Enzyme agent

Abstract

Provided is a technique capable of increasing the amount of water-soluble dietary fibers in a plant-based liquid composition. In the present technology, provided is an enzyme agent containing two or more cellulase enzymes from different sources. The enzyme agent involved in the technology contains exo-cellulase and endo-cellulase. The enzyme agent according to the present technology can be used as an agent for increasing the amount of water-soluble dietary fibers in a dietary fiber-containing liquid processing composition. The enzyme agent according to the present technology can be used as a residue-reducing agent in a liquid processing composition containing dietary fibers.

Description

Technical Field

[0001] The present technology relates to an enzyme agent. More specifically, the present technology relates to an enzyme agent containing two or more cellulases from different sources, an agent for increasing the amount of water-soluble dietary fiber in a liquid processing composition containing dietary fiber, and an agent for reducing residue in the liquid processing composition containing dietary fiber, as well as dietary fiber-containing processed food and beverages using the enzyme agent, a method for producing the liquid processing composition containing dietary fiber, a method for increasing the amount of water-soluble dietary fiber in the liquid processing composition containing dietary fiber, and a method for reducing residue in the liquid processing composition containing dietary fiber. Background Art

[0002] Beverages rich in nutrients like protein have long been popular because they are easy to consume. Meanwhile, beverages made with plant-based proteins like soy, oats, and almonds have become increasingly popular as alternatives to animal milk, driven by the growing number of vegetarians, allergies, and religious concerns. There is a growing demand for liquid compositions containing processed plant proteins to achieve physiological benefits not achieved with cow's milk. For example, the market for oat milk has been expanding in recent years due to its high content of beta-glucan, a water-soluble dietary fiber.

[0003] For example, Patent Document 1 discloses a method for producing oat milk with high nutritional value, stability, and excellent aroma and taste by treating oat flour with α-amylase and cellulase. Patent Document 2 discloses a method for obtaining cellooligosaccharides in high yield by treating natural cellulose (natural water-insoluble fiber containing cellulose) with cellulase. Patent Document 3 discloses a dough composition with improved texture by adding cellooligosaccharides to plant-derived powder.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: CN107821600A

[0007] Patent Document 2: International Publication No. 2006 / 011479

[0008] Patent Document 3: Japanese Patent Application Laid-Open No. 2009-095252 Summary of the Invention

[0009] Plant-based processed food and beverages are highly useful from the perspective of nutritional value and storage stability, and are expected to be utilized in various applications. On the other hand, due to the inclusion of a filtration step when preparing plant-based liquid compositions such as oat milk, residues containing insoluble dietary fiber are produced. There is no sufficient research on reducing this residue and increasing the amount of water-soluble dietary fiber.

[0010] Therefore, the present technology aims to provide a technology capable of increasing the amount of water-soluble dietary fiber in a plant-based liquid composition.

[0011] The inventors of the present application conducted intensive research and found that the amount of water-soluble dietary fiber in a plant-based liquid composition can be significantly increased by allowing two or more cellulases from different sources to act on a plant material. The present technology was completed through further research based on this finding.

[0012] That is, in the present technology, first, an enzyme agent is provided, which contains two or more cellulases from different sources.

[0013] The enzyme agent involved in the present technology can contain exo-type cellulase and endo-type cellulase.

[0014] As the exo-cellulase that can be used in the enzyme agent according to the present technique, cellulase derived from a microorganism belonging to the genus Trichoderma can be used.

[0015] As the endo-cellulase used in the enzyme agent according to the present technique, cellulase derived from a microorganism belonging to the genus Aspergillus can be used.

[0016] The enzyme agent according to the present technology can be used as an agent for increasing the amount of water-soluble dietary fiber in a liquid processing composition containing dietary fiber.

[0017] The increasing agent of the present technology may contain exo-cellulase and endo-cellulase. In this case, the activity ratio of the exo-cellulase to the endo-cellulase may be exo-cellulase activity:endo-cellulase activity = 1:99 to 99:1.

[0018] The enzyme agent according to the present technology can be used as a residue reducing agent in a liquid processing composition containing dietary fiber.

[0019] The residue reducing agent of the present technology may contain exo-cellulase and endo-cellulase. In this case, the activity ratio of the exo-cellulase to the endo-cellulase may be exo-cellulase activity:endo-cellulase activity = 1:99 to 99:1.

[0020] The enzyme agent according to the present technology can be used for the production of processed foods and drinks containing dietary fiber.

[0021] Next, the present technology provides a method for producing a dietary fiber-containing liquid processing composition, which includes a cellulase treatment step of treating the dietary fiber-containing composition with two or more cellulases from different sources.

[0022] Cereals can be used as the dietary fiber-containing composition used in the production method according to the present technique.

[0023] The two or more cellulases from different sources used in the production method according to the present technology may contain exo-type cellulase and endo-type cellulase.

[0024] As the exo-cellulase that can be used in the production method according to the present technology, cellulase derived from a microorganism belonging to the genus Trichoderma can be used.

[0025] As the endo-cellulase that can be used in the production method according to the present technology, cellulase derived from a microorganism belonging to the genus Aspergillus can be used.

[0026] In the cellulase treatment step of the production method according to the present technology, the exo-cellulase and the endo-cellulase can be allowed to act at an activity ratio of exo-cellulase activity:endo-cellulase activity = 1:99 to 99:1.

[0027] In the present technology, a method for increasing the amount of water-soluble dietary fiber in a liquid processing composition containing dietary fiber and a method for reducing residue in a liquid processing composition containing dietary fiber are further provided, which include a cellulase treatment step in which the liquid processing composition containing dietary fiber is treated with two or more cellulases from different sources.

[0028] According to the present technology, the amount of water-soluble dietary fiber in a plant-based liquid composition can be increased. DETAILED DESCRIPTION

[0029] Hereinafter, preferred modes for implementing the present technology will be described. In addition, the embodiment described below shows an example of a representative embodiment of the present technology, but the scope of the present technology should not be narrowly interpreted thereby.

[0030] 1. Enzymes

[0031] The enzyme agent involved in the present technology contains two or more cellulases from different sources. In the present technology, as demonstrated in the examples described below, by treating a dietary fiber-containing composition with two or more cellulases from different sources, the amount of water-soluble dietary fiber in the produced liquid processed dietary fiber-containing composition is synergistically increased to an amount greater than that anticipated by those skilled in the art, and the amount of residue is synergistically reduced to an amount greater than that anticipated by those skilled in the art.

[0032] Dietary fiber refers to high-molecular-weight substances such as polysaccharides and polysaccharide derivatives, and is an indigestible component of food that cannot be digested by human digestive enzymes.

[0033] Water-soluble dietary fiber refers to substances that dissolve easily in water-soluble solvents but are not easily digested by human digestive enzymes. Specific examples include indigestible dextrin, inulin, polydextrose, indigestible glucan, isomaltodextrin, guar gum hydrolysate, and cellooligosaccharides. Cellooligosaccharides are oligosaccharides with a structure consisting of one to six β-1,4-linked glucopyranose units. This refers to cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose. In recent years, like other oligosaccharides, the physiological functions of cellooligosaccharides have become increasingly clear, and they hold promise as new ingredients for functional foods.

[0034] Cellulase is an enzyme that hydrolyzes the glycosidic bonds of β-1,4-glucan. Cellulase that can be used in the present technology may also have other functions as long as it has cellulase activity.

[0035] Cellulases are classified into exo-type, which breaks down cellulose chains sequentially into cellobiose units from the non-reducing end, and endo-type, which randomly cuts cellulose chains from the inside. In this technology, both exo-type and endo-type cellulases can be used, and it is preferred to use both exo-type and endo-type cellulases.

[0036] In the present technology, as an exo-cellulase, an enzyme that acts as an endo-cellulase may also be used as long as the exo-cellulase activity is the main activity. Similarly, in the present technology, as an endo-cellulase, an enzyme that acts as an exo-cellulase may also be used as long as the endo-cellulase activity is the main activity.

[0037] The origin of the cellulase that can be used in the present technology is not particularly limited as long as the function and effect of the present technology are not impaired, and examples thereof include cellulase derived from microorganisms of the genus Trichoderma and cellulase derived from microorganisms of the genus Aspergillus.

[0038] Cellulases derived from microorganisms of the genus Trichoderma include a large number of exo-type cellulases (American Chemical Society, June 1, 1979, p. 237-260). Therefore, in the present technology, cellulases derived from microorganisms of the genus Trichoderma can be preferably used as exo-type cellulases.

[0039] Cellulases derived from microorganisms of the genus Aspergillus include a large number of endo-type cellulases (Acta Cryst. (2002). D58, 660-667). Therefore, in the present technology, cellulases derived from microorganisms of the genus Aspergillus can be preferably used as endo-type cellulases.

[0040] Examples of microorganisms belonging to the genus Trichoderma include Trichoderma reesei and Trichoderma viride. In the present technology, cellulase derived from Trichoderma viride is preferably used.

[0041] Examples of microorganisms belonging to the genus Aspergillus include Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus saitoi, Aspergillus awamori, and Aspergillus flavus. In the present technology, cellulase derived from Aspergillus niger is preferably used.

[0042] In the present technology, the present invention is not limited to natural (wild-type) cellulases, and recombinant cellulases may also be used. Commercially available cellulases or cellulase preparations may also be used. Examples of commercially available cellulases or cellulase preparations include cellulases derived from Trichoderma viride, manufactured by Amano Enzyme Products Co., Ltd., as cellulases derived from microorganisms of the genus Trichoderma, and cellulases derived from Aspergillus niger, manufactured by Amano Enzyme Products Co., Ltd., as cellulases derived from microorganisms of the genus Aspergillus.

[0043] The cellulase used in the present technology can be prepared from the culture broth of the microorganisms that serve as the source of the above-mentioned cellulase. As a specific preparation method, a method of recovering the cellulase from the culture broth or bacterial cells of the above-mentioned microorganisms can be cited. For example, when using a cellulase-secreting microorganism, the bacterial cells can be recovered from the culture broth by prefiltration, centrifugation, etc. as needed, and the enzyme can be separated and / or purified. In addition, when using a non-cellulase-secreting microorganism, the bacterial cells can be recovered from the culture broth in advance as needed, and then the bacterial cells can be crushed by pressure treatment, ultrasonic treatment, etc. to extract the enzyme, and the enzyme can be separated and / or purified. As the enzyme separation and / or purification method, it is possible to use a publicly available protein separation and / or purification method without particular limitation, and examples include: centrifugation, UF concentration method, salting-out method, various chromatography methods using ion exchange resins, etc. The separated and / or purified enzyme can be powdered by a drying method such as freeze drying or reduced pressure drying. In addition, the drying method can also use an appropriate excipient and / or drying aid to powderize it. Alternatively, the separated and / or purified enzyme may be liquefied by adding appropriate additives and sterilizing by filtration.

[0044] When an exo-cellulase and an endo-cellulase are used in combination, the mixing ratio is not particularly limited and can be appropriately set according to the source of the cellulase used, the type of raw materials, etc. The activity ratio of the exo-cellulase to the endo-cellulase can be set, for example, to exo-cellulase activity:endo-cellulase activity = 1:99 to 99:1, preferably 2:98 to 90:10, 3:97 to 20:80, 4:96 to 30:70, 5:95 to 40:60, 10:90 to 40:60, 20:80 to 40:60, and more preferably 30:70 to 40:60.

[0045] The amount of enzyme that increases the cellulase activity by an amount equivalent to the reducing power of 1 μmole of glucose in 1 minute is defined as 1 unit (1 U). In this technology, the cellulase activity is the value measured by the cellulase activity assay described in the Examples below.

[0046] When using exo-cellulase, its coordination amount is not particularly limited and can be suitably set according to the source of the cellulase used and the kind of raw material etc. As the lower limit of the coordination amount of the exo-cellulase that becomes the dietary fiber of enzyme treatment object per 1g, can be set to, for example, more than 0.1U, preferably set to more than 0.3U, more than 0.6U, more than 1U, more than 1.5U, more than 2U, more preferably set to more than 3U, more than 6U, further preferably set to more than 9U.

[0047] The upper limit of the amount of exo-cellulase incorporated can be set to, for example, 200 U or less, 100 U or less, 50 U or less, or 30 U or less, preferably 20 U or less, or 15 U or less, more preferably 13 U or less, and even more preferably 11 U or less.

[0048] When using endo-cellulase, its coordination amount is not particularly limited and can be suitably set according to the source of the cellulase used and the kind of raw material etc. As the lower limit of the coordination amount of endo-cellulase that becomes the dietary fiber of enzyme treatment object, 1g can be set to, for example, more than 1U, preferably more than 2U, more than 3U, more than 5U, more preferably more than 10U, further preferably more than 15U.

[0049] The upper limit of the amount of endo-cellulase incorporated can be set, for example, to 500 U or less, 300 U or less, or 200 U or less, preferably to 150 U or less, or 120 U or less, more preferably to 100 U or less, or 80 U or less, and still more preferably to 60 U or less, or 40 U or less, or 20 U or less.

[0050] In the present technology, two or more cellulases from different sources may be used in combination with other enzymes and other components as long as the effects and effects of the present technology are not impaired. As other enzymes, for example, α-amylase may be mentioned. As other components, for example, excipients, pH regulators, colorants, flavorings, disintegrants, lubricants, stabilizers, etc. used in conventional formulations may be used. Furthermore, components with functions disclosed or discovered in the future may be appropriately used in combination according to the purpose.

[0051] 2. Agents for increasing the amount of water-soluble dietary fiber and reducing residues in liquid processing compositions containing dietary fiber

[0052] The enzyme agent according to the present technology can be preferably used as an agent for increasing the amount of water-soluble dietary fiber in a liquid processing composition containing dietary fiber and as an agent for reducing residues in a liquid processing composition containing dietary fiber.

[0053] There are no particular limitations on the dietary fiber-containing compositions that are intended to increase the amount of water-soluble dietary fiber and the dietary fiber-containing compositions that are intended to reduce residue, as long as they do not impair the effects and benefits of the present technology. Examples of dietary fiber-containing compositions that can be used in the present invention include liquid compositions, powdered compositions, slurry compositions, and texturized compositions. Specific examples of liquid compositions containing dietary fiber include: (i) a liquid obtained by dispersing a dry powder of a food material containing dietary fiber in water; (ii) a liquid obtained by crushing and dispersing a food material containing dietary fiber in water and, if necessary, removing unnecessary substances by centrifugation, filtration, a filter bag, a sieve, or any other method; (iii) a liquid obtained by increasing the dietary fiber content by removing components other than dietary fiber from the liquids of (i) or (ii); and (iv) a liquid obtained by mixing a dried powder prepared from any of the liquids (i) to (iii) with water. Preferred examples of liquid compositions containing dietary fiber include plant-based milk.

[0054] Examples of food materials containing dietary fiber include legumes such as soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupines, and kidney beans; cereals such as wheat, barley, oats, sorghum, rice, rye, buckwheat, millet, sorghum, teff, corn, and potatoes; nuts such as almonds, coconuts, peanuts, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, pili nuts, chestnuts, sesame seeds, and pine nuts; and seeds such as chia seeds, quinoa, amaranth seeds, canary seeds, and flax seeds.

[0055] As the water-soluble dietary fiber amount in the composition containing vegetable dietary fiber, it is not particularly limited, and its lower limit is for example more than 0.01 weight %, more than 0.05 weight %, more than 0.1 weight %, preferably more than 0.25 weight %, more than 0.5 weight %, more preferably more than 1.0 weight %, more than 1.2 weight %.As the upper limit of the dietary fiber content in the liquid composition containing vegetable dietary fiber, for example, below 50 weight %, below 40 weight %, below 35 weight %, preferably below 30 weight %, below 25 weight %, more preferably below 20 weight %, below 15 weight %.

[0056] By treating the dietary fiber-containing composition exemplified above with the enzyme agent according to the present technology, the amount of water-soluble dietary fiber in the liquid composition containing plant dietary fiber can be increased or the residue can be reduced.

[0057] In addition, since the details of the enzyme agent used for the water-soluble dietary fiber increasing agent and the residue reducing agent according to the present technology are as described above, the description thereof is omitted here.

[0058] 3. Processed foods and drinks containing dietary fiber

[0059] The enzyme agent involved in the present technology, the agent increasing the amount of water-soluble dietary fiber or the residue reducing agent can be preferably used in the manufacture of processed food and beverages containing dietary fiber. The processed food and beverages containing dietary fiber manufactured using the enzyme agent involved in the present technology, the agent increasing the amount of water-soluble dietary fiber or the residue reducing agent are characterized in that, compared with processed food and beverages containing dietary fiber manufactured without using these agents, the residue is less and the amount of water-soluble dietary fiber is more.

[0060] The water-soluble dietary fiber amount in the processed diet product containing dietary fiber is not particularly limited according to the raw material and the kind of diet product, as its lower limit, for example, be more than 0.01 weight %, more than 0.05 weight %, more than 0.1 weight %, preferably more than 0.25 weight %, more than 0.5 weight %, more preferably more than 1.0 weight %.In addition, as the upper limit of the water-soluble dietary fiber amount in the processed diet product containing dietary fiber, for example, be below 50 weight %, below 40 weight %, below 35 weight %, preferably below 30 weight %, below 25 weight %, more preferably below 20 weight %, below 15 weight %.

[0061] Specific examples of the dietary fiber-containing processed food and beverage according to the present technique include plant-based milks such as oat milk (also called “Oat milk”) and rice milk, vegetable juice, fruit juice, and plant-based yogurt.

[0062] 4. Method for producing a liquid processing composition containing dietary fiber

[0063] The method for producing a liquid processed composition containing dietary fiber involved in the present technology is a method including a cellulase treatment step. In addition, in the present technology, depending on the physical properties of the target liquid processed composition containing dietary fiber, for example, a liquefaction step, a recovery step, etc. may also be performed. Below, each step is described in detail.

[0064] (1) Cellulase treatment process

[0065] The cellulase treatment step is a step in which a dietary fiber-containing composition (which may also be a liquid processed dietary fiber-containing composition) is treated with two or more cellulases from different sources. Details of the cellulases and dietary fiber-containing composition used are as described above, and therefore, description thereof is omitted here.

[0066] Then the various conditions in the cellulase treatment process can be freely set as long as they do not damage the effect of the present technology. For example, pH, temperature, action time, etc. can be set according to the physicochemical properties such as the optimal pH, stable pH range, optimal temperature, and temperature stability of the cellulase used. pH can be set to, for example, pH4.0 to 9.5, pH5.0 to 8.5, preferably set to pH5.5 to 7.5, and more preferably set to pH6.0 to 7.0. Temperature can be set to, for example, 10°C to 80°C, preferably set to 25°C to 75°C, 35°C to 70°C, and more preferably set to 55°C to 65°C. Action time can be set to, for example, 5 minutes to 48 hours, preferably set to 30 minutes to 24 hours, 1 to 12 hours, 2 to 8 hours, and more preferably set to 3 hours to 5 hours. In addition, the optimal reaction conditions can be determined by preliminary experiments.

[0067] (2) Liquefaction process

[0068] The liquefaction step is a step for liquefying the dietary fiber-containing composition. While this step is not essential in the present technology, it may be appropriately performed if the dietary fiber-containing composition used as a raw material contains ingredients required for liquefaction, such as starch. The liquefaction method used in the liquefaction step is also not particularly limited; one or a combination of two or more common liquefaction methods may be used. Examples include methods using a liquefaction enzyme such as α-amylase.

[0069] The order of the cellulase treatment step and the liquefaction step is not particularly limited. The liquefaction step can be performed before or after the cellulase treatment step, or the cellulase treatment step and the liquefaction step can be performed simultaneously. In the present technology, the cellulase treatment step is preferably performed after the liquefaction step.

[0070] (3) Recycling process

[0071] The recovery step is a step of recovering the dietary fiber-containing liquid processed composition produced through the cellulase treatment step and, if necessary, the liquefaction step. The specific recovery method can be used by using one or a combination of two or more recovery methods commonly used in the production of dietary fiber-containing liquid processed compositions, depending on the type of dietary fiber-containing liquid processed composition produced.

[0072] The dietary fiber-containing liquid processed composition manufactured through the cellulase treatment step is characterized in that, compared to a dietary fiber-containing liquid processed composition manufactured without the cellulase treatment step, the residue is reduced and the amount of water-soluble dietary fiber is increased. That is, in the recovery step of the present technology, a dietary fiber-containing liquid processed composition with reduced residue and / or a dietary fiber-containing liquid processed composition with increased water-soluble dietary fiber can be recovered.

[0073] One embodiment of the method for producing a liquid processed composition containing dietary fiber according to the present technology includes the following steps (1) and (2). In addition, an enzyme inactivation step may be added after step (2).

[0074] (1) Step of preparing a composition containing dietary fiber

[0075] (2) A step of treating the prepared dietary fiber-containing composition with two or more cellulases from different sources

[0076] One embodiment of the method for producing a liquid processed composition containing dietary fiber according to the present technology includes the following steps (1) to (4). In addition, an enzyme inactivation step may be added after step (2) and / or (3).

[0077] (1) Step of preparing a composition containing dietary fiber

[0078] (2) A step of treating the prepared dietary fiber-containing composition with a liquefying enzyme such as α-amylase

[0079] (3) A step of treating the dietary fiber-containing composition treated with liquefying enzyme with two or more cellulases from different sources

[0080] (4) Step of recovering the liquid processing composition containing dietary fiber

[0081] One embodiment of the method for producing a plant-based food or beverage or a plant-based food or beverage raw material of the present technology includes the following steps (1) to (3). In addition, an enzyme inactivation step may be added after step (2).

[0082] (1) Step of preparing a composition containing dietary fiber

[0083] (2) A step of adding the prepared dietary fiber-containing composition to a liquefying enzyme such as α-amylase and treating it with two or more cellulases from different sources

[0084] (3) Step of recovering the liquid processing composition containing dietary fiber

[0085] The enzymes used in step (2) may be treated simultaneously or separately. The order of treatment in the case of separate treatments is not particularly limited. Furthermore, in the case of separate treatments, an enzyme inactivation step may be added between each enzyme treatment as needed.

[0086] 5. Method for increasing the amount of water-soluble dietary fiber in a dietary fiber-containing liquid processing composition and reducing the residue

[0087] The method for increasing the amount of water-soluble dietary fiber in the liquid processed composition containing dietary fiber involved in the present technology and the method for reducing residue in the liquid processed composition containing dietary fiber are methods including a cellulase treatment step. In addition, in the present technology, depending on the physical properties of the target liquid processed composition containing dietary fiber, for example, a liquefaction step, a recovery step, etc. can also be performed. In addition, the details of each step are as described above, so the description is omitted here.

[0088] In this technology, the following configurations can be adopted.

[0089] (1) An enzyme agent comprising two or more cellulases from different sources.

[0090] (2) The enzyme agent according to (1), comprising exo-cellulase and endo-cellulase.

[0091] (3) The enzyme agent according to (2), wherein the exo-cellulase is a cellulase derived from a microorganism belonging to the genus Trichoderma.

[0092] (4) The enzyme agent according to (2) or (3), wherein the endo-cellulase is a cellulase derived from a microorganism belonging to the genus Aspergillus.

[0093] (5) An agent for increasing the amount of water-soluble dietary fiber in a liquid processing composition containing dietary fiber, comprising the enzyme agent according to any one of (1) to (4).

[0094] (6) The increasing agent according to (5), comprising exo-cellulase and endo-cellulase,

[0095] The activity ratio of the exo-cellulase to the endo-cellulase is exo-cellulase activity:endo-cellulase activity=1:99-99:1.

[0096] (7) A residue reducing agent in a liquid processing composition containing dietary fiber, comprising the enzyme agent according to any one of (1) to (4).

[0097] (8) The residue reducing agent according to (7), comprising exo-cellulase and endo-cellulase,

[0098] The activity ratio of the exo-cellulase to the endo-cellulase is exo-cellulase activity:endo-cellulase activity=1:99-99:1.

[0099] (9) A processed food or beverage containing dietary fiber, comprising the enzyme agent according to any one of (1) to (4).

[0100] (10) A method for producing a liquid processed composition containing dietary fiber, comprising a cellulase treatment step of treating the dietary fiber-containing composition with two or more cellulases of different origins.

[0101] (11) The method for producing a dietary fiber-containing liquid processing composition according to (10), wherein the dietary fiber-containing composition is a cereal.

[0102] (12) The method for producing a dietary fiber-containing liquid processed composition according to (10) or (11), wherein the two or more cellulases from different sources include exo-type cellulase and endo-type cellulase.

[0103] (13) The method for producing a dietary fiber-containing liquid processing composition according to (12), wherein the exo-cellulase is a cellulase derived from a microorganism of the genus Trichoderma.

[0104] (14) The method for producing a dietary fiber-containing liquid processing composition according to (12) or (13), wherein the endo-cellulase is a cellulase derived from a microorganism belonging to the genus Aspergillus.

[0105] (15) A method for producing a liquid processing composition containing dietary fiber according to any one of (12) to (14), wherein, in the cellulase treatment step, the exo-cellulase and the endo-cellulase are allowed to act according to an activity ratio of exo-cellulase activity: endo-cellulase activity = 1:99 to 99:1.

[0106] (16) A method for increasing the amount of water-soluble dietary fiber in a liquid processing composition containing dietary fiber, comprising a cellulase treatment step of treating the liquid processing composition containing dietary fiber with two or more cellulases of different origins.

[0107] (17) A method for reducing residue in a liquid processing composition containing dietary fiber, comprising a cellulase treatment step of treating the liquid processing composition containing dietary fiber with two or more cellulases from different sources.

[0108] Example

[0109] The present invention will be described in more detail below based on examples. The examples described below are representative examples of the present invention, but the scope of the present invention should not be construed as being limited thereby.

[0110] 1. Raw materials

[0111] The enzymes and plant-based raw materials used in the Examples are shown in Table 1 below.

[0112] [Table 1]

[0113]

[0114] 2. Enzyme Activity Assay

[0115] [Method for measuring cellulase activity]

[0116] The cellulase activity can be measured by a method based on the cellulase activity test method in the 9th edition of the Food Additive Standards.

[0117] Pre-weigh approximately 1 g of sodium carboxymethyl ether cellulose (also known as sodium carboxymethyl cellulose) (degree of etherification 0.62-0.68) and dry it at 105°C for 4 hours. Measure the weight loss. Accurately weigh 0.625 g of the dried sodium carboxymethyl ether cellulose and place it in a 100 mL Erlenmeyer flask. Add 50 mL of water and heat to dissolve. After cooling, add 10 mL of 1 mol / L acetic acid / sodium acetate buffer (pH 4.5) and water to make a total of 100 mL of this solution, which serves as the substrate solution.

[0118] Accurately weigh 4 mL of substrate solution and place it into a 50 mL Nessler colorimetric tube. After incubating at 37 ± 0.5°C for at least 10 minutes, accurately weigh and add 1 mL of sample solution and immediately shake to mix. This solution should be incubated at 37 ± 0.5°C for 30 minutes. Accurately add 2 mL of alkaline copper solution and shake to mix. The Nessler colorimetric tube should be stoppered and heated in a water bath for 30 minutes. After cooling, accurately add 2 mL of Nelson's solution and shake thoroughly. Then, accurately add 3 mL of 0.5 mol / L sodium hydroxide solution and shake to dissolve the precipitate. After incubating for 20 minutes, accurately add 13 mL of 1 mol / L acetic acid / sodium acetate buffer (pH 4.5). Accurately weigh 1 mL of this solution and add 9 mL of 1 mol / L acetic acid / sodium acetate buffer (pH 4.5) and shake thoroughly to mix. Measure the absorbance (A1) of this solution at 750 nm against water.

[0119] Separately, accurately weigh 1 mL of the sample solution and place it in a 50 mL Nessler colorimetric tube. Accurately add 2 mL of alkaline copper test solution and shake to mix. Next, accurately weigh and add 4 mL of the substrate solution and shake to mix. Repeat the same procedure to measure the absorbance (A2).

[0120] Under these conditions, the amount of enzyme that increases the reducing power of 1 μmole of glucose in 1 minute is defined as 1 unit, and the cellulase activity per 1 g or 1 mL of sample (U / g, U / mL) is calculated using the following formula.

[0121] Cellulase activity (U / g, U / mL) = G × (1 / 30) × (1 / 0.180) × n

[0122] G: The amount of glucose generated (mg) calculated from the glucose standard curve based on the (A1-A2 / a) value

[0123] 1 / 30: Conversion factor to 1 minute

[0124] 1 / 0.180: 1 μmole of glucose = 0.180 mg

[0125] a: Slope of the glucose standard curve

[0126] n: dilution factor per 1g or 1mL of sample

[0127] [α-Amylase activity determination method]

[0128] After heating 10 mL of a 1% potato starch substrate solution (0.1 mol / L acetic acid, pH 5.0) at 37°C for 10 minutes, 1 mL of a sample solution containing α-amylase was added and immediately shaken to mix. After standing at 37°C for 10 minutes, 1 mL of this solution was added to 10 mL of a 0.1 mol / L hydrochloric acid test solution and immediately shaken to mix. Next, 0.5 mL of this solution was weighed, and 10 mL of a 0.0002 mol / L iodine test solution (Japanese) was added. After shaking to mix, the absorbance (AT) at a wavelength of 660 nm was measured using water as a control. Furthermore, 1 mL of water was added in place of the sample solution, and the absorbance (AB) was measured in the same manner. Under these conditions, 1 unit (1 U) is defined as the amount of enzyme that reduces the iodine-induced color of potato starch by 10% within 1 minute. The α-amylase activity was calculated using the following formula.

[0129] α-amylase activity (U / g, U / mL) = {(AB-AT) / AB} × 1 / W

[0130] AT: absorbance of the reaction solution

[0131] AB: absorbance of blank solution

[0132] W: The amount of sample in 1 mL of sample solution (g or mL)

[0133] 3. Experimental Examples

[0134] <Experimental Example 1>

[0135] In Experimental Example 1, the effects of differences in the enzymes used on the amount of residue in a liquid processed composition containing dietary fiber were investigated. In Experimental Example 1, oat flour was used as an example of a dietary fiber-containing composition, and processed oat milk was produced as an example of a liquid processed composition containing dietary fiber. Furthermore, the content of cellooligosaccharides, an example of water-soluble dietary fiber, was quantified.

[0136] (1) Manufacturing of processed oat milk

[0137] 18.1g of oat flour and 150g of water were mixed and suspended in a 300mL Erlenmeyer flask. 0.01% α-amylase was added to the suspension and reacted at 60°C for 0.5 hours. Subsequently, enzymes were added according to the combinations shown in Table 2 below and reacted at 60°C for 2 or 4 hours. After the reaction, the mixture was heated at 95°C for 5 minutes to inactivate the enzymes, thereby producing processed oat milk.

[0138] (2) Measurement and evaluation

[0139] [Residue reduction rate]

[0140] The processed oat milk was centrifuged (9,000×g, 5 minutes), the supernatant was removed, and the resulting residue was dried at 80° C. for 6 hours to obtain a dried residue.

[0141] The amount of dry residue obtained was measured as actual measurement data, and the ratio to the residue amount of the target example was calculated. The residue reduction rate was calculated using the following calculation formula.

[0142] Residue reduction rate = 100 - {(residue amount of each comparative example or each embodiment / residue amount of the control example) × 100}

[0143] As theoretical value data, the theoretical value of the residue reduction rate was determined based on the drying residue rates of Comparative Examples 1 and 2 and the enzyme mixing ratios of the respective Examples.

[0144] [Quantitative determination of water-soluble dietary fiber]

[0145] To processed oat milk produced after a 4-hour cellulase treatment, saccharifying enzyme was added to a final concentration of 1.0 mg / mL, and the reaction was allowed to proceed at 50°C for 2 hours. The resulting reaction solution was heated at 95°C for 5 minutes to inactivate the enzyme. Following inactivation, 190 μL of 1 mol / L trichloroacetic acid was added to 1.5 mL of the inactivated oat milk solution, and the solution was allowed to stand at room temperature for 10 minutes. After 10 minutes, the solution was centrifuged (12,000 rpm for 10 minutes), and the supernatant was recovered. An equal amount of diethyl ether was added to the recovered supernatant and the solution was suspended. The solution was centrifuged (12,000 rpm for 10 minutes), the organic solvent layer was discarded, and the aqueous layer was recovered. This procedure was repeated twice. An equal amount of 99.5% ethanol (for HPLC, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the recovered aqueous layer, stirred, and filtered through a membrane filter for analysis. Analysis was performed using high-performance liquid chromatography (HPLC) using a Mitsubishi Chemical MCL GEL CK04S column. The total amount of each cellooligosaccharide (C2: cellobiose, C3: cellotriose, C4: cellotetraose, C5: cellopentaose, C6: cellohexaose) produced was calculated based on the peak area and used as the sum of the peak areas to evaluate the amount of water-soluble dietary fiber produced.

[0146] (3) Results

[0147] The results are shown in Table 2.

[0148] [Table 2]

[0149]

[0150] The numerical values ​​in parentheses for Cellulase 1 represent relative values ​​of the added activities of Examples 1 to 3, with the added activity of Comparative Example 2 being 100.

[0151] The numerical values ​​in parentheses for Cellulase 2 represent relative values ​​of the added activities of Examples 1 to 3, with the added activity of Comparative Example 1 being 100.

[0152] The addition amount of the cellulase 2 in Comparative Example 1 and the cellulase 1 in Comparative Example 2 adopts the addition amount that makes the generation amount of water-soluble dietary fiber consistent.In addition, because the generation amount of the water-soluble dietary fiber of Comparative Examples 1 and 2 is identical, the theoretical value of the generation amount of the water-soluble dietary fiber in each embodiment is also the value identical with Comparative Examples 1 and 2.

[0153] (4) Investigation

[0154] As shown in Table 2, the measured values ​​of the residue reduction rates of Examples 1 to 3 all exceeded the theoretical values.

[0155] These results indicate that the use of two or more cellulases from different sources synergistically reduces the residue in the dietary fiber-containing liquid processing composition to an amount greater than that anticipated by those skilled in the art.

[0156] In addition, the measured values ​​of the generation amount of the water-soluble dietary fiber of embodiment 1~3 also all exceed theoretical value.Show from these results, by using two or more cellulases different from source, the water-soluble dietary fiber amount in the liquid processed composition containing dietary fiber increases synergistically to more than the amount that those skilled in the art can expect.

Claims

An enzyme agent comprising two or more cellulases from different sources.

2. The enzyme agent according to claim 1, wherein Contains exo-cellulase and endo-cellulase.

3. The enzyme agent according to claim 2, wherein The exo-cellulase is a cellulase derived from a microorganism of the genus Trichoderma.

4. The enzyme agent according to claim 2, wherein The endo-cellulase is a cellulase derived from a microorganism of the genus Aspergillus. 5 . An agent for increasing the amount of water-soluble dietary fiber in a dietary fiber-containing liquid processing composition, comprising the enzyme agent according to claim 1 .

6. The increasing agent according to claim 5, wherein Contains exo-cellulase and endo-cellulase, The activity ratio of the exo-cellulase to the endo-cellulase is exo-cellulase activity:endo-cellulase activity=1:99-99:

1. 7 . A residue reducing agent for a liquid processing composition containing dietary fiber, comprising the enzyme agent according to claim 1 .

8. The residue reducing agent according to claim 7, wherein Contains exo-cellulase and endo-cellulase, The activity ratio of the exo-cellulase to the endo-cellulase is exo-cellulase activity:endo-cellulase activity=1:99-99:

1. 9 . A processed food or beverage containing dietary fiber, comprising the enzyme agent according to claim 1 .

10. A method for producing a liquid processed composition containing dietary fiber, comprising a cellulase treatment step of treating the dietary fiber-containing composition with two or more cellulases from different sources.

11. The method for producing a dietary fiber-containing liquid processing composition according to claim 10, wherein The dietary fiber-containing composition is cereals.

12. The method for producing a dietary fiber-containing liquid processing composition according to claim 10, wherein The two or more cellulases from different sources contain exo-cellulase and endo-cellulase.

13. The method for producing a dietary fiber-containing liquid processing composition according to claim 12, wherein: The exo-cellulase is a cellulase derived from a microorganism of the genus Trichoderma.

14. The method for producing a dietary fiber-containing liquid processing composition according to claim 12, wherein The endo-cellulase is a cellulase derived from a microorganism of the genus Aspergillus.

15. The method for producing a dietary fiber-containing liquid processing composition according to claim 12, wherein In the cellulase treatment step, the exo-cellulase and the endo-cellulase are allowed to act at an activity ratio of exo-cellulase activity:endo-cellulase activity = 1:99 to 99:

1.

16. A method for increasing the amount of water-soluble dietary fiber in a liquid processed composition containing dietary fiber, comprising a cellulase treatment step of treating the liquid processed composition containing dietary fiber with two or more cellulases from different sources.

17. A method for reducing residue in a liquid processing composition containing dietary fiber, comprising a cellulase treatment step of treating the liquid processing composition containing dietary fiber with two or more cellulases from different sources.

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