the cellulase reactivity is increased by using esterase in combination with the cellulase preparation. In particular, the amount of produced reducing sugar in the 4) section of Reference Example 4 in which three kinds of enzymes are used in combination is about 1.69 times that of the 1) section of the cellulase preparation alone treatment. In addition, in the section 4), the overall decomposition of the decomposition liquid progressed, and the filtration speed at the time of filter paper filtration was high, and a complete transparent liquid was obtained.

Reference Example 4
(6) Tea leaf decomposition-6
Three types of tea leaves (raw) (tea leaves-1, tea leaves-2, tea leaves-3) were used. 30 ml of pure water was added to 1 g of this tea leaf, and 10 mg of a mixed preparation containing 6 mg (36 units) of cellulase, 2 mg (0.0075 units) of cinnamic acid esterase and 2 mg of tannase (0.2 units) was added and reacted. As a control, 10 mg (60 units) of cellulase preparation (TP2-Kyowa) was used alone. The mixture was incubated at 40 ° C. for reaction, sampled after 24 hours, filtered through a membrane filter having a pore size of 0.2 μm, and the total amount of polyphenols in the filtrate was measured. The results are shown in Table 3.

In the mixed enzyme preparation use group used in Reference Example 4 of the present invention, the total amount of polyphenols greatly exceeds the control (single use group). Although the enzyme action varies depending on the type of tea leaf, the combined effect of the three types of enzymes was observed in each leaf.

Example 3
(7) Effect on taste Using the same three types of tea leaves (tea leaves-1, tea leaves-2, tea leaves-3) used in Example 2, free amino acids related to taste were examined. . 30 ml of pure water was added to 1 g of tea leaves, and 10 mg of the same mixed enzyme preparation as that used in Example 2 was added thereto and reacted. As a control, 10 mg of cellulase preparation (TP2-Kyowa Kyowa Kasei) was used alone. The reaction was carried out by incubating at 40 ° C., and the amounts of aspartic acid, glutamic acid and theanine were measured over time. As a result, the amino acid amount peaked at 2 hours in any of the test sections, and no difference in enzyme action was observed. This is probably because water-soluble, low-molecular free amino acids are relatively easily dissolved in water. Although there were differences in the elution amount of each amino acid depending on the type of tea leaf, the elution amount pattern over time was the same, and after 2 hours peaked, it remained flat.

Example 4
(8) Decomposition of rice straw 100 ml of water was added to 5 g of rice straw (ground or non-ground), 200 mg of the same mixed enzyme preparation used in Example 2 was added, and the mixture was reacted at 40 ° C. for 48 hours. . As a control, 200 mg of cellulase preparation (TP2-Kyowa Kyowa Kasei) was used alone. After completion of the reaction, the reducing sugar produced in the reaction solution was measured. The results are shown below.
result:
Reducing sugar (per rice straw dry matter)
Rice straw (grinding): The present invention (mixed enzyme preparation use zone) 35.2%
: Control (cellulase single use section) 28.9%
Reducing sugar (per rice straw dry matter)
Rice straw (unground): The present invention (mixed enzyme preparation use zone) 19.6%
: Control (cellulase preparation single use section) 15.5%
In both cases of crushed straw and non-pulverized straw, the decomposing ability of the mixed enzyme preparation of the present invention exceeded the control (single use section). The crushed straw showed good results when pulverized and unpulverized. In the case of cellulosic solid substrates, there is generally a correlation between particle size and degradation rate. The difference in decomposition rate between ground and unground rice straw is related to particle size. The lignin content of rice straw is 6-7%.

Example 5
(9) Decomposition of sweet potato vines (raw) 200ml of water was added to 60g of sweet potato vines (raw), and juiced using a mixer. The whole was made up to 300 ml with a small amount of water and divided into two equal parts. To this, 0.1% by mass of enzyme was added and the enzyme reaction was carried out at 40 ° C. for 24 hours. As the enzyme, the same mixed enzyme preparation as that used in Example 2 was used. As a control, a cellulase preparation (TP2-Kyowa Kyowa Kasei Co., Ltd.) was used alone. After completion of the reaction, it was naturally filtered with a filter paper. The produced reducing sugar was measured for this filtrate. The results are shown below.
The total amount of produced reducing sugar was 590 mg in the control (single use group), whereas it was 940 mg in the mixed enzyme preparation use group of the present invention. This is 1.6 times the control plot. Due to this influence, the viscosity of the enzyme-treated solution was different, and the viscosity was 32 cp (30 ° C.) in the control (single use group) and 11 cp (30 ° C.) in the group using the mixed enzyme preparation of the present invention. This was also reflected in the filterability, and the filtration rate of the mixed enzyme preparation use group of the present invention was three times that of the control group (single use group). By reducing the molecular weight of this polysaccharide, the filterability is improved, and the filtration process time is shortened.

Example 6
(10) Decomposition of corn core 30 ml of water is added to 1 g of corn core, and after autoclaving, cellulase preparation (TP2-Kyowa Kyowa Kasei), ferulic acid esterase (Kyowa Kasei), cinnamic acid esterase (Kyowa Kasei) Manufactured), pectinase preparation (manufactured by Shin Nippon Chemical Co., Ltd.), hemicellulase preparation (manufactured by Shin Nippon Chemical Co., Ltd.) 15 mg each was added as shown in Table 2. As test plots, eight test plots shown in Table 4 were prepared. After standing at 37 ° C. for 4 days, the glucose concentration in the sample was measured. The glucose concentration was measured using a glucose measurement kit (manufactured by Magazyme).

FIG. 1 shows the glucose concentration in the sample of each test section. Compared to test group 5 and test group 6 to which a pectinase preparation or hemicellulase preparation generally used as a plant tissue degrading enzyme was added, it exceeded that in test group 4 to which a ferulic acid esterase preparation was added. In addition, a three results enzyme combination with further glucose concentrations in test groups 8 a higher that need use in the present invention.

As described above, the present invention allows cellulase and ferulic acid esterase to act on plant tissues in any order, either sequentially or simultaneously. Therefore, in the method for solubilizing plant tissues using cellulase, the cellulase can be used without costly pretreatment. Plant tissue can be efficiently solubilized by increasing the degradability.
In addition, since cellulase and cinnamic acid esterase are allowed to act on plant tissues sequentially or simultaneously in any order, in the solubilization method of plant tissues using cellulase, the cellulase and the cinnamate esterase can be produced by increasing the degradability of cellulase without incurring pretreatment costs. Tissue can be solubilized efficiently.

In addition, cellulase, ferulic acid esterase, and tannase are allowed to act on plant tissues in any order, either sequentially or simultaneously. Therefore, in the method for solubilizing plant tissues using cellulase, the cellulase degradability can be reduced without incurring pretreatment costs. The plant tissue can be efficiently solubilized by increasing it.
In addition, cellulase, cinnamic acid esterase, and tannase are allowed to act on plant tissues in any order, either sequentially or simultaneously, so that the cellulase degradability can be reduced without incurring pretreatment costs in the method for solubilizing plant tissues using cellulase. The plant tissue can be efficiently solubilized by increasing it.

In addition, cellulase, ferulic acid esterase, and cinnamic acid esterase are allowed to act on plant tissues sequentially or simultaneously in any order. Therefore, cellulase degradation can be performed without costly pretreatment in the solubilization method of plant tissues using cellulase. Plant tissue can be efficiently solubilized by increasing the force.
Moreover, since 0.126 to 126 units of cellulase, 0.00006 to 0.06 unit of ferulic acid esterase, and 0.000075 to 0.075 unit of cinnamic acid esterase are allowed to act on 1 g of plant tissue, the degradability of cellulase can be increased efficiently.

In addition, it is a mixed enzyme preparation for solubilizing plant tissue by acting on plant tissue, and since it contains cellulase and ferulic acid esterase, by increasing the degradability of cellulase without costing pretreatment Plant tissue can be solubilized efficiently.
Moreover, it is a mixed enzyme preparation for acting on plant tissue to solubilize plant tissue, and contains cellulase and cinnamic acid esterase, so that by increasing the degradability of cellulase without costing pretreatment Plant tissue can be solubilized efficiently.

Moreover, it is a mixed enzyme preparation for acting on plant tissue to solubilize plant tissue, and it contains cellulase, ferulic acid esterase, and tannase, so that it can decompose cellulase without costing pretreatment. The plant tissue can be efficiently solubilized by increasing.
Moreover, it is a mixed enzyme preparation for acting on plant tissue to solubilize plant tissue, and contains cellulase, cinnamic acid esterase and tannase. The plant tissue can be efficiently solubilized by increasing.

Moreover, it is a mixed enzyme preparation for acting on plant tissue to solubilize plant tissue, and since it contains cellulase, ferulic acid esterase, and cinnamic acid esterase, cellulase Plant tissue can be efficiently solubilized by increasing the degradability.
In addition, since the blending ratio of cellulase, ferulic acid esterase, and cinnamic acid esterase is 100-30 parts by mass of cellulase, ferulic acid esterase is 20-30 parts by mass, and cinnamic acid esterase is 20-30 parts by mass, Cellulase degradability can be increased efficiently.
In addition, since the cellulase activity is 100 to 10,000 units, the ferulic acid esterase activity is 1 to 100 units, and the cinnamic acid esterase activity is 1.2 to 120 units, the degradability of cellulase can be increased efficiently.

Claims (2)

Cellulase, full Erura acid esterase, and a method of solubilizing a plant tissue to act sequentially or simultaneously plant tissue cinnamic acid esterase in any order,
A method for solubilizing plant tissue, which comprises subjecting cellulase to 0.126 to 126 units, ferulic acid esterase to 0.00006 to 0.06 unit, and cinnamic acid esterase to 0.000075 to 0.075 unit per 1 g of plant tissue . Is allowed to act on plant tissue comprising a mixed enzyme preparation to solubilize the plant tissue, cellulases, ferulic acid esterase, and a mixed enzyme preparation containing cinnamic acid esterase,
Cellulase, ferulic acid esterase and cinnamic acid esterase are blended in proportions of 100 parts by mass of cellulase, 20-30 parts by mass of ferulic acid esterase, and 20-30 parts by mass of cinnamic acid esterase,
And cellulase activity from 100 to 10,000 units, ferulic acid esterase activity 1-100 units and, mixed enzyme preparation cinnamic acid esterase activity, characterized in 120 units der Rukoto 1.2.

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