Enzyme agent for reducing carbohydrates in vegetable foods and drinks

Abstract

Provided is a technique for reducing carbohydrates in vegetable foods and drinks. The present technology provides: an enzyme agent for reducing saccharides in a vegetable food or beverage, said enzyme agent containing a cyclodextrin-producing enzyme; and a vegetable food or beverage using the enzyme agent for reducing saccharides. In addition, the present technology provides a method for producing a vegetable food or beverage, which includes a step for causing a cyclodextrin-generating enzyme to act in a step for liquefying a vegetable raw material, and a method for reducing saccharides in a vegetable food or beverage, which includes a step for causing a cyclodextrin-generating enzyme to act on a vegetable raw material.

Description

Technical Field

[0001] The present invention relates to an enzyme agent for reducing the sugar content in plant-based food and beverages. More specifically, the present invention relates to a technology for reducing the sugar content in plant-based food and beverages using a cyclodextrin-producing enzyme. Background Art

[0002] In recent years, due to the improvement of health intention, plant-based food and beverage products have attracted attention. For example, oat beverages are prepared by suspending oat materials in an aqueous medium and using one or more amylases to decompose the starch of the oat materials. The amylase used in this case is β-amylase or a mixture of α-amylase and β-amylase. The oat beverage prepared by this method generates maltose through the action of β-amylase, thereby presenting a suitable sweet taste (Patent Document 1).

[0003] Here, the enzyme used in the present technology is described. Cyclodextrin-producing enzyme is an enzyme that acts on starch to produce α-, β-, and γ-cyclodextrin. In addition, it is known that cyclodextrin-producing enzyme acts on starch to produce cyclodextrin without liquefying starch granules (Patent Document 2).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2018-075029

[0007] Patent Document 2: Japanese Patent Application Laid-Open No. 09-262091 Summary of the invention

[0008] As mentioned above, in recent years, due to the improvement of health intention, plant-based food and beverage products have received attention, for example, in plant-based food and beverage products such as oat drinks obtained by treating with β-amylase in addition to α-amylase using the technology shown in Patent Document 1, a large amount of sugars represented by glucose and maltose are contained. Therefore, the requirement for reducing sugars cannot be met.

[0009] Therefore, the main object of the present technology is to provide a technology for reducing sugars in plant-based food and beverages.

[0010] The inventors of the present application conducted intensive research on technologies for reducing sugars in plant-based foods and beverages, and as a result, found that sugars in plant-based foods and beverages can be reduced by allowing cyclodextrin-generating enzymes to act on plant-based raw materials in plant-based foods and beverages, thereby completing the present technology.

[0011] That is, in the present technology, first, an enzyme agent for reducing sugars in plant-based food and beverages is provided, the enzyme agent comprising a cyclodextrin-producing enzyme.

[0012] The enzyme agent for reducing sugars according to the present technique may contain maltotriose-producing enzyme.

[0013] The present technology also provides a plant-based food or drink using the sugar-reducing enzyme agent according to the present technology.

[0014] The plant-based food and beverage according to the present technology may also be oat milk.

[0015] Next, the present technology provides a method for producing a plant-based food and drink, the method comprising the step of allowing a cyclodextrin-producing enzyme to act in the step of liquefying a plant-based raw material.

[0016] The method for producing a plant-based food and drink according to the present technique may further include a step of allowing maltotriose-producing enzyme to act on a plant-based raw material.

[0017] The present technology further provides a method for reducing sugars in a plant-based food or drink, the method comprising the step of allowing a cyclodextrin-producing enzyme to act on a plant-based raw material.

[0018] The method for reducing the carbohydrate content of a plant-based food or drink according to the present technique may include a step of allowing maltotriose-producing enzyme to act on a plant-based raw material.

[0019] In the present technology, "sugar" is a general term for monosaccharides and disaccharides.

[0020] According to the present technology, it is possible to reduce the sugar content in plant-based food and beverages. DETAILED DESCRIPTION

[0021] Hereinafter, a preferred mode for carrying out the present invention will be described. In addition, the embodiment described below is an example of a representative embodiment of the present invention, and the scope of the present invention is not to be interpreted narrowly thereby.

[0022] 1. Enzymes for reducing sugars

[0023] The enzyme agent for reducing carbohydrates involved in the present technology contains a cyclodextrin-generating enzyme as an active ingredient. In addition, it is preferred that α-amylase is substantially not contained. "Substantially not containing α-amylase" means that α-amylase is allowed to be contained within a range that can achieve the purpose, that is, within a range that does not affect the production amount of carbohydrates.

[0024] In addition, as the content of the alpha-amylase that does not affect the production amount of carbohydrates, it can be freely set according to the kind of the plant-based food and drink products manufactured, the kind of the raw materials used, etc. Relative to the plant-based raw materials used in the plant-based food and drink products manufactured per 1g, it can be preferably set to below 0.01U, more preferably set to below 0.001U, and further preferably set to below 0.0001.

[0025] (1) Cyclodextrin-generating enzyme

[0026] The cyclodextrin-producing enzyme (EC 2.4.1.19) that can be used in the present technology is an enzyme that has the activity of producing cyclodextrins having a degree of polymerization of 6 to 8 using a polymer composed of α-1,4-linked glucose such as starch as a substrate. As the cyclodextrin-producing enzyme that can be used in the present technology, as long as it has the activity of producing cyclodextrin, it may be an enzyme that further has other functions.

[0027] In the present technology, by making cyclodextrin generating enzyme act on plant raw material, starch in plant raw material can be liquefied, and sugar (monosaccharide and disaccharide) in manufactured plant food and drink can be reduced. In addition, by making cyclodextrin generating enzyme act on plant raw material, cyclodextrin in manufactured plant food and drink can be utilized to cover peculiar smell.

[0028] The source of the cyclodextrin-producing enzyme that can be used in the present technology is not particularly limited, and examples thereof include enzymes from the genus Bacillus, such as Bacillus stearothermophilus, Bacillus megaterium, Bacillus circulans, Bacillus macerans, Bacillus ohbensis, and Bacillus clarkii; from the genus Geobacillus, such as Geobacillus stearothermophilus; from the genus Paenibacillus, such as Paenibacillus macerans; macerans); Klebsiella, such as Klebsiella pneumoniae; Thremoanaerobactor; Brevibacterium. These cyclodextrin-producing enzymes may be used alone or in combination. Among them, from the viewpoint of improving the flavor of the plant raw material, the cyclodextrin-producing enzyme is preferably a cyclodextrin glucanotransferase from Paenibacillus and / or a cyclodextrin glucanotransferase from Geobacillus, and more preferably a cyclodextrin glucanotransferase from Paenibacillus macerans and / or a cyclodextrin glucanotransferase from Anoxybacillus caldiproteolyticus (also including formerly known as Geobacillus stearothermophilus).

[0029] Here, the "cyclodextrin-producing enzyme derived from Paenibacillus macerans" and / or "cyclodextrin-producing enzyme from Geobacillus stearothermophilus" refers to a cyclodextrin-producing enzyme produced by a microorganism (which may be a wild strain or a mutant strain) classified as Paenibacillus macerans and / or Geobacillus stearothermophilus, or a cyclodextrin-producing enzyme obtained by a genetic engineering method using a cyclodextrin-producing enzyme gene. Therefore, a recombinant produced by a host microorganism into which a cyclodextrin-producing enzyme gene obtained from Paenibacillus macerans and / or Geobacillus stearothermophilus (or a gene modified from the gene) is introduced also corresponds to the "cyclodextrin-producing enzyme derived from Paenibacillus macerans" and / or "cyclodextrin-producing enzyme from Geobacillus stearothermophilus".

[0030] The cyclodextrin generating enzyme used in the present technology can be prepared from the culture solution of the microorganism that is the source of the above-mentioned cyclodextrin generating enzyme. As a specific preparation method, a method for recovering the cyclodextrin generating enzyme from the culture solution or bacterial cell of the above-mentioned microorganism can be cited. For example, in the case of using a cyclodextrin generating enzyme secretory microorganism, the bacterial cell can be recovered from the culture solution by filtering, centrifugation, etc. in advance as needed, and then the enzyme is separated and / or purified. In addition, in the case of using a cyclodextrin generating enzyme non-secreting microorganism, the bacterial cell can be recovered from the culture solution in advance as needed, and the enzyme can be exposed by crushing the bacterial cell by pressure treatment, ultrasonic treatment, etc., and then the enzyme is separated and / or purified. As the separation and / or purification method of the enzyme, the disclosed protein separation and / or purification method can be used without particular limitation, for example, centrifugal separation method, UF concentration method, salting-out method, various chromatography methods using ion exchange resins, etc. can be cited. The separated and / or purified enzyme can be powdered by drying methods such as freeze drying and reduced pressure drying. In addition, it can also be powdered using appropriate excipients and / or drying aids in the drying method. Alternatively, the separated and / or purified enzyme may be liquefied by adding appropriate additives and sterilizing by filtration.

[0031] In the present technology, commercially available products may be used as cyclodextrin-producing enzymes. Examples of preferred commercially available products include cyclodextrin glucanotransferase derived from Paenibacillus macerans manufactured by Amano Enzyme Co., Ltd. and cyclodextrin glucanotransferase derived from Geobacillus stearothermophilus manufactured by Amano Enzyme Co., Ltd.

[0032] As long as the effect of the present technology is not damaged, the content of the cyclodextrin generating enzyme in the enzyme agent for reducing sugars involved in the present technology can be freely set. The content of the cyclodextrin generating enzyme can be set to more than 0.01U relative to the plant raw material used in the plant food and drink manufactured by 1g, for example, from the viewpoint of further improving the effect of reducing sugars, it can be preferably set to more than 0.05U, more preferably set to more than 0.1U, further preferably set to more than 0.3U, and further preferably set to more than 0.5U.

[0033] In addition, the content of cyclodextrin generating enzyme can be set to, for example, 0.02U or more relative to 1g of polysaccharides (preferably starch) used in the manufactured plant-based food and beverage. From the viewpoint of further improving the sugar reducing effect, it can be preferably set to 0.08U or more, more preferably set to 0.2U or more, further preferably set to 0.5U or more, and further preferably set to 0.8U or more.

[0034] As long as the effect of the present technology is not impaired, the upper limit of the content of cyclodextrin generating enzyme is not particularly limited, and can be set to, for example, less than 300U, less than 200U, less than 100U, less than 50U, less than 40U or less than 30U relative to each 1g of plant raw materials used in the manufactured plant-based food and beverage.

[0035] The upper limit of the cyclodextrin generating enzyme content can be set to, for example, 500 U or less, 300 U or less, 150 U or less, 80 U or less, 60 U or less, or 40 U or less per 1 g of the polysaccharide (preferably starch) used in the produced plant-based food and drink.

[0036] In the present technology, the activity of the cyclodextrin-producing enzyme is a value measured by the measurement method described in the Examples described later.

[0037] (2) Others

[0038] As long as the sugar reduction enzyme agent involved in the present technology contains the aforementioned cyclodextrin generating enzyme, it can also be composed of the aforementioned cyclodextrin generating enzyme only, and as long as the effect of the present technology is not damaged, it is also possible to freely choose to contain one or more other components. As other components, for example, components such as excipients, pH adjusting agents, colorants, flavoring agents, disintegrants, lubricants, stabilizers commonly used in formulations can be used. Furthermore, it is also possible to use a disclosed or future discovered component with function according to an appropriate purpose.

[0039] (3) Maltotriose synthase

[0040] The enzyme agent for reducing carbohydrates involved in the present technology may also contain maltotriose generating enzyme. The maltotriose generating enzyme that can be used in the present technology is an enzyme that has the activity of acting on starch and mainly generating maltotriose. As the maltotriose generating enzyme that can be used in the present technology, as long as it has the activity of generating maltotriose, it may also be an enzyme that has other functions.

[0041] In the present technology, by allowing maltotriose-producing enzyme to act on the plant raw material treated with cyclodextrin-producing enzyme, trisaccharides can be increased while keeping the sugars (monosaccharides and disaccharides) in the produced plant food and drink at a low level, thereby imparting moderate sweetness.

[0042] The source of the maltotriose generating enzyme that can be used in the present technology is not particularly limited, and for example, maltotriose generating enzymes from organisms belonging to the genus Streptomyces, the genus Bacillus, the genus Microbacterium, and the genus Cellulosimicrobium can be cited. These maltotriose generating enzymes can be used alone or in combination. Among these maltotriose generating enzymes, maltotriose generating enzymes from organisms belonging to the genus Microbacterium or from organisms belonging to the genus Cellulosimicrobium can be preferably cited, and maltotriose generating enzymes from the genus Microbacterium can be more preferably cited. Specifically, enzymes that show substrate specificity for acting on amylose, amylopectin, glycogen, and starch can be used.

[0043] Here, "maltotriose-producing enzyme derived from Microbacterium" refers to maltotriose-producing enzyme produced by a microorganism classified as Microbacterium (which may be a wild strain or a mutant strain), or maltotriose-producing enzyme obtained by genetic engineering using a maltotriose-producing enzyme gene. Therefore, a recombinant produced by a host microorganism into which a maltotriose-producing enzyme gene obtained from Microbacterium (or a gene in which the gene is modified) is introduced also corresponds to "maltotriose-producing enzyme derived from Microbacterium".

[0044] The maltotriose generating enzyme used in the present technology can be prepared from the culture solution of the microorganism that is the source of the above-mentioned maltotriose generating enzyme. As a specific preparation method, a method of recovering maltotriose generating enzyme from the culture solution or bacterial cell of the above-mentioned microorganism can be cited. For example, in the case of using a maltotriose generating enzyme secreting microorganism, the enzyme can be separated and / or purified after the bacterial cell is recovered from the culture solution by filtering, centrifugation, etc. as needed. In addition, in the case of using a maltotriose generating enzyme non-secreting microorganism, the bacterial cell can be recovered from the culture solution in advance as needed, and then the enzyme is exposed by crushing the bacterial cell by pressure treatment, ultrasonic treatment, etc., and then the enzyme is separated and / or purified. As an enzyme separation and / or purification method, a disclosed protein separation and / or purification method can be used without particular limitation, for example, centrifugation, UF concentration method, salting-out method, various chromatography methods using ion exchange resins, etc. can be cited. The separated and / or purified enzyme can be powdered by a drying method such as freeze drying and reduced pressure drying. In addition, it can also be powdered using an appropriate excipient and / or drying aid in the drying method. Alternatively, the separated and / or purified enzyme may be liquefied by adding appropriate additives and sterilizing by filtration.

[0045] In the present technique, a commercially available product may be used as the maltotriose-producing enzyme, and a preferred example of the commercially available product is maltotriose-producing enzyme manufactured by Amano Enzyme Products Co., Ltd.

[0046] 2. Plant-based food

[0047] The plant-based food and drink involved in the present technology is a plant-based food and drink produced using the aforementioned sugar-reducing enzyme. As a specific example of a plant-based beverage, oat beverages, more specifically oat milk (also referred to as "oat milk"), can be cited. As a specific example of a plant-based food, plant-based yogurt can be cited.

[0048] The amount of sugars (monosaccharides and disaccharides) included in the plant-based food and drink products involved in the present technology is not particularly limited. For example, the amount of sugars included in the plant-based food and drink products is 5 mg or less, 4 mg or less, preferably 3 mg or less, more preferably 2 mg or less, and further preferably 1.5 mg or less relative to each 1g of the plant-based food and drink products.

[0049] In addition to reducing sugars (monosaccharides and disaccharides), the plant-based food and beverage involved in the present technology can also increase the amount of cyclodextrin. By increasing cyclodextrin, it is possible to mask odors. The amount of cyclodextrin contained in the plant-based food and beverage involved in the present technology is not particularly limited. The amount of cyclodextrin contained in the plant-based food and beverage, as the total amount of α-cyclodextrin, is, for example, 1 mg or more, preferably 2 mg or more, more preferably 3 mg or more, further preferably 4 mg or more, and further preferably 5 mg or more per 1g of the plant-based food and beverage.

[0050] In addition, the ratio of cyclodextrin to sugars (monosaccharides + disaccharides) (cyclodextrin / sugars (monosaccharides + disaccharides)) is not particularly limited, but in order to obtain both the sugar reduction effect and the masking effect, it is, for example, 3 or more, preferably 4 or more, and more preferably 5 or more.

[0051] In the case of implementing the maltotriose generating enzyme treatment process, the plant-based food and beverage involved in the present technology can also include increasing trisaccharides in addition to reducing sugars (monosaccharides and disaccharides). By increasing trisaccharides, sweetness can be imparted. The amount of trisaccharides contained in the plant-based food and beverage involved in the present technology is not particularly limited. The amount of trisaccharides contained in the plant-based food and beverage is, for example, more than 2 mg, preferably more than 4 mg, more preferably more than 6 mg, further preferably more than 8 mg, and further preferably more than 10 mg, relative to every 1g of the plant-based food and beverage.

[0052] 3. Method for producing plant-based food and beverages, method for reducing sugar content in plant-based food and beverages

[0053] The manufacturing method of the plant-based food and drink involved in the present technology is a method including a process of causing a cyclodextrin generating enzyme to act in the liquefaction process of the plant-based raw material (hereinafter also referred to as a "cyclodextrin generating enzyme action process"). The carbohydrate reduction method of the plant-based food and drink involved in the present technology is a method including a process of causing a cyclodextrin generating enzyme to act on the plant-based raw material (hereinafter also referred to as a "cyclodextrin generating enzyme action process"). In addition, depending on the type of the plant-based food and drink, within the scope of not damaging the effect of the present technology, a general food manufacturing process may also be performed before and after each process or simultaneously with each process. In the above-mentioned manufacturing method of the plant-based food and drink, the carbohydrate reduction method of the plant-based food and drink, a maltotriose generating enzyme action process and a recovery process may also be further implemented.

[0054] In the manufacturing method and the method for reducing sugars involved in the present technology, it is preferred not to perform a process (hereinafter also referred to as "α-amylase action process") in which α-amylase is allowed to act on a plant raw material for the purpose of liquefaction of starch. In addition, the α-amylase action process can be performed as long as it does not affect the amount of sugar produced, but when the α-amylase action process is performed, it is preferably performed before the cyclodextrin generating enzyme action process described later, or simultaneously with the cyclodextrin generating enzyme action process. As long as the effect of the present technology is not impaired, the amount of α-amylase added when the α-amylase action process is performed can be freely set. The specific amount of α-amylase added in the present technology is the same as the content when α-amylase is contained in the aforementioned enzyme agent for reducing sugars within a range that does not affect the amount of sugar produced, so the description is omitted here. Below, each process is described in detail.

[0055] (1) Plant-based raw materials

[0056] As long as the effect of the present technology is not damaged, the origin and type of the plant raw materials that can be used in the present technology are not particularly limited, and can be freely selected according to the target plant food and beverage. For example, beans such as soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupines, kidney beans, etc. can be cited; cereals such as wheat, barley, oats, rice, rye, buckwheat, barnyard grass, millet, and teff; nuts such as almonds, coconuts, peanuts, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, pili nuts, chestnuts, sesame seeds, and pine nuts; chia seeds, quinoa, amaranth, canary seeds, linseed, etc. In the present technology, they can be used alone or in combination of two or more. Among these raw materials, cereals can be preferably cited, and oats can be more preferably cited.

[0057] In the present technology, the properties of the plant raw material when subjected to various enzyme treatments are not particularly limited unless the effects of the present technology are impaired, and preferred examples include liquid, slurry, and paste.

[0058] The plant raw material that can be used in the present technology contains sugars. The content of the plant sugar contained in the plant raw material (based on the weight of the plant raw material in a dry state) is not particularly limited, and for example, 20% by weight or more can be cited. Preferably, 30% by weight or more can be cited, more preferably 40% by weight or more, and further preferably 50% by weight or more can be cited. The upper limit of the content range is not particularly limited, and for example, 90% by weight or less, 80% by weight or less, and 70% by weight or less can be cited.

[0059] In the present technology, the vegetable raw material when carrying out the cyclodextrin generating enzyme action step described later preferably uses the vegetable raw material that has not been treated with α-amylase. When the starch contained in the vegetable raw material is liquefied, α-amylase is not treated, and cyclodextrin generating enzyme is utilized to liquefy, thereby the sugars in the plant food and drink after liquefaction can be reduced.

[0060] In the present technology, the plant raw material used in the maltotriose generating enzyme action step described later is not particularly limited, and preferably a plant raw material treated with a cyclodextrin generating enzyme is used. It is more preferred to use a plant raw material in which the cyclodextrin generating enzyme is inactivated after being treated with a cyclodextrin generating enzyme. By treating with a maltotriose generating enzyme, the trisaccharides of the plant food and drink after the sugar content is reduced can be increased, thereby imparting sweetness.

[0061] (2) Cyclodextrin production enzyme action process

[0062] The cyclodextrin generating enzyme action step is a step of allowing the cyclodextrin generating enzyme to act on the plant raw material. As long as the effect of the present technology is not impaired, the amount of cyclodextrin generating enzyme added in the cyclodextrin generating enzyme action step can be freely set. The specific amount of cyclodextrin generating enzyme added in the present technology is the same as the content of cyclodextrin generating enzyme in the aforementioned carbohydrate reducing enzyme agent, so the description is omitted here.

[0063] As long as the effect of the present technology is not impaired, the various conditions of the cyclodextrin generating enzyme action process can be freely set. 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 cyclodextrin generating enzyme used. It can be that pH is, for example, set to pH5.0~8.0, preferably set to pH5.5~7.5, and more preferably set to pH5.5~7.0. The temperature can be, for example, set to 30°C~80°C, preferably set to 40°C~75°C, and more preferably set to 50°C~70°C. The action time can be, for example, set to 10 minutes~48 hours, preferably set to 30 minutes~24 hours, and more preferably set to 1 hour~12 hours. In addition, the best reaction conditions can be determined by preliminary experiments.

[0064] (3) Maltotriose production enzyme action process

[0065] The maltotriose generating enzyme action process is a process in which maltotriose generating enzyme acts on a plant material. As long as the effect of the present technology is not impaired, the amount of maltotriose generating enzyme added in the maltotriose generating enzyme action process can be freely set. From the viewpoint of further improving the sugar reduction effect and the sweetness enhancement effect, the specific amount of maltotriose generating enzyme added in the present technology can be preferably set to more than 0.5U, more preferably set to more than 1U, and further preferably set to more than 5U as the amount of plant material used per 1g of maltotriose generating enzyme. The upper limit of the range of the amount of plant material used as per 1g of maltotriose generating enzyme is not particularly limited, for example, it can be set to less than 1500U, less than 300U, less than 150U, less than 100U, less than 50U, less than 20U or less than 15U.

[0066] As long as the effect of the present technology is not impaired, various conditions of the maltotriose generating enzyme action process can be freely set. 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 maltotriose generating enzyme used. pH can be set to pH5.0-8.0, preferably pH5.5-7.5, and more preferably pH6.0-7.0. The temperature can be set to 30°C-80°C, preferably 40°C-75°C, and more preferably 45°C-65°C. The action time can be set to 10 minutes-12 hours, preferably 30 minutes-6 hours, and more preferably 1 hour-3 hours. In addition, the optimal reaction conditions can be determined by preliminary experiments.

[0067] (4) Recycling process

[0068] The recovery process is a process for recovering the plant food and drink that has passed the cyclodextrin generating enzyme action process. In the case of the aforementioned maltotriose generating enzyme action process, in the recovery process, the plant food and drink that has passed the cyclodextrin generating enzyme action process and the maltotriose generating enzyme action process are recovered. The specific recovery method can be used in the manufacture of general plant food and drink in one or a combination of two or more according to the type of the manufactured plant food and drink.

[0069] The plant-based food and drink that has undergone the cyclodextrin generating enzyme action step is characterized in that the amount of sugars (monosaccharides + disaccharides) contained is reduced. Specifically, for example, the content of sugars (monosaccharides + disaccharides) in the plant-based food and drink that has undergone the cyclodextrin generating enzyme action step is 5 mg or less per 1g of the plant-based food and drink. That is, in the present technology, a recovery step can be performed, and the recovery step recovers a plant-based food and drink with a content of sugars (monosaccharides + disaccharides) of 5 mg or less per 1g of the plant-based food and drink.

[0070] In addition, in the present technology, by performing the maltotriose generating enzyme action step, the trisaccharides of the plant food and drink with reduced sugars (monosaccharides + disaccharides) can be increased to impart sweetness. Specifically, for example, the content of trisaccharides in the plant food and drink that has undergone the maltotriose generating enzyme action step is 2 mg or more per 1 g of the plant food and drink. That is, in the present technology, a recovery step can be performed, and the recovery step recovers the plant food and drink with a content of trisaccharides of 2 mg or more per 1 g of the plant food and drink.

[0071] In summary, in the present technology, a recovery process can be performed to recover plant-based foods and beverages having a sugar (monosaccharide + disaccharide) content of less than 5 mg per 1 gram of plant-based foods and beverages and a trisaccharide content of more than 2 mg per 1 gram of plant-based foods and beverages.

[0072] One embodiment of the method for producing a plant-based food or beverage and the method for reducing sugar content of the present technology includes the following steps (1) and (2). In addition, an enzyme inactivation step may be added after step (2).

[0073] (1) Process of preparing raw materials containing plant sugars

[0074] (2) Process of treating the prepared raw materials with cyclodextrin-generating enzyme

[0075] One embodiment of the method for producing a plant-based food or beverage and the method for reducing sugar content of the present technology includes the following steps (1) to (3). In addition, an enzyme inactivation step may be added after step (2).

[0076] (1) Process of preparing raw materials containing plant sugars

[0077] (2) Process of treating the prepared raw material with cyclodextrin-generating enzyme instead of α-amylase treatment

[0078] (3) Process for recovering a plant-based food or beverage having a sugar (monosaccharide + disaccharide) content of 5 mg or less per 1 g of the plant-based food or beverage

[0079] In the step (3), a plant-based food or beverage having a trisaccharide content of 2 mg or more per 1 g of the plant-based food or beverage may be recovered.

[0080] One embodiment of the method for producing a plant-based food or drink and the method for reducing sugars of the present technology includes the following steps (1) to (3).

[0081] (1) Process of preparing raw materials containing plant sugars

[0082] (2) Process of treating the prepared raw materials with cyclodextrin-generating enzyme

[0083] (3) Process of treating the prepared raw materials with maltotriose generating enzyme

[0084] In addition, step (3) may be performed simultaneously with step (2) or after step (2). It is preferred that step (3) be performed after step (2), and it is more preferred that an enzyme inactivation step be added after step (2). That is, it is preferred that the following steps (1) to (4) are included.

[0085] (1) Process of preparing raw materials containing plant sugars

[0086] (2) Process of treating the prepared raw materials with cyclodextrin-generating enzyme

[0087] (3) A step of inactivating the cyclodextrin-producing enzyme in the raw material treated in step (2)

[0088] (4) A step of treating the raw material obtained in step (3) with maltotriose generating enzyme

[0089] Another embodiment of the method for producing a plant-based food and drink and the method for reducing sugar content of the present technology includes the following steps (1) and (2). In addition, an enzyme inactivation step may be added after step (1) and / or step (2).

[0090] (1) Step of preparing a raw material containing plant sugars treated with cyclodextrin-generating enzyme

[0091] (2) Process of treating the prepared raw materials with maltotriose generating enzyme

[0092] Example

[0093] Hereinafter, the present invention will be described in more detail based on examples. In addition, the examples described below are examples showing representative examples of the present invention, and the scope of the present invention is not to be construed narrowly thereby.

[0094] 1. Use of enzymes

[0095] Cyclodextrin-generating enzyme

[0096] CGT-1: Cyclodextrin glucanotransferase from Paenibacillus macerans, manufactured by Amano Enzyme Co., Ltd.

[0097] CGT-2: Cyclodextrin glucanotransferase from Geobacillus stearothermophilus, manufactured by Amano Enzyme Co., Ltd.

[0098] Maltotriose synthase

[0099] Maltotriose-producing enzyme from Microbacterium sp. manufactured by Amano Enzyme Co., Ltd.

[0100] 2. Enzyme activity assay

[0101] [Method for measuring the activity of cyclodextrin-generating enzyme]

[0102] 1g of potato starch was dissolved in 100mL of distilled water (adjusted to pH 5.5 with dilute acetic acid) to make it completely gelatinized. 10mL of the gelatinized solution was measured, and 1mL of the enzyme solution was added and mixed. The mixture was incubated at 40°C for exactly 10 minutes, and the reaction was stopped with 0.1N hydrochloric acid. 10mL of iodine-potassium iodide test solution (0.4mmol / L) was added to 1mL of the reaction solution and mixed to prepare a test solution. In addition, the iodine-potassium iodide test solution was prepared by dissolving 10.0g of potassium iodide and 1.0g of iodine in water to make 100mL, and then diluted 200 times with water. Next, the absorption wavelength of 660nm of the mixed solution was measured. As a control, a mixed solution of potato starch that was not treated with enzymes was prepared in the same manner, and the absorption wavelength of 660nm was measured. Then, the amount of enzyme that reduced the blue iodine color of starch by 1% within 1 minute was taken as 1 unit (1U).

[0103] [Method for measuring maltotriose-producing enzyme activity]

[0104] The enzyme is allowed to act on soluble starch as a substrate, and the resulting reducing sugar is quantitatively measured by the Somogyi-Nelson method. 0.5 mL of soluble starch solution and 0.4 mL of 0.1 mol / L acetic acid-sodium acetate buffer (pH 6.0) (containing 0.05 mol / L CaCl2) are measured in a 50 mL Nessler colorimetric tube, and the mixture is thoroughly shaken and mixed. After being placed at 40±0.5°C for 10 to 15 minutes, 0.1 mL of the sample solution is added and immediately shaken and mixed. After the solution is accurately placed at 40±0.5°C for 15 minutes, 1 mL of alkaline copper test solution is added, shaken and mixed, and the stopper is covered. The solution is accurately heated in a boiling water bath for 20 minutes, and immediately cooled. After cooling, 1 mL of Nelson's solution is added, and after being shaken thoroughly until the red precipitate of cuprous oxide is completely dissolved, the solution is placed at room temperature for 20 minutes, and 22 mL of water is added and shaken thoroughly. For this solution, the absorbance at a wavelength of 520 nm is measured with water as a reference. One unit is defined as the time when reducing sugar equivalent to 1 μmol of glucose is generated in 1 minute.

[0105] 3. Experimental Examples

[0106] <Experimental Example 1>

[0107] In Experimental Example 1, the sugar-reducing effect of cyclodextrin-producing enzyme was investigated.

[0108] (1) Experimental methods

[0109] 100 mL of water was added to 12.5 g of oat flour (PREMIUM OAT FLOUR, sugar content 67.7 g / 100 g, manufactured by SLOW FOOD KITCHEN) and suspended, and cyclodextrin generating enzyme was added thereto in an amount of 0 to 30 U relative to the raw material. After adding the enzyme, the enzyme was allowed to act for 2 to 24 hours while stirring in a 60°C water bath, and after the starch contained in the oat flour was liquefied, the enzyme was inactivated by treating at 95°C for 10 minutes. 190 μL of 1 mol / L trichloroacetic acid was added to 1.5 mL of the enzyme-inactivated oat solution, and the solution was allowed to stand at room temperature for 10 minutes. After 10 minutes, centrifugation was performed (12,000 rpm, 10 minutes), and the supernatant was recovered. An equal amount of diethyl ether was added to the recovered supernatant and suspended. Centrifugation was performed (12,000 rpm, 10 minutes), the organic solvent layer was discarded, and the water layer was recovered. This operation was repeated twice. An equal amount of 99.5% ethanol (for high performance liquid chromatography, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the recovered water layer and stirred, and the substance filtered with a membrane filter was provided for analysis. For the quantification of α-cyclodextrin, after the cyclodextrin generating enzyme treatment, 1% glucoamylase was added and allowed to act at 60°C for 2 hours to prepare a sample that had undergone the same treatment. The analysis was performed using high performance liquid chromatography, and the columns used were MCL GEL CK04S manufactured by Mitsubishi Chemical Corporation and TSK gel Amide-80 manufactured by Tosoh Corporation. The amount of each component generated was quantified by the peak area. As a comparative example, α-amylase was used instead of the cyclodextrin generating enzyme, and the same operation was performed.

[0110] (2) Results

[0111] The results are shown in Tables 1 and 2 below.

[0112] [Table 1]

[0113]

[0114] α-CD: α-cyclodextrin

[0115] [Table 2]

[0116]

[0117] α-CD: α-cyclodextrin

[0118] (3) Investigation

[0119] As shown in Tables 1 and 2, the sugar content of the oat milk of Comparative Example 1 treated with α-amylase was 5.4 mg / mL. On the other hand, as shown in Examples 1 to 30, it was confirmed that the amount of sugars could be reduced to 1.2 mg / mL or less by using a cyclodextrin-producing enzyme.

[0120] <Experimental Example 2>

[0121] In Experimental Example 2, the sugar-reducing effect and viscosity-reducing effect of the cyclodextrin-producing enzyme were investigated.

[0122] (1) Experimental methods

[0123] 100 mL of water was added to 12.5 g of oat flour (PREMIUM OAT FLOUR, sugar content 67.7 g / 100 g, manufactured by SLOW FOOD KITCHEN) and suspended, and cyclodextrin generating enzyme was added thereto in an amount of 0 to 18 U relative to the raw material. After adding the enzyme, the enzyme was allowed to act for 2 or 4 hours while stirring in a 60°C water bath to liquefy the starch contained in the oat flour. The enzyme was inactivated by treating at 95°C for 10 minutes. 190 μL of 1 mol / L trichloroacetic acid was added to 1.5 mL of the enzyme-inactivated oat solution and allowed to stand at room temperature for 10 minutes. After 10 minutes, centrifuge (12,000 rpm, 10 minutes) and recover the supernatant. An equal amount of diethyl ether was added to the recovered supernatant and suspended. Centrifuge (12,000 rpm, 10 minutes), discard the organic solvent layer, and recover the water layer. Repeat this operation twice. An equal amount of 99.5% ethanol (for high performance liquid chromatography, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the recovered water layer and stirred, and the substance filtered with a membrane filter was provided for analysis. The analysis used high performance liquid chromatography, and the columns used were MCL GEL CK04S manufactured by Mitsubishi Chemical Corporation and TSK gel Amide-80 manufactured by Tosoh Corporation. The production of glucose and maltose was quantified by peak area. In addition, the viscosity of the oatmeal solution treated with enzyme inactivation was measured. 50 mL of the oatmeal solution treated with enzyme inactivation was placed in a tube, heated to 60°C using a viscometer (TVB-15 viscometer, rotor No. 4 or No. 2, Toki Sangyo Co., Ltd.), and the viscosity was measured. As a comparative example, α-amylase was used instead of cyclodextrin generating enzyme, and the same operation was performed. As a reference example, the same operation was performed without adding enzyme.

[0124] (2) Results

[0125] The results are shown in Table 3 below.

[0126] [Table 3]

[0127]

[0128] (3) Investigation

[0129] As shown in Table 3, the viscosity of the oat milk of Comparative Examples 2 and 3 treated with α-amylase was significantly reduced, while the sugar content was 6 to 7 mg / mL. On the other hand, as shown in Examples 31 to 42, by using a cyclodextrin-forming enzyme, it was confirmed that the amount of sugar could be reduced to 1 mg / mL or less while reducing the viscosity to a certain extent.

[0130] <Experimental Example 3>

[0131] The amount of sugars (monosaccharides + disaccharides) in the enzyme-treated oat milk obtained above was significantly reduced, and as a result, the sweetness was also reduced. Therefore, with the purpose of reducing the amount of sugars (monosaccharides + disaccharides) and imparting sweetness, the sugar reduction effect and trisaccharide production effect brought about by the combined use of cyclodextrin-generating enzyme and maltotriose-generating enzyme were investigated.

[0132] (1) Experimental methods

[0133] 100 mL of water was added to 12.5 g of oat flour (PREMIUM OAT FLOUR, sugar content 67.7 g / 100 g, manufactured by SLOW FOOD KITCHEN) and suspended. Cyclodextrin-generating enzyme (CGT-1: 6 U / g-raw material, CGT-2: 4 U / g-raw material) and maltotriose-generating enzyme (12 U / g-raw material, 36 U / g-raw material, 60 U / g-raw material) were added to the raw materials. After adding the enzyme, the enzyme was allowed to act for 2 hours while stirring in a 60°C water bath, and then treated at 95°C for 10 minutes to inactivate the enzyme. 190 μL of 1 mol / L trichloroacetic acid was added to 1.5 mL of the enzyme-inactivated oat solution, and the solution was allowed to stand at room temperature for 10 minutes. After 10 minutes, the solution was centrifuged (12,000 rpm, 10 minutes) and the supernatant was recovered. An equal amount of diethyl ether was added to the recovered supernatant and suspended. Centrifugal separation (12,000 rpm, 10 minutes), discard the organic solvent layer, and recover the water layer. Repeat this operation twice. Add an equal amount of 99.5% ethanol (for high performance liquid chromatography, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to the recovered water layer and stir, and the material filtered with a membrane filter is provided for analysis. The analysis uses high performance liquid chromatography, and the columns used are MCL GEL CK04S manufactured by Mitsubishi Chemical Corporation and TSK gel Amide-80 manufactured by Tosoh Corporation. The amount of each component generated is quantified by the peak area.

[0134] (2) Results

[0135] The results are shown in Table 4 below.

[0136] [Table 4]

[0137]

[0138] (3) Investigation

[0139] As shown in Table 4, the production of trisaccharides was confirmed in the oat milk obtained by using a cyclodextrin-producing enzyme and a maltotriose-producing enzyme in combination. Therefore, it was found that the cyclodextrin-producing enzyme and the maltotriose-producing enzyme in combination could reduce sugars (monosaccharides + disaccharides) and increase trisaccharides compared with Comparative Example 1 of Experimental Example 1.

[0140] <Experimental Example 4>

[0141] In Experimental Example 4, the sugar-reducing effect and trisaccharide-generating effect were investigated in the case where the cyclodextrin-generating enzyme treatment was followed by the maltotriose-generating enzyme treatment.

[0142] (1) Experimental methods

[0143] 100 mL of water was added to 12.5 g of oat flour (PREMIUM OAT FLOUR, sugar content 67.7 g / 100 g, manufactured by SLOW FOOD KITCHEN) and suspended, and cyclodextrin-generating enzyme (CGT-1: 6 U / g-raw material, CGT-2: 4 U / g-raw material) was added to the raw material. After adding the enzyme, the enzyme was allowed to act for 2 hours while stirring in a 60°C water bath. After cooling to 50°C, maltotriose-generating enzyme (1.2 U / g-raw material, 6 U / g-raw material, 12 U / g-raw material, 36 U / g-raw material) was added. After adding the enzyme, the enzyme was allowed to act for 2 hours while stirring in a 50°C water bath, and then treated at 95°C for 10 minutes to inactivate the enzyme. 190 μL of 1 mol / L trichloroacetic acid was added to 1.5 mL of the oat solution that had been inactivated, and the mixture was allowed to stand at room temperature for 10 minutes. After 10 minutes, centrifuge (12,000 rpm, 10 minutes) and recover the supernatant. Add an equal amount of diethyl ether to the recovered supernatant and suspend it. Centrifuge (12,000 rpm, 10 minutes), discard the organic solvent layer, and recover the water layer. Repeat this operation twice. Add an equal amount of 99.5% ethanol (for high performance liquid chromatography, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to the recovered water layer and stir, and the substance filtered with a membrane filter is provided for analysis. The analysis uses high performance liquid chromatography, and the columns used are MCL GEL CK04S manufactured by Mitsubishi Chemical Corporation and TSK gel Amide-80 manufactured by Tosoh Corporation. The amount of each component generated is quantified by the peak area.

[0144] (2) Results

[0145] The results are shown in Table 5 below.

[0146] [Table 5]

[0147]

[0148] (3) Investigation

[0149] As shown in Table 5, it was found that when maltotriose-producing enzyme was allowed to act on the cyclodextrin-producing enzyme-treated cells, the amount of trisaccharide produced increased compared to when the two enzymes acted simultaneously.

[0150] <Experimental Example 5>

[0151] In Experimental Example 5, the sugar-reducing effect and trisaccharide-generating effect were investigated in the case where the cyclodextrin-generating enzyme was inactivated and then treated with maltotriose-generating enzyme.

[0152] (1) Experimental methods

[0153] 100 mL of water was added to 12.5 g of oat flour (PREMIUM OAT FLOUR, sugar content 67.7 g / 100 g, manufactured by SLOW FOOD KITCHEN) and suspended, and cyclodextrin-generating enzyme (CGT-1: 6 U / g-raw material, CGT-2: 4 U / g-raw material) was added to the raw material. After adding the enzyme, the enzyme was allowed to act for 2 hours while stirring in a 60°C water bath, and then treated at 95°C for 10 minutes to inactivate the enzyme. After cooling to 50°C, maltotriose-generating enzyme (6 U / g-raw material, 12 U / g-raw material) was added. After adding the enzyme, the enzyme was allowed to act for 2 hours while stirring in a 50°C water bath, and then treated at 95°C for 10 minutes to inactivate the enzyme. 190 μL of 1 mol / L trichloroacetic acid was added to 1.5 mL of the oat solution that had been inactivated, and the mixture was allowed to stand at room temperature for 10 minutes. After 10 minutes, centrifuge (12,000 rpm, 10 minutes) and recover the supernatant. Add an equal amount of diethyl ether to the recovered supernatant and suspend it. Centrifuge (12,000 rpm, 10 minutes), discard the organic solvent layer, and recover the water layer. Repeat this operation twice. Add an equal amount of 99.5% ethanol (for high performance liquid chromatography, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to the recovered water layer and stir, and the substance filtered with a membrane filter is provided for analysis. The analysis uses high performance liquid chromatography, and the columns used are MCL GEL CK04S manufactured by Mitsubishi Chemical Corporation and TSK gel Amide-80 manufactured by Tosoh Corporation. The amount of each component generated is quantified by the peak area.

[0154] (2) Results

[0155] The results are shown in Table 6 below.

[0156] [Table 6]

[0157]

[0158] (3) Investigation

[0159] As shown in Table 6, by inactivating the cyclodextrin-generating enzyme and then treating the maltotriose-generating enzyme, it was possible to achieve both an increase in the production of trisaccharides and a decrease in the production of sugars (monosaccharides + disaccharides). Furthermore, the production of α-cyclodextrin was also confirmed. From this result, it can be inferred that the oat milk obtained this time has a sweet taste and a reduced grain flavor despite the small amount of sugars (monosaccharides + disaccharides).

Claims

1. An enzyme agent for reducing carbohydrates in a plant-based food or beverage, comprising a cyclodextrin-producing enzyme. 2 . The enzyme agent for reducing sugars in a plant-based food or beverage according to claim 1 , comprising maltotriose-producing enzyme.

3. A plant-based food or beverage comprising the enzyme agent for reducing sugar content according to claim 1 or 2.

4. The plant-based food and drink according to claim 3, wherein A plant-based dietary product is oat milk.

5. A method for producing a plant-based food or beverage, comprising the step of allowing a cyclodextrin-producing enzyme to act during a liquefaction step of a plant-based raw material. 6 . The method for producing a plant-based food and drink according to claim 5 , comprising the step of allowing maltotriose-producing enzyme to act on the plant-based raw material.

7. A method for reducing the sugar content of a plant-based food or beverage, comprising the step of allowing a cyclodextrin-producing enzyme to act on a plant-based raw material. 8 . The method for reducing sugars in a plant-based food and drink according to claim 7 , comprising the step of allowing maltotriose-producing enzyme to act on the plant-based raw material.

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