Carbohydrate-reducing agent for carbohydrate-containing beverages and foods

Abstract

Provided is a technique for reducing saccharides in a saccharide-containing food or drink. In the present technique, saccharides in a saccharide-containing food or drink are reduced by allowing glucose oxidase, α-glucosidase, and / or β-D-fructofuranosidase to act on the saccharide-containing food or drink or a raw material of the saccharide-containing food or drink, simultaneously or in different orders. In the present technique, monosaccharides and disaccharides, particularly glucose and sucrose, in a saccharide-containing food or drink can be reduced.

Description

Technical Field

[0001] This technology relates to a sugar-reducing agent for sugary foods and beverages. More specifically, this invention relates to a sugar-reducing agent capable of reducing monosaccharides and disaccharides in sugary foods and beverages, a method for manufacturing the foods and beverages, and a method for reducing the sugar content of sugary foods and beverages. Background Technology

[0002] In recent years, increased health awareness has led to a surge in demand for low-sugar beverages and reduced-sugar foods. Fruit and vegetable juices, in particular, are highly nutritious and popular, but their sugar content is a concern. Specifically, excessive sugar intake can lead to health risks, increased calorie intake, adverse effects on dental health, a sharp rise in blood sugar levels, and disruption of the nutritional balance of beverages. Against this backdrop, the development of technologies to reduce the sugar content in sugary drinks and beverages is booming.

[0003] For example, Patent Document 1 discloses the following technology: a citrus juice beverage is prepared by mixing a citrus juice source with a low Brix value (low sugar content) with additional settled pulp (or bottom pulp) and a sweetener that improves the perceived sweetness of the juice beverage without significantly increasing the calorie content of the beverage, thereby producing a sugar-reduced citrus juice beverage.

[0004] Furthermore, Patent Document 2 discloses a technique for reducing the sugar content of food materials by contacting them with a glucosyltransferase having a specific sequence. Patent Document 2 describes a method that can reduce the total content of monosaccharides and disaccharides in food materials by 25%.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Publication No. 2007-504821

[0008] Patent Document 2: International Publication No. 2016 / 173929 Summary of the Invention

[0009] As mentioned above, in recent years, due to increased health awareness, low-sugar beverages and reduced-sugar foods have received attention. However, the actual situation is that there is a desire to further develop technologies to reduce the sugar content in sugary drinks and foods.

[0010] Therefore, the main objective of this technology is to provide a technique for reducing the sugar content in sugary foods and beverages.

[0011] The inventors of this application have conducted in-depth research on the technology of reducing sugar content in sugary foods and beverages, and as a result, discovered a new technology for reducing sugar content in sugary foods and beverages using two or more specific enzymes, thus completing this technology.

[0012] That is, in this technology, firstly, a sugar-reducing agent for sugary foods is provided, which contains:

[0013] glucose oxidase; and

[0014] α-glucosidase and / or β-D-fructofuranosidase.

[0015] The sugar-reducing agent involved in this technology can reduce glucose and sucrose in sugary foods and beverages.

[0016] The sugar-reducing agent involved in this technology can reduce the sugar content in sucrose-containing beverages.

[0017] The sugar-reducing agent involved in this technology can reduce the sugar content in beverages made from fruits and / or vegetables.

[0018] In this technology, a food product is then provided that uses the sugar-reducing agent involved in this technology.

[0019] Furthermore, this technology provides a method for manufacturing a food product, comprising:

[0020] A process in which glucose oxidase acts on a sugary food or its raw material; and

[0021] The process of acting α-glucosidase and / or β-D-fructofuranosidase on sugary food products or raw materials of such sugary food products, and

[0022] A method for reducing the sugar content of sugary foods and beverages is provided, comprising:

[0023] A process in which glucose oxidase acts on a sugary food or its raw material; and

[0024] A process of applying α-glucosidase and / or β-D-fructofuranosidase to a sugary food or the raw material of such a food.

[0025] In this technology, "carbohydrates" refers to the collective term for monosaccharides and disaccharides (excluding sugar alcohols) as defined in the nutritional guidelines (Ministry of Health, Labour and Welfare Notice No. 176). Monosaccharides are sugars that do not undergo further breakdown and include glucose, fructose, and galactose. Disaccharides include sucrose (glucose + fructose), maltose (glucose + glucose), and lactose (glucose + galactose), etc. Detailed Implementation

[0026] The preferred embodiments for carrying out the present invention will be described below. Furthermore, the embodiments described below illustrate an example of a representative embodiment of the present invention, but the scope of the present invention should not be interpreted narrowly as a result.

[0027] 1. Carbohydrate-lowering agents

[0028] The sugar-reducing agent involved in this technology contains glucose oxidase, α-glucosidase, and / or β-D-fructofuranosidase as active ingredients. While glucose oxidase, α-glucosidase, and / or β-D-fructofuranosidase, even when used alone, are effective in reducing sugars in sugary foods and drinks, the sugar-reducing effect can be significantly enhanced by using them in combination, as shown in the examples described later. In particular, the sugar-reducing agent involved in this technology can reduce both glucose and sucrose in sugary foods and drinks.

[0029] The reduction rate of sugars (monosaccharides and disaccharides, hereinafter the same) in food and beverages manufactured using the sugar-reducing agent involved in this technology is not particularly limited, but is, for example, 30% or more, preferably 35% or more, more preferably 40% or more, and even more preferably 43% or more, relative to the sugars in food and beverages manufactured without the sugar-reducing agent involved in this technology.

[0030] The reduction rate of glucose and sucrose in food and beverage products manufactured using the sugar-reducing agent of this technology is not particularly limited. Compared to food and beverage products manufactured without the sugar-reducing agent of this technology, the reduction rate is, for example, 30% or more, preferably 35% or more, more preferably 40% or more, and even more preferably 43% or more. While the individual reduction rates are not particularly limited, the reduction rate of glucose in food and beverage products manufactured using the sugar-reducing agent of this technology, compared to glucose in food and beverage products manufactured without the sugar-reducing agent of this technology, is, for example, 10% or more, 20% or more, preferably 30% or more, and more preferably 40% or more. Furthermore, the reduction rate of sucrose in food and beverage products manufactured using the sugar-reducing agent of this technology, compared to sucrose in food and beverage products manufactured without the sugar-reducing agent of this technology, is, for example, 40% or more, preferably 50% or more, and more preferably 60% or more.

[0031] The following provides a detailed description of the ingredients that can be used in the sugar-reducing agents involved in this technology.

[0032] (1) Glucose oxidase

[0033] The glucose oxidase (EC1.1.3.4) that can be used in this technology is an enzyme that oxidizes glucose to produce gluconic acid and hydrogen peroxide. In addition, as long as the glucose oxidase that can be used in this technology has glucose oxidase activity, it can also be an enzyme that has other functions.

[0034] The source of glucose oxidase that can be used in this technology is not particularly limited, and examples include glucose oxidases from filamentous fungi (e.g., *Acremonium chrysogenum*, *Aspergillus aculeatus*, *Aspergillus niger*, and *Penicillium*). Glucose oxidases derived from *Aspergillus* microorganisms are preferred, and glucose oxidases derived from *Aspergillus niger* are more preferred.

[0035] The term "glucose oxidase derived from Aspergillus niger" here refers to glucose oxidase produced by microorganisms classified as Aspergillus niger (which can be wild-type or variant strains), or glucose oxidase obtained through genetic engineering using a glucose oxidase gene. Therefore, recombinants produced by host microorganisms that have introduced a glucose oxidase gene obtained from Aspergillus niger (or a gene modified from that gene) are also equivalent to "glucose oxidase derived from Aspergillus niger".

[0036] The glucose oxidase used in this technology can be prepared from the culture medium of microorganisms that serve as the source of the glucose oxidase. Specific preparation methods include recovering glucose oxidase from the culture medium or cells of the aforementioned microorganisms. For example, when using glucose oxidase-secreting microorganisms, the enzyme can be separated and / or purified after recovering the cells from the culture medium by pre-filtration, centrifugation, etc., as needed. Furthermore, when using non-glucose oxidase-secreting microorganisms, the enzyme can be separated and / or purified after recovering the cells from the culture medium beforehand by pressure treatment, ultrasonic treatment, etc., to extract the enzyme. As a method for enzyme separation and / or purification, publicly available protein separation and / or purification methods can be used without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatographic methods using ion exchange resins. The separated and / or purified enzyme can be powdered by freeze-drying, vacuum drying, or other drying methods. Furthermore, appropriate excipients and / or drying aids can be used in these drying methods to achieve powdering. In addition, isolated and / or purified enzymes can also be made into liquids by adding appropriate additives and then filtering and sterilizing.

[0037] In this technology, commercially available products can also be used as glucose oxidases. Examples of preferred commercially available products include glucose oxidases derived from Aspergillus niger and Acremonium chrysogenum manufactured by Amano Enzyme Products Co., Ltd.

[0038] The content of glucose oxidase in the sugar-reducing agent involved in this technology can be freely set, provided that the effectiveness of this technology is not compromised. The content of glucose oxidase relative to 1g of sugar contained in a sugary food or beverage or 1g of sugar contained in the raw materials of the sugary food or beverage can be set to, for example, 20U or more. From the viewpoint of further improving the sugar-reducing effect, it can be set to preferably 50U or more, more preferably 100U or more, 150U or more, and even more preferably 180U or more.

[0039] The upper limit of glucose oxidase content is not particularly limited as long as it does not impair the effectiveness of this technology. It can be set to, for example, less than 25,000U, less than 20,000U, less than 10,000U, less than 5,000U, less than 3,000U, less than 2,500U, less than 2,000U, less than 1,400U, less than 1,200U, less than 1,050U, less than 900U, less than 800U, or less than 770U, relative to 1g of sugar in sugary drinks or beverages or the raw materials of such sugary drinks or beverages.

[0040] In addition, in this technology, the amount of enzyme required to oxidize 1 μmol of glucose within 1 minute is defined as 1 unit (1 U).

[0041] (2) α-glucosidase

[0042] The α-glucosidase (EC.3.2.1.20) that can be used in this technology is an enzyme that primarily hydrolyzes the α-1,4-glycosidic bonds on the non-reducing terminal side of maltodextrins. Some α-glucosidases have strong glycotransfer activity and include enzymes that use maltose as a substrate to generate isomaltoligosaccharides with α-1,6-glycosidic bonds. Furthermore, the α-glucosidase that can be used in this technology can also be an enzyme with other functions, provided it possesses α-glucosidase activity.

[0043] The sources of α-glucosidases that can be used in this technology are not particularly limited, and examples include filamentous fungi (e.g., *Absidia*, *Acremonium*, and *Aspergillus*), yeasts (e.g., *Saccharomyces*), actinomycetes (e.g., *Streptomyces avermitilis*, *Streptomyces griseus*, and *Streptomyces violaceoruber*), or bacteria (e.g., *Bacillus*, *Burkholderiaginsengisoli*, *Halomonas aquamarina*, and *Pseudomonas*). Preferably, α-glucosidases derived from *Aspergillus* microorganisms are included, and more preferably, α-glucosidases derived from *Aspergillus niger* are included.

[0044] The term "α-glucosidase derived from Aspergillus niger" here refers to α-glucosidase produced by microorganisms classified as Aspergillus niger (which can be wild-type or variant strains), or α-glucosidase obtained through genetic engineering using the α-glucosidase gene. Therefore, recombinants produced by host microorganisms that have introduced the α-glucosidase gene obtained from Aspergillus niger (or a gene derived from that gene by modification) are also equivalent to "α-glucosidase derived from Aspergillus niger".

[0045] The α-glucosidase used in this technology can be prepared from the culture medium of microorganisms that serve as the source of the aforementioned α-glucosidase. Specific preparation methods include recovering α-glucosidase from the culture medium or cells of the aforementioned microorganisms. For example, when using α-glucosidase-secreting microorganisms, the enzyme can be separated and / or purified after recovering the cells from the culture medium by pre-filtration, centrifugation, etc., as needed. Furthermore, when using α-glucosidase-non-secreting microorganisms, the enzyme can be separated and / or purified after recovering the cells from the culture medium beforehand by pressure treatment, ultrasonic treatment, etc., and then extracting the enzyme. As a method for enzyme separation and / or purification, publicly available protein separation and / or purification methods can be used without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatographic methods using ion exchange resins, etc. The separated and / or purified enzyme can be powdered by freeze-drying, vacuum drying, etc., and furthermore, appropriate excipients and / or drying aids can be used in this drying method to achieve powdering. In addition, isolated and / or purified enzymes can also be made into liquids by adding appropriate additives and then filtering and sterilizing.

[0046] In this technology, commercially available products can also be used as α-glucosidases. As a preferred example of a commercially available product, an α-glucosidase derived from Aspergillus niger manufactured by Amano Enzyme Products Co., Ltd. can be cited.

[0047] The content of α-glucosidase in the sugar-reducing agent involved in this technology can be freely set, provided that the effectiveness of this technology is not compromised. The content of α-glucosidase relative to 1g of sugar contained in a sugary food or beverage or 1g of sugar contained in the raw materials of such a sugary food or beverage can be set to, for example, 2000U or more. From the viewpoint of further improving the sugar-reducing effect, it can be set to preferably 4000U or more, 6000U or more, 8000U or more, more preferably 10000U or more, 15000U or more, and even more preferably 18000U or more.

[0048] As long as the effectiveness of this technology is not compromised, the upper limit of the α-glucosidase content is not particularly limited. It can be set to, for example, below 1,600,000 U, below 1,300,000 U, below 1,000,000 U, below 500,000 U, below 320,000 U, below 240,000 U, below 200,000 U, below 170,000 U, below 130,000 U, below 100,000 U, below 70,000 U, or below 50,000 U, relative to 1g of sugar in sugary drinks or the raw materials of such sugary drinks.

[0049] In addition, in this technology, the activity of α-glucosidase is defined as the amount of enzyme that generates 1 μg of glucose within 1 minute using α-methyl-D-glucosidase as a substrate, which is defined as 1 unit (1U).

[0050] (3) β-D-fructofuranosidase

[0051] The β-D-fructofuranosidase that can be used in this technology is an enzyme that hydrolyzes the residues on the non-reducing terminal side of β-D-fructofuranosides. Furthermore, the β-D-fructofuranosidase that can be used in this technology can also be an enzyme with other functions, as long as it possesses β-D-fructofuranosidase activity.

[0052] There are no particular limitations on the sources of β-D-fructofuranosidases that can be used in this technology, and examples include filamentous fungi (e.g., Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, and Aspergillus japonicus), yeasts (e.g., Kluyveromyces lactis and Saccharomyces cerevisiae), or bacteria (e.g., Arthrobacter and Bacillus).

[0053] The term "β-D-fructofuranosidase derived from Saccharomyces cerevisiae" here refers to β-D-fructofuranosidase produced by microorganisms classified as yeast (which can be wild-type or variant strains), or β-D-fructofuranosidase obtained through genetic engineering using the β-D-fructofuranosidase gene. Therefore, recombinants produced by host microorganisms that have been introduced with the β-D-fructofuranosidase gene obtained from Saccharomyces cerevisiae (or genes derived from modified Saccharomyces cerevisiae) are also equivalent to "β-D-fructofuranosidase derived from Saccharomyces cerevisiae".

[0054] The β-D-fructofuranosidase used in this technology can be prepared from the culture medium of microorganisms that serve as the source of the aforementioned β-D-fructofuranosidase. Specific preparation methods include recovering β-D-fructofuranosidase from the culture medium or cells of the aforementioned microorganisms. For example, when using β-D-fructofuranosidase-secreting microorganisms, the enzyme can be separated and / or purified after recovering the cells from the culture medium by pre-filtration, centrifugation, etc., as needed. Furthermore, when using β-D-fructofuranosidase-non-secreting microorganisms, the enzyme can be separated and / or purified after recovering the cells from the culture medium beforehand by pressure treatment, ultrasonic treatment, etc., and extracting the enzyme. As methods for enzyme separation and / or purification, publicly available protein separation and / or purification methods can be used without particular limitation, such as centrifugation, UF concentration, salting out, and various chromatographic methods using ion exchange resins, etc. Separated and / or purified enzymes can be powdered by drying methods such as freeze-drying and vacuum drying. Alternatively, appropriate excipients and / or drying aids can be used in these drying methods to achieve powder formation. Furthermore, separated and / or purified enzymes can also be prepared into liquids by adding appropriate additives and then sterilizing by filtration.

[0055] In this technology, commercially available products can also be used as β-D-fructofuranosidases. As a preferred example of a commercially available product, β-D-fructofuranosidase derived from Saccharomyces cerevisiae manufactured by Sigma-Aldrich can be cited.

[0056] The content of β-D-fructofuranosidase in the sugar-reducing agent involved in this technology can be freely set, provided that the effectiveness of this technology is not compromised. The content of β-D-fructofuranosidase relative to 1g of sugar in a sugary food or beverage or 1g of sugar in the raw materials of such a sugary food or beverage can be set to, for example, 0.15U or more. From the viewpoint of further improving the sugar-reducing effect, it can be set to preferably 7U or more, 8U or more, 9U or more, 10U or more, more preferably 15U or more, 30U or more, 50U or more, 100U or more, 200U or more, 300U or more, 400U or more, 500U or more, 600U or more, 700U or more, 800U or more, 900U or more, 1000U or more, 1500U or more, 2000U or more, 2500U or more, and more preferably 2700U or more.

[0057] The upper limit of β-D-fructofuranosidase content is not particularly limited as long as it does not impair the effectiveness of this technology. It can be set to, for example, below 3,000,000 U, below 1,000,000 U, below 600,000 U, below 300,000 U, below 100,000 U, below 50,000 U, below 10,000 U, below 6,000 U, below 4,500 U, or below 3,300 U, relative to 1g of sugar in sugary drinks or beverages or 1g of sugar in the raw materials of such sugary drinks or beverages.

[0058] In addition, in this technology, the activity of β-D-fructofuranosidase is defined as the amount of enzyme required to hydrolyze 1 μmol of sucrose into invert sugar within 1 minute at pH 4.5 and 55°C using 1.0% w / v sucrose as a substrate.

[0059] (4) Other

[0060] The sugar-reducing agents involved in this technology can be composed of the aforementioned glucose oxidase, α-glucosidase, and / or β-D-fructofuranosidase, provided they contain these enzymes. They can also freely contain one or more other components, provided this does not impair the effectiveness of the technology. These other components can include, for example, excipients, pH adjusters, colorants, flavoring agents, disintegrants, lubricants, and stabilizers used in conventional formulations. Furthermore, disclosed or future-discovered functional components or enzymes can be appropriately used, depending on the intended purpose.

[0061] 2. Food and beverages

[0062] The food and beverage products involved in this technology are those manufactured using the aforementioned sugar-reducing agents.

[0063] (1) Raw materials

[0064] The sugary foods and beverages used in this technology, or any raw materials that can be used in the foods and beverages covered by this technology, are not particularly limited in terms of origin or type, and can be freely selected according to the target food or beverage, as long as they do not impair the effect of this technology. In this technology, raw materials containing sucrose are particularly preferred.

[0065] As for the source of raw materials, there are no particular restrictions, and the selection is free, as long as it does not impair the function and effect of this technology. Examples of fruit juices used in fruit juice beverages include: juices of oranges (including tangerines), pineapples, grapefruits, apples, grapes, peaches, strawberries, bananas, mangoes, melons, apricots, lemons, kiwis, blueberries, pears, grapes, pomegranates, limes, and other fruit juices listed in the quality display standards for fruit beverages.

[0066] In addition, the vegetables used as raw materials for the vegetable juice beverage of the present invention include: carrots, spinach, onions, tomatoes, celery, bell peppers, pumpkins, corn, carrots, cucumbers, beets, parsley, lettuce, spinach, cauliflower, broccoli, kale, red bell peppers, ginger, green peppers, purple cabbage, and other vegetable juices.

[0067] Examples of raw material forms include liquid, paste, and slurry, with liquid being more preferred. Furthermore, the sugary food and beverages used in this technology, or the raw materials that can be used in the food and beverages involved in this technology, can be used directly or processed into liquid, paste, or slurry forms. They can also preferably be used in the form of pressed juice (freshly squeezed juice), concentrated juice, slurry-like juice, or powdered juice.

[0068] (2) The amount of sugar contained in food and beverages

[0069] The amount of sugars (monosaccharides and disaccharides, hereinafter the same) contained in the food and beverage products involved in this technology is not particularly limited, but it is preferable to reduce the sugar content in food and beverage products manufactured without using the sugar reducing agent involved in this technology. Specifically, the total sugar content contained in the food and beverage products involved in this technology is, for example, 70% or less, preferably 65% ​​or less, more preferably 60% or less, and even more preferably 57%, 50%, or 40% or less, relative to the total sugar content in food and beverage products manufactured without using the sugar reducing agent involved in this technology.

[0070] In particular, the amount of glucose and sucrose contained in the food and beverage products involved in this technology is preferably reduced compared to the glucose and sucrose content in food and beverage products manufactured without the use of the sugar-reducing agent involved in this technology. Specifically, the glucose and sucrose content contained in the food and beverage products involved in this technology, compared to the glucose and sucrose content in food and beverage products manufactured without the use of the sugar-reducing agent involved in this technology, is, for example, 70% or less, preferably 65% ​​or less, more preferably 60% or less, and even more preferably 57% or less. In various cases, the glucose content contained in the food and beverage products involved in this technology, compared to the glucose content in food and beverage products manufactured without the use of the sugar-reducing agent involved in this technology, is, for example, 95% or less, preferably 93% or less, more preferably 90%, 80%, 70%, or 60% or less. Furthermore, the sucrose content in the food and beverage products involved in this technology is, for example, 60% or less, preferably 50% or less, and more preferably 40% or less, relative to the sucrose content in food and beverage products manufactured without using the sugar-reducing agent involved in this technology.

[0071] (3) Types of food and drink

[0072] Provided that the effectiveness of this technology is not impaired, there are no particular limitations on the types of food and beverages involved in this technology, but beverages are preferred. Examples of beverages include vegetable drinks and fruit juices. In addition to beverages, examples include Japanese sweets (mochi, steamed buns, daifuku, Nagasaki cake, red bean paste, etc.), Western sweets (cream, cake, ice cream, etc.), soups, sauces, salad dressings, etc.

[0073] 3. Manufacturing methods of food and beverage products, and methods for reducing the sugar content of sugary food and beverage products.

[0074] The manufacturing method of the food and beverage and the method for reducing the sugar content of the sugary food and beverage involved in this technology are as follows, comprising: a step of acting glucose oxidase on the sugary food and beverage or the raw material of the sugary food and beverage (hereinafter also referred to as the "glucose oxidase action step"); and a step of acting α-glucosidase and / or β-D-fructofuranosidase on the sugary food and beverage or the raw material of the sugary food and beverage (hereinafter also referred to as the "α-glucosidase action step" and / or the "β-D-fructofuranosidase action step").

[0075] The glucose oxidase action, α-glucosidase action, and / or β-D-fructofuranosidase action can be performed simultaneously or in different orders. That is, glucose oxidase and α-glucosidase and / or β-D-fructofuranosidase can act simultaneously on carbohydrate-containing foods or their raw materials, or they can act separately on the carbohydrate-containing foods or their raw materials. When acting separately, the order is not particularly limited, and an enzyme inactivation step can be performed between the actions of each enzyme, as needed.

[0076] Furthermore, depending on the type of food or beverage, without compromising the effectiveness of this technology, general food manufacturing processes such as recycling processes can be performed before, after, or simultaneously with each process. The following provides a detailed description of each process.

[0077] (1) Glucose oxidase action process

[0078] The glucose oxidase action step is a process in which glucose oxidase acts on sugary foods or their raw materials. The amount of glucose oxidase added in the glucose oxidase action step can be freely set as long as it does not impair the effectiveness of this technology. The specific amount of glucose oxidase added in this technology is the same as the glucose oxidase content in the aforementioned sugar-reducing agent, therefore, it is omitted here.

[0079] The various conditions of the glucose oxidase reaction process can be freely set without compromising the effectiveness of this technology. For example, the pH, temperature, and reaction time can be set based on the physicochemical properties of the glucose oxidase used, such as its optimal pH, stable pH range, optimal temperature, and temperature stability. The pH can be set to, for example, pH 2.0–11.0, preferably pH 2.5–10.0, and more preferably pH 3.0–5.5. The temperature can be set to, for example, 10°C–70°C, preferably 20°C–50°C, and more preferably 25°C–45°C. The reaction time can be set to, for example, 15 minutes–120 hours, preferably 30 minutes–72 hours, and more preferably 12 hours–48 hours. Furthermore, the optimal reaction conditions can be determined through preliminary experiments.

[0080] (2) α-glucosidase action process

[0081] The α-glucosidase action step is a process in which α-glucosidase acts on sugary foods or the raw materials of such sugary foods. The amount of α-glucosidase added in the α-glucosidase action step can be freely set as long as it does not impair the effectiveness of this technology. The specific amount of α-glucosidase added in this technology is the same as the α-glucosidase content in the aforementioned sugar-reducing agent, therefore, it is omitted here.

[0082] The various conditions of the α-glucosidase reaction process can be freely set without compromising the effectiveness of this technology. For example, the pH, temperature, and reaction time can be set based on the physicochemical properties of the α-glucosidase used, such as its optimal pH, stable pH range, optimal temperature, and temperature stability. The pH can be set, for example, to pH 2.0–11.0, preferably to pH 2.5–10.0, and more preferably to pH 3.0–5.5. The temperature can be set, for example, to 10°C–70°C, preferably to 20°C–50°C, and more preferably to 25°C–45°C. The reaction time can be set, for example, to 15 minutes–120 hours, preferably to 30 minutes–72 hours, and more preferably to 12 hours–48 hours. Furthermore, the optimal reaction conditions can be determined through preliminary experiments.

[0083] (3) β-D-fructofuranosidase action process

[0084] The β-D-fructofuranosidase action step is a process in which β-D-fructofuranosidase acts on sugary foods or the raw materials of such sugary foods. The amount of β-D-fructofuranosidase added in the β-D-fructofuranosidase action step can be freely set as long as it does not impair the effectiveness of this technology. The specific amount of β-D-fructofuranosidase added in this technology is the same as the content of β-D-fructofuranosidase in the aforementioned sugar-reducing agent, therefore, description is omitted here.

[0085] As long as the effectiveness of this technology is not compromised, various conditions of the β-D-fructofuranosidase reaction process can be freely set. For example, pH, temperature, reaction time, etc., can be set according to the physicochemical properties of the β-D-fructofuranosidase used, such as optimal pH, stable pH range, optimal temperature, and temperature stability. pH can be set to, for example, pH 2.0 to 11.0, preferably pH 2.5 to 10.0, more preferably pH 3.0 to 5.5. Temperature can be set to, for example, 10°C to 70°C, preferably 20°C to 65°C, more preferably 25°C to 60°C. Reaction time can be set to, for example, 15 minutes to 120 hours, preferably 30 minutes to 72 hours, more preferably 12 hours to 48 hours. In addition, the optimal reaction conditions can be determined through preliminary experiments.

[0086] (4) Recycling process

[0087] The recycling process involves recovering food and beverage products that have undergone glucose oxidase, α-glucosidase, and / or β-D-fructofuranosidase processes. Specific recycling methods can be tailored to the type of food or beverage being manufactured, freely combining one or more common recycling methods used in food and beverage production.

[0088] The characteristic of food and beverage products manufactured through the glucose oxidase action step, the α-glucosidase action step, and / or the β-D-fructofuranosidase action step is that the amount of sugars (monosaccharides + disaccharides) contained is reduced. That is, in this technology, compared to food and beverage products manufactured without the glucose oxidase action step, the α-glucosidase action step, and / or the β-D-fructofuranosidase action step, a recovery step is possible to recover the reduced sugar (monosaccharide + disaccharide) content of the food and beverage.

[0089] One method of manufacturing food products and reducing sugar content in sugary food products according to this technology includes the following steps (1), (2), (3) and / or (4) and (5). Alternatively, an enzyme inactivation step may be added as needed after one or more steps selected from steps (2), (3), and (4). Steps (2), (3), and (4) may be performed simultaneously or in different orders.

[0090] (1) The process of preparing sugary drinks or the raw materials for sugary drinks.

[0091] (2) The process of causing glucose oxidase to act on the prepared sugary food or the raw materials of the sugary food.

[0092] (3) The process of causing α-glucosidase to act on the prepared sugary food or the raw materials of the sugary food.

[0093] (4) The process of causing β-D-fructofuranosidase to act on the prepared sugary food or the raw materials of the sugary food.

[0094] (5) The process of recycling food and beverage products manufactured through processes (1), (2), and (3) and / or (4).

[0095] Example

[0096] The present invention will now be described in more detail based on embodiments. Furthermore, the embodiments described below illustrate one example of a representative embodiment of the invention, but the scope of the invention should not be interpreted narrowly as a result.

[0097] 1. Use of raw materials / enzymes

[0098] The raw materials and enzymes used in the examples are shown in Table 1 below.

[0099] [Table 1]

[0100]

[0101] 2. Enzyme activity assay methods

[0102] [Methods for Assaying Glucose Oxidase Activity]

[0103] Measure an appropriate amount of enzyme and add it to cooled pH 7.0 potassium phosphate-sodium hydroxide buffer (0.1 mol / L) to dissolve or uniformly disperse to 50 mL as the sample solution. Measure 2.50 g of D(+)-glucose and dissolve it in water to 25 mL as the substrate solution. Add 0.5 mL of the substrate solution, 2 mL of potassium phosphate-sodium hydroxide buffer (0.1 mol / L, pH 7.0, containing phenol), 0.5 mL of peroxidase reagent (25 units / mL), and 0.1 mL of 4-aminoantipyrine solution (1→250) to a quartz cell and heat at 37°C for 10 minutes. Add 0.1 mL of the sample solution to this liquid and mix thoroughly, then heat at 37°C to prepare the test solution. Alternatively, use pH 7.0 potassium phosphate-sodium hydroxide buffer (0.1 mol / L) or water instead of the sample solution, following the same procedure as preparing the test solution, to prepare the comparison solution. For both the test and control solutions, the absorbance at 500 nm was measured 2 and 5 minutes after the sample solution was added. The amount of oxidized glucose was quantified using the molar absorptivity of the generated quinone imine pigment. The amount of enzyme required to oxidize 1 μmol of glucose within 1 minute was defined as 1 unit (1 U).

[0104] [Methods for Assaying α-Glucosidase Activity]

[0105] The activity was determined using Method 2 of the α-glucosidase activity test method in the 9th edition of the food additive standard.

[0106] (1) Measure 2.0 g of α-methyl-D-glucoside and dissolve it in water to 100 mL to prepare an α-methyl-D-glucoside solution. Measure 1 mL of the α-methyl-D-glucoside solution and 1 mL of 0.02 mol / L acetate-sodium acetate buffer (pH 5.0) into a test tube. After standing at 40 °C for 10–15 minutes, add 0.5 mL of an aqueous solution (enzyme solution) containing transglucosidase and shake thoroughly to mix. Place it accurately at 40 °C for 60 minutes. After 60 minutes, place it in a boiling water bath and heat accurately for 5 minutes, then cool it under running water.

[0107] (2) Measure 3 mL of the colorimetric solution from the glucose assay kit (Fujifilm and Kojun Pharmaceutical Co., Ltd., hereinafter the same) into a test tube, add 0.2 mL of the reaction solution obtained in (1), shake thoroughly to mix, and incubate at 40°C for 5 minutes. For this liquid, use water as a control and measure the absorbance (E60) at a wavelength of 505 nm. In addition, measure 1 mL of 0.02 mol / L acetate-sodium acetate buffer (pH 5.0) and 0.5 mL of an aqueous solution (enzyme solution) containing transglucosidase into a test tube as a blank, heat accurately in a boiling water bath for 5 minutes, and then cool in running water. After cooling, add 1 mL of α-methyl-D-glucosidase solution, and perform the same operation as above to measure the absorbance (E0).

[0108] (3) Dilute glucose standard solution I or II of the glucose assay kit with water to the specified concentration (20 mg / dL, 40 mg / dL). Measure 3 mL of the colorimetric solution of the glucose assay kit into a test tube, add 0.2 mL of the above glucose solution, shake thoroughly to mix, and incubate accurately at 40°C for 5 minutes. For this liquid, using water as a control, measure the absorbance (ES) at a wavelength of 505 nm. Alternatively, use 0.2 mL of water instead of the glucose solution as a blank, and perform the same operation as above to measure the absorbance (EB). Based on the obtained absorbance, construct a calibration line for glucose and determine the amount of glucose (μg) (G) when the absorbance difference is 1.000.

[0109] (4) The amount of enzyme that produces 1 μg of glucose within 60 minutes is taken as 1 unit (1U) and calculated by the following formula.

[0110] α-Glucosidase activity (U / g, U / mL) = (E60 - E0) × G × 2.5 / 0.1 × n / 0.5

[0111] E60: Absorbance of the reaction solution

[0112] E0: Absorbance of blank solution

[0113] G: Amount of glucose (μg) at an absorbance difference of 1.000 (calculated based on the glucose calibration line)

[0114] 2.5: Volume of the reaction system (mL)

[0115] 0.1: Volume of reaction solution collected (mL)

[0116] 0.5: Enzyme solution collection volume (mL)

[0117] n: Dilution factor per 1g or 1mL of sample

[0118] [Method for determining the activity of β-D-fructofuranosidase]

[0119] (1) Preparation of reagents

[0120] A 100 mM sodium acetate solution (sodium acetate trihydrate (Sigma-Aldrich "#S8625")) adjusted to pH 4.5 with 1 M HCl at 55 °C was used as a buffer.

[0121] The solution of sucrose (Sigma-Aldrich "#S7903") dissolved in 100 mM sodium acetate buffer (pH 4.5) was used as a 1.0% w / v sucrose substrate solution.

[0122] Just before use, dissolve a solution containing 0.3–0.4 U / mL of β-D-fructofuranosidase in cold purified water to prepare an enzyme solution.

[0123] Dilute 1N NaOH (Supelco "#S2567") with purified water to a ratio of 1:2 to prepare a 0.5M NaOH dilution solution.

[0124] Prepare a 0.5% w / v PAHBAH reagent by dissolving p-hydroxybenzoyl hydrazine (PAHBAH) (Sigma-Aldrich "#H9882") in 0.5M NaOH.

[0125] Glucose (Sigma-Aldrich "#G8270") was dissolved in purified water to prepare a 5.56 mM glucose standard solution.

[0126] (2) Measurement method

[0127] (2-1) Preparation of glucose standard solution

[0128] Use 1.0 mL of purified water as a blank for the glucose standard solution.

[0129] The purified water (0.95 mL, 0.90 mL, 0.80 mL, 0.70 mL, 0.60 mL, 0.50 mL) and the prepared glucose standard solution (0.05 mL, 0.10 mL, 0.20 mL, 0.30 mL, 0.40 mL, 0.50 mL) were mixed to prepare the glucose standard solution, and then equilibrated at 55 °C.

[0130] (2-2) Preparation of blank test solution

[0131] The solution obtained by mixing 0.10 mL of purified water and 0.90 mL of the prepared sucrose substrate solution was used as a blank test solution and equilibrated at 55 °C.

[0132] (2-3) Preparation of test solution

[0133] The solution obtained by mixing 0.90 mL of the prepared sucrose substrate solution and 0.10 mL of the prepared enzyme solution was used as the test solution and incubated accurately at 55°C for 20 minutes.

[0134] (2-4) Determination of absorbance

[0135] Measure 0.10 mL from each of the prepared glucose standard solutions, blank test solutions, and test solutions, and add them to a tube containing 2.90 mL of the prepared 0.5% w / v PAHBAH reagent. Mix rapidly, place in a boiling water bath, heat for 5 minutes, and cool to room temperature. After cooling, mix again and transfer the solution to an appropriate cuvette. Measure the absorbance of all reaction mixtures at 410 nm using an appropriate spectrophotometer.

[0136] The amount of enzyme required to hydrolyze 1 μmol of sucrose into invert sugar within 1 minute is defined as 1 unit (1U).

[0137] 3. Experimental Example

[0138] <Experimental Example 1>

[0139] (1) Experimental methods

[0140] 50 mL of commercially available fruit juice (V8 Pomegranate Blueberry Juice) was stirred at 35°C for 10 minutes for pre-culturing. Next, the enzymes listed in Table 3 were added, and the mixture was stirred at 35°C for 24 hours for enzyme treatment. After the enzyme reaction was complete, the mixture was treated at 90°C for 20 minutes to inactivate the enzymes. The mixture was then cooled to room temperature to obtain fruit juice 1.

[0141] (2) Sugar composition analysis

[0142] The obtained fruit juice 1 was diluted 5 times with deionized water and centrifuged at 13300G for 10 minutes. The supernatant was diluted 2 times with ethanol (final ethanol concentration ≥ 50%) and passed through a membrane filter (0.22 μm) for analysis using high performance liquid chromatography (HPLC) with a differential refractive index detector (RI) "RID-20A" Shimadzu Corporation. Detailed column specifications and conditions are shown in Table 2 below. Sugar composition was analyzed using standards for each sugar (Sigma). Furthermore, the ratio of glucose and sucrose to total sugar content (sugar composition) was calculated based on the obtained sugar composition.

[0143] [Table 2]

[0144]

[0145] (3) Results

[0146] The results are shown in Table 3 below.

[0147] [Table 3]

[0148]

[0149] (4) Investigation

[0150] As shown in Comparative Examples 1-3 and 6, the reduction rate of carbohydrates was less than 30% when glucose oxidase, catalase, β-D-fructofuranosidase, or α-glucosidase were used alone. Furthermore, as shown in Comparative Examples 4 and 5, the reduction rate of carbohydrates was also less than 35% when glucose oxidase and catalase were used in combination. On the other hand, Example 1, which used glucose oxidase and α-glucosidase, and Example 2, which used glucose oxidase and β-D-fructofuranosidase, showed a high reduction rate of over 40%.

[0151] <Experimental Example 2>

[0152] (1) Experimental methods

[0153] 50 mL of commercially available fruit juice (Ceres Peach Juice) was stirred at 35°C for 10 minutes for pre-culturing. The enzymes listed in Table 4 below were added, and the mixture was treated at 35°C for 24 hours. After the enzyme reaction was complete, the mixture was treated at 90°C for 20 minutes to inactivate the enzymes. The mixture was then cooled to room temperature to obtain fruit juice 2.

[0154] (2) Sugar composition analysis

[0155] The sugar composition of the obtained fruit juice 2 was analyzed using the same method as in Experiment 1.

[0156] (3) Results

[0157] The results are shown in Table 4 below.

[0158] [Table 4]

[0159]

[0160] (4) Investigation

[0161] As shown in Table 4, compared with Comparative Examples 7-10 which used glucose oxidase or α-glucosidase alone and Comparative Examples 11-13 which used both glucose oxidase and catalase, Example 3, which used both glucose oxidase and α-glucosidase, showed a high reduction rate.

[0162] <Experimental Example 3>

[0163] (1) Experimental methods

[0164] 50 mL of commercially available fruit juice (TRADERJOE'S Orange Peach Mango Juice) was stirred at 35°C for 10 minutes for pre-culturing. The enzymes listed in Table 5 below were added, and the mixture was treated at 35°C for 24 hours. After the enzyme reaction was complete, the mixture was treated at 90°C for 20 minutes to inactivate the enzymes. The mixture was then cooled to room temperature to obtain fruit juice 3.

[0165] (2) Sugar composition analysis

[0166] The sugar composition of the obtained fruit juice 3 was analyzed using the same method as in Experimental Example 1.

[0167] (3) Results

[0168] The results are shown in Table 5 below.

[0169] [Table 5]

[0170]

[0171] (4) Investigation

[0172] As shown in Table 5, compared with Comparative Examples 14 and 15 which used glucose oxidase or α-glucosidase alone, Examples 4 to 6 which used both glucose oxidase and α-glucosidase showed a high reduction rate.

[0173] <Experimental Example 4>

[0174] (1) Experimental methods

[0175] 50 mL of commercially available fruit juice (V8 vegetable juice) was stirred at 35°C for 10 minutes for pre-culturing. The enzymes listed in Table 6 below were added, and the mixture was treated at 35°C for 24 hours. After the enzyme reaction was complete, the mixture was treated at 90°C for 20 minutes to inactivate the enzymes. The mixture was then cooled to room temperature to obtain fruit juice 4.

[0176] (2) Sugar composition analysis

[0177] The sugar composition of the obtained fruit juice 4 was analyzed using the same method as in Experimental Example 1.

[0178] (3) Results

[0179] The results are shown in Table 6 below.

[0180] [Table 6]

[0181]

[0182] (4) Investigation

[0183] As shown in Table 6, it can be seen that the reduction rate of carbohydrates increases with the increase of the amount of glucose oxidase used in conjunction with α-glucosidase.

Claims

1. A sugar-reducing agent for a sugary food or beverage, comprising: glucose oxidase; and α-glucosidase and / or β-D-fructofuranosidase.

2. The sugar-reducing agent according to claim 1 reduces glucose and sucrose in sugary foods and beverages.

3. The sugar-reducing agent according to claim 1, wherein the sugar-containing beverage is a sucrose-containing beverage.

4. The sugar-reducing agent according to claim 1, wherein the sugar-containing beverage is a beverage made from fruits and / or vegetables.

5. A food product that uses the sugar-reducing agent according to any one of claims 1 to 4.

6. A method for manufacturing a food product, comprising: A process in which glucose oxidase acts on a sugary food or its raw material; and A process of applying α-glucosidase and / or β-D-fructofuranosidase to a sugary food or the raw material of such a food.

7. A method for reducing the sugar content of a sugary food or beverage, comprising: A process in which glucose oxidase acts on a sugary food or its raw material; and A process of applying α-glucosidase and / or β-D-fructofuranosidase to a sugary food or the raw material of such a food.

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