Saccharide-depletion agent for saccharide-containing food / beverage

Abstract

Provided is a technique for depleting saccharides in a saccharide-containing food / beverage. The provided technique causes the saccharides in a saccharide-containing food / beverage to be depleted by causing glucose oxidase, α-glucosidase, and / or β-D-fructofuranosidase to act concurrently or in no particular order on the saccharide-containing food / beverage or a feedstock for the saccharide-containing food / beverage. This technique allows the monosaccharides and disaccharides in a saccharide-containing food / beverage, in particular glucose and sucrose, to be depleted.

Description

Title of the Invention: SACCHARIDE-REDUCING AGENT FOR SACCHARIDE-CONTAINING FOODS AND BEVERAGES Technical Field

[0001] This technology relates to a saccharide-reducing agent for saccharide-containing foods and beverages. More specifically, the present invention relates to a saccharide-reducing agent capable of reducing monosaccharides and disaccharides in saccharide-containing foods and beverages, a method for producing foods and beverages, and a method for reducing saccharides in saccharide-containing foods and beverages. Background Art

[0002] In recent years, the growing health consciousness has led to increased demand for low-saccharide beverages and reduced-saccharide foods. Fruit and vegetable juices, in particular, are popular due to their high nutritional value, but their saccharide content is a concern. Specifically, excessive saccharide intake can lead to health risks, increased calorie intake, negative impacts on dental health, rapid spikes in blood glucose levels, and an imbalance in the nutritional content of beverages. Against this background, technologies to reduce the saccharide content in saccharide-containing foods and beverages are being developed.

[0003] For example, Patent Literature 1 discloses a technology for producing a reduced-saccharide citrus juice beverage by mixing a citrus juice source having a low Brix value (low saccharide content) with additional settled pulp (or bottom pulp) and a sweetener that enhances the perceived sweetness of the juice beverage without significantly increasing the calorie content of the beverage.

[0004] Furthermore, Patent Literature 2 discloses a technology for reducing the saccharide content in food materials by contacting the food materials with a glucosyltransferase having a specific sequence. Patent Literature 2 states that the total content of monosaccharides and disaccharides combined in the food materials can be reduced by 25%. Citation List Patent Literature

[0005] Patent Literature 1: Japanese Translation of PCT Publication No. JP-T-2007-504821 Patent Literature 2: International Publication No. WO 2016 / 173929 Summary of Invention Technical Problem

[0006] As mentioned above, in recent years, low-saccharide beverages and reduced-saccharide foods have attracted attention due to the growing health consciousness, but the reality is that further development of technologies to reduce the saccharide content in saccharide-containing foods and beverages is desired.

[0007] Therefore, the primary objective of this technology is to provide a technology for reducing saccharide content in saccharide-containing foods and beverages. Solution to Problem

[0008] The inventors of this application have diligently researched technologies for reducing saccharide content in saccharide-containing foods and beverages, and as a result have discovered a novel technology for reducing saccharide content in saccharide-containing foods and beverages by using two or more specific enzymes in combination, and have completed this technology.

[0009] In other words, this technology first provides a saccharide-reducing agent for saccharide-containing foods and beverages, containing glucose oxidase, and a-glucosidase and / or P-D-fructofuranosidase. The saccharide-reducing agent according to this technology can reduce glucose and sucrose in saccharide-containing foods and beverages. The saccharide-reducing agent according to this technology can reduce the amount of saccharides in sucrose-containing foods and beverages. The saccharide-reducing agent according to this technology can reduce the amount of saccharides in beverages made from fruits and / or vegetables as raw materials.

[0010] This technology then provides foods and beverages using the saccharide-reducing agent according to this technology.

[0011] This technology further provides a method for producing foods and beverages, including a step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with glucose oxidase, and a step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with a-glucosidase and / or P-D-fructofuranosidase, and a method for reducing saccharides in saccharide-containing foods and beverages, including a step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with glucose oxidase, and a step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with a-glucosidase and / or P-D-fructofuranosidase.

[0012] In this technology, the term “saccharides” refers to the general term for monosaccharides and disaccharides (excluding saccharide alcohols) as defined in the Nutrition Labeling Standards (Ministry of Health, Labour and Welfare Notification No. 176). Monosaccharides are saccharides that cannot be broken down further, and include glucose, fructose, and galactose. Disaccharides include sucrose (glucose + fructose), maltose (glucose + glucose), lactose (glucose + galactose), etc. Description of Embodiments

[0013] The following describes preferred embodiments for carrying out the present invention. The embodiments described below are merely examples of typical embodiments of the present invention, and this should not be interpreted as narrowing the scope of the invention.

[0014] 1. Saccharide-reducing agent The saccharide-reducing agent according to this technology contains glucose oxidase and a-glucosidase and / or P-D-fructofuranosidase as active ingredients. Although glucose oxidase and a-glucosidase and / or P-D-fructofuranosidase are effective in reducing saccharides in saccharide-containing foods and beverages on their own, as shown in the examples described later, the saccharide-reducing effect can be greatly improved by using glucose oxidase and a-glucosidase and / or P-D-fructofuranosidase in combination. In particular, the saccharide-reducing agent according to this technology can reduce glucose and sucrose in saccharide-containing foods and beverages.

[0015] The reduction rate of saccharides (monosaccharides and disaccharides, hereinafter the same) when foods and beverages are produced using the saccharide-reducing agent according to 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, compared to the saccharides in foods and beverages produced without using the saccharide-reducing agent according to this technology.

[0016] The reduction rates of glucose and sucrose when producing foods and beverages using the saccharide-reducing agent according to this technology are not particularly limited, but are, for example, 30% or more, preferably 35% or more, more preferably 40% or more, and even more preferably 43% or more, compared to glucose and sucrose in foods and beverages produced without using the saccharide-reducing agent according to this technology. The reduction rates of each are not particularly limited, but the reduction rate of glucose when foods and beverages are produced using the saccharide-reducing agent according to this technology is, for example, 10% or more, 20 % or more, preferably 30% or more, and more preferably 40% or more, compared to glucose in foods and beverages produced without using the saccharide-reducing agent according to this technology. Furthermore, the reduction rate of sucrose when foods and beverages are produced using the saccharide-reducing agent according to this technology is, for example, 40% or more, preferably 50% or more, and more preferably 60% or more, compared to sucrose in foods and beverages produced without using the saccharide-reducing agent according to this technology.

[0017] The components and the like that can be used in the saccharide-reducing agent according to this technology are described in detail below.

[0018] (1) Glucose oxidase The glucose oxidase (EC 1.1.3.4) that can be used in this technology is an enzyme that oxidizes glucose to produce gluconic acid and hydrogen peroxide. In addition, the glucose oxidase that can be used in this technology may also be an enzyme that has other actions, as long as it possesses glucose oxidase activity.

[0019] The origin of the glucose oxidase that can be used in this technology is not particularly limited, but examples include glucose oxidase from filamentous fungi (e.g., Acremonium chrysogenum, Aspergillus aculeatus, Aspergillus niger, and the genus Penicillium). Preferably, glucose oxidase derived from genus Aspergillus microorganisms is used, and more preferably, glucose oxidase derived from Aspergillus niger is used.

[0020] Here, “glucose oxidase derived from Aspergillus niger” refers to glucose oxidase produced by microorganisms classified as Aspergillus niger (whether wild-type or mutant strains), or glucose oxidase obtained through genetic engineering using the glucose oxidase gene. Therefore, recombinants produced by host microorganisms into which the glucose oxidase gene obtained from Aspergillus niger (or a modified version of that gene) has been introduced also fall under the category of “glucose oxidase derived from Aspergillus niger”.

[0021] The glucose oxidase used in this technology can be prepared from the culture solution of the above-mentioned microorganism from which the glucose oxidase originates. Specific preparation methods include recovering glucose oxidase from the culture solution or microbial bodies of the above-mentioned microorganisms. For example, when using glucose oxidase-secreting microorganisms, the microbial bodies can be previously recovered from the culture solution by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using nonglucose oxidase-secreting microorganisms, the microbial bodies can be previously recovered from the culture solution, as needed, and then the microbial bodies can be disrupted by pressurization, sonication, etc. to extract the enzyme, and then the enzyme can be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method without particular limitation, such as centrifugation, UF concentration, salting-out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying method. Furthermore, the separated and / or purified enzymes can be formulated as a liquid by adding appropriate additives and sterilizing by filtration.

[0022] In this technology, commercially available glucose oxidases can also be used. Preferred examples of commercially available glucose oxidases include those derived from Aspergillus niger and Acremonium chrysogenum, manufactured by Amano Enzyme Inc.

[0023] The glucose oxidase content in the saccharide-reducing agent according to this technology can be freely set as long as it does not impair the effect of this technology. The glucose oxidase content can be set to, for example, 20 U or more per 1 g of saccharide contained in saccharide-containing foods and beverages or per 1 g of saccharide contained in raw materials for the saccharide-containing foods and beverages, and from the viewpoint of further enhancing the saccharide-reducing effect, it can be preferably set to 50 U or more, more preferably 100 U or more, 150 U or more, and even more preferably 180 U or more.

[0024] The upper limit of the glucose oxidase content also is not particularly limited as long as it does not impair the effect of this technology, but it can be set to, for example, 25,000 U or less, 20,000 U or less, 10,000 U or less, 5,000 U or less, 3,000 U or less, 2,500 U or less, 2,000 U or less, 1,400 U or less, 1,200 U or less, 1,050 U or less, 900 U or less, 800 U or less, or 770 U or less per 1 g of saccharide contained in saccharide-containing foods and beverages or per 1 g of saccharide contained in raw materials for the saccharide-containing foods and beverages.

[0025] In this technology, regarding glucose oxidase activity, the amount of enzyme required to oxidize 1 umol of glucose per minute is defined as 1 unit (1 U).

[0026] (2) a-Glucosidase The a-glucosidase (EC. 3.2.1.20) that can be used in this technology is an enzyme that primarily hydrolyzes the a-1,4-glucosidic bond at the non-reducing end of maltooligosaccharides. Some a-glucosidases have strong transglycosylation activity and some enzymes produce isomaltoligosaccharides containing an a-1,6-glucosidic bond using maltose as a substrate. In addition, the a-glucosidase that can be used in this technology may also have other actions, as long as it possesses a-glucosidase activity.

[0027] The origin of the a-glucosidase that can be used in this technology is not particularly limited, but may include, for example, a-glucosidases derived from filamentous fungi (e.g., genera Absidia, Acremonium, and Aspergillus), yeasts (e.g., genus Saccharomyces), actinomycetes (e.g., Streptomyces avermitilis, Streptomyces griseus, and Streptomyces violaceoruber), or bacteria (e.g., genus Bacillus, Burkholderia ginsengisoli, Halomonas aquamarina and genus Pseudomonas). Preferably, a-glucosidases derived from microorganisms of the genus Aspergillus are used, and more preferably, a-glucosidases derived from Aspergillus niger are used.

[0028] Here, “a-glucosidase derived from Aspergillus niger” refers to a-glucosidase produced by microorganisms classified as Aspergillus niger (whether wild-type or mutant), or a-glucosidase obtained through genetic engineering using the a-glucosidase gene. Therefore, recombinant organisms produced by host microorganisms into which the a-glucosidase gene obtained from Aspergillus niger (or a modified version of that gene) has been introduced also fall under the category of “a-glucosidase derived from Aspergillus niger”.

[0029] The a-glucosidase used in this technology can be prepared from the culture solution of the above-mentioned microorganism from which the a-glucosidase is derived. Specific preparation methods include recovering the a-glucosidase from the culture solution or microbial bodies of the above-mentioned microorganism. For example, when using a-glucosidase-secreting microorganisms, the microbial bodies can be previously recovered from the culture solution by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using a-glucosidase-non-secreting microorganisms, the microbial bodies can be previously recovered from the culture solution, as needed, and then the microbial bodies can be disrupted by pressurization, sonication, etc., to extract the enzyme, and then the enzyme can be separated and / or purified. As for the enzyme separation and / or purification method, any known protein separation and / or purification method 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 enzymes can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying process. Furthermore, the separated and / or purified enzymes can be formulated as a liquid by adding appropriate additives and sterilizing by filtration.

[0030] In this technology, commercially available a-glucosidase can also be used, and a preferred example of such a product is a-glucosidase derived from Aspergillus niger, manufactured by Amano Enzyme Inc.

[0031] The a-glucosidase content in the saccharide-reducing agent according to this technology can be freely set as long as it does not impair the effect of this technology. The a-glucosidase content can be set to, for example, 2,000 U or more per 1 g of saccharide contained in saccharide-containing foods and beverages or per 1 g of saccharide contained in raw materials for the saccharide-containing foods and beverages. From the viewpoint of further enhancing the saccharide-reducing effect, it can preferably be set to 4,000 U or more, 6,000 U or more, 8,000 U or more, more preferably 10,000 U or more, 15,000 U or more, and even more preferably 18,000 U or more.

[0032] The upper limit of the a-glucosidase content is also not particularly limited as long as it does not impair the effect of this technology, but it can be set to, for example, 1,600,000 U or less, 1,300,000 U or less, 1,000,000 U or less, 500,000 U or less, 320,000 U or less, 240,000 U or less, 200,000 U or less, 170,000 U or less, 130,000 U or less, 100,000 U or less, 70,000 U or less, or 50,000 U or less per 1 g of saccharide contained in saccharide-containing foods and beverages or per 1 g of saccharide contained in raw materials for the saccharide-containing foods and beverages.

[0033] In this technology, regarding the activity of a-glucosidase, the amount of enzyme that produces 1 pg of glucose per minute using a-methyl-D-glucoside as a substrate is defined as 1 unit (1 U).

[0034] (3) P-D-fructofuranosidase The P-D-fructofuranosidase that can be used in this technology is an enzyme that hydrolyzes the residue at the non-reducing end of P-D-fructofuranoside. Furthermore, the P-D-fructofuranosidase that can be used in this technology may also have other actions, as long as it possesses P-D-fructofuranosidase activity.

[0035] The origin of the P-D-fructofuranosidase that can be used in this technology is not particularly limited, but examples thereof include P-D-fructofuranosidases derived from 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., genera Arthrobacter and Bacillus).

[0036] Here, “P-D-fructofuranosidase derived from Saccharomyces cerevisiae” refers to P-D-fructofuranosidase produced by a microorganism classified as yeast (whether wild-type or mutant), or P-D-fructofuranosidase obtained by genetic engineering using the P-D-fructofuranosidase gene. Therefore, recombinant organisms produced by host microorganisms into which the P-D-fructofuranosidase gene (or a modified version thereof) obtained from Saccharomyces cerevisiae has been introduced also fall under the category of “P-D-fructofuranosidase derived from Saccharomyces cerevisiae”.

[0037] The P-D-fructofuranosidase used in this technology can be prepared from the culture solution of the above-mentioned microorganism from which the P-D-fructofuranosidase is derived. Specific preparation methods include recovering the P-D-fructofuranosidase from the culture solution or microbial bodies of the above-mentioned microorganism. For example, when using P-D-fructofuranosidase-secreting microorganisms, the microbial bodies can be previously recovered from the culture solution by filtration, centrifugation, etc., as needed, and then the enzyme can be separated and / or purified. When using P-D-fructofuranosidase-non-secreting microorganisms, the microbial bodies can be previously recovered from the culture solution, as needed, and then the enzyme can be extracted by disrupting the microbial bodies by pressurization, sonication, etc., and then the enzyme can be separated and / or purified. As for the enzyme separation and / or purification method, any known protein separation and / or purification method can be used without particular limitation, such as centrifugation, UF concentration, salting-out, and various chromatography methods using ion exchange resins. The separated and / or purified enzymes can be powdered by drying methods such as freeze-drying or vacuum drying, and can also be powdered using appropriate excipients and / or drying aids in the drying process. Furthermore, the separated and / or purified enzymes can be formulated as a liquid by adding appropriate additives and sterilizing by filtration.

[0038] In this technology, commercially available P-D-fructofuranosidase can also be used, and a preferred example of a commercially available product is P-D- fructofuranosidase derived from Saccharomyces cerevisiae, manufactured by Sigma-Aldrich.

[0039] The P-D-fructofuranosidase content in the saccharide-reducing agent according to this technology can be freely set as long as the effect of this technology are not impaired. The P-D-fructofuranosidase content can be set to, for example, 0.15 U or more per 1 g of saccharide contained in saccharide-containing foods and beverages or per 1 g of saccharide contained in raw materials for the saccharide-containing foods and beverages, and from the viewpoint of further enhancing the saccharide-reducing effect, it can be preferably set to 7 U or more, 8 U or more, 9 U or more, 10 U or more, more preferably 15 U or more, 30 U or more, 50 U or more, 100 U or more, 200 U or more, 300 U or more, 400 U or more, 500 U or more, 600 U or more, 700 U or more, 800 U or more, 900 U or more, 1,000 U or more, 1,500 U or more, 2,000 U or more, 2,500 U or more, and even more preferably 2,700 U or more.

[0040] The upper limit of the P-D-fructofuranosidase content also is not particularly limited as long as it does not impair the effect of this technology, but it can be set to, for example, 3,000,000 U or less, 1,000,000 U or less, 600,000 U or less, 300,000 U or less, 100,000 U or less, 50,000 U or less, 10,000 U or less, 6,000 U or less, 4,500 U or less, or 3,300 U or less per 1 g of saccharide contained in saccharide-containing foods and beverages or per 1 g of saccharide contained in raw materials for the saccharide-containing foods and beverages.

[0041] Note that, in the present technology, regarding the activity of P-D-fructofuranosidase, the amount of enzyme required to hydrolyze 1 pmol of sucrose to invert sugar per minute at pH 4.5 and 55°C using 1.0% w / v sucrose as a substrate is defined as 1 unit (1 U).

[0042] (4) Others The saccharide-reducing agent according to this technology may consist of the aforementioned glucose oxidase and a-glucosidase and / or p-D-fructofuranosidase, as long as the aforementioned glucose oxidase and a-glucosidase and / or P—D— fructofuranosidase are contained, or it may contain one or more other components freely selected as long as the effect of this technology is not impaired. Other components may include, for example, excipients, pH adjusters, colorants, flavoring agents, disintegrants, lubricants, stabilizers, etc., that are commonly used in formulations. Furthermore, known or future functional components and enzymes may be used in combination as appropriate for the purpose.

[0043] 2. Foods and beverages The foods and beverages according to the present technology are those produced using the aforementioned saccharide-reducing agent.

[0044] (1) Raw materials The saccharide-containing foods and beverages used in this technology, or the raw materials that can be used in foods and beverages according to this technology, are not particularly limited in terms of origin, type, etc., as long as they do not impair the effect of this technology, and can be freely selected according to the target foods and beverages. In this technology, raw materials containing sucrose (sucrose-containing raw materials) are particularly preferred.

[0045] The origin of the raw materials is not particularly limited as long as it does not impair the action or effect of this technology, and can be freely selected. Examples of raw materials for fruit juice used in fruit juice beverages include orange (including mandarin), pineapple, grapefruit, apple, grape, peach, strawberry, banana, mango, melon, apricot, lemon, kiwi, blueberry, pear, grape, pomegranate, lime juice, and other fruit juices listed in the Fruit Beverage Quality Labeling Standards.

[0046] Furthermore, examples of vegetable raw materials used in the vegetable juice beverage of the present invention include vegetable juices from carrots, spinach, onions, tomatoes, celery, bell peppers, pumpkins, corn, carrots, cucumbers, beets, parsley, lettuce, spinach, cauliflower, broccoli, kale, red bell peppers, ginger, green peppers, and red cabbage.

[0047] Examples of raw material forms include liquid, paste, and slurry, with liquid being more preferable. Furthermore, the raw materials that can be used in the saccharide-containing foods and beverages used in this technology, or in foods and beverages according to this technology, can be used as is, or in liquid, paste, or slurry form. Preferably, they can also be used in the form of fresh juice, concentrated juice, paste-like juice, powdered juice, etc.

[0048] (2) Amount of saccharides contained in foods and beverages The amount of saccharides (monosaccharides and disaccharides, hereinafter the same) contained in foods and beverages according to this technology is not particularly limited, but it is preferable that the amount is reduced compared to the amount of saccharides in foods and beverages produced without using the saccharide-reducing agent according to this technology. Specifically, the total amount of saccharides contained in foods and beverages according to this technology is, for example, 70% or less, preferably 65% or less, more preferably 60% or less, and even more preferably 57% or less, 50% or less, or 40% or less compared to the total amount of saccharides in foods and beverages produced without using the saccharide-reducing agent according to this technology.

[0049] In particular, it is preferable that the amount of glucose and sucrose contained in the foods and beverages according to this technology is reduced compared to the amount of glucose and sucrose contained in foods and beverages produced without using the saccharide-reducing agent according to this technology. Specifically, the amount of glucose and sucrose contained in the foods and beverages according to this technology is, for example, 70% or less, preferably 65% or less, more preferably 60% or less, and even more preferably 57% or less compared to the amount of glucose and sucrose contained in foods and beverages produced without using the saccharide-reducing agent according to this technology. For each of these, the amount of glucose contained in the foods and beverages according to this technology is, for example, 95% or less, preferably 93% or less, more preferably 90% or less, 80% or less, 70% or less, and 60% or less compared to the amount of glucose contained in foods and beverages produced without using the saccharide-reducing agent according to this technology. Also, the amount of sucrose contained in the foods and beverages according to this technology is, for example, 60% or less, preferably 50% or less, and more preferably 40% or less compared to the amount of sucrose contained in foods and beverages produced without using the saccharide-reducing agent according to this technology.

[0050] (3) Types of foods and beverages The types of foods and beverages according to this technology are not particularly limited as long as they do not impair the effect of this technology, but beverages are preferred. Examples of beverages include vegetable juices and fruit juices. Other examples of foods according to this technology include Japanese sweets (mochi, manju, daifuku, castella, anko, etc.), Western sweets (cream, cake, ice cream, etc.), soups, sauces, dressings, etc.

[0051] 3. Method for producing foods and beverages, and method for reducing saccharides in saccharide-containing foods and beverages The method for producing foods and beverages and the method for reducing the saccharide in saccharide-containing foods and beverages according to this technology are methods including a step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with glucose oxidase (hereinafter also referred to as the “glucose oxidase treatment step”) and a step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with a-glucosidase and / or P-D-fructofuranosidase (hereinafter also referred to as the “a-glucosidase treatment step” and / or the “P-D-fructofuranosidase treatment step”).

[0052] The glucose oxidase treatment step and the a-glucosidase treatment step and / or the P-D-fructofuranosidase treatment step can be carried out simultaneously or in any order. That is, saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages may be treated with glucose oxidase and a- glucosidase and / or p-D-fructofuranosidase simultaneously or separately. If treated separately, the order is not particularly limited, and an enzyme deactivation step may be performed between the treatments with each enzyme as needed.

[0053] In addition, depending on the type of foods and beverages or the like, it is possible to perform general food producing steps such as a recovery step before or after each process, or simultaneously with each step, to the extent that it does not impair the effect of this technology. The following describes each step in detail.

[0054] (1) Glucose oxidase treatment step The glucose oxidase treatment step is a step in which saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages are treated with glucose oxidase. The amount of glucose oxidase added in the glucose oxidase treatment step can be freely set as long as it does not impair the effect of this technology. The specific amount of glucose oxidase added in this technology is the same as the glucose oxidase content in the saccharide-reducing agent mentioned above, so the explanation is omitted here.

[0055] The various conditions of the glucose oxidase treatment step can be freely set as long as they do not impair the effect of this technology. For example, the pH, temperature, treatment time, etc. can be set according to the physicochemical properties of the glucose oxidase used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. The pH can be set, for example, to pH 2.0 to 11.0, preferably to pH 2.5 to 10.0, and more preferably to pH 3.0 to 5.5. The temperature can be set, for example, to 10°C to 70°C, preferably to 20°C to 50°C, and more preferably to 25°C to 45°C. The treatment time can be set, for example, to 15 minutes to 120 hours, preferably to 30 minutes to 72 hours, and more preferably to 12 hours to 48 hours. The optimal reaction conditions can be determined through preliminary experiments.

[0056] (2) a-Glucosidase treatment step The a-glucosidase treatment step is a step in which saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages are treated with a-glucosidase. The amount of a-glucosidase added in the a-glucosidase treatment step can be freely set as long as it does not impair the effect of this technology. The specific amount of a-glucosidase added in this technology is the same as the amount of a-glucosidase contained in the saccharide-reducing agent mentioned above, so the explanation is omitted here.

[0057] The various conditions for the a-glucosidase treatment step can be freely set as long as they do not impair the effect of this technology. For example, the pH, temperature, treatment time, etc. can be set according to the physicochemical properties of the a-glucosidase used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. The pH can be set, for example, to pH 2.0 to 11.0, preferably to pH 2.5 to 10.0, and more preferably to pH 3.0 to 5.5. The temperature can be set, for example, to 10°C to 70°C, preferably to 20°C to 50°C, and more preferably to 25°C to 45°C. The treatment time can be set, for example, to 15 minutes to 120 hours, preferably to 30 minutes to 72 hours, and more preferably to 12 hours to 48 hours. The optimal reaction conditions can be determined through preliminary experiments.

[0058] (3) P-D-fructofuranosidase treatment step The P-D-fructofuranosidase treatment step is a step in which saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages are treated with P-D-fructofuranosidase. The amount of P-D-fructofuranosidase added in the P-D-fructofuranosidase treatment step can be freely set as long as it does not impair the effect of this technology. The specific amount of P-D-fructofuranosidase added in this technology is the same as the amount of P-D-fructofuranosidase contained in the saccharide-reducing agent mentioned above, so the explanation is omitted here.

[0059] The various conditions for the P-D-fructofuranosidase treatment step can be freely set as long as they do not impair the effect of this technology. For example, the pH, temperature, treatment time, etc. can be set according to the physicochemical properties of the P-D-fructofuranosidase used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. The pH can be set, for example, to pH 2.0 to 11.0, preferably to pH 2.5 to 10.0, and more preferably to pH 3.0 to 5.5. The temperature can be set, for example, to 10°C to 70°C, preferably to 20°C to 65°C, and more preferably to 25°C to 60°C. The treatment time can be set, for example, to 15 minutes to 120 hours, preferably to 30 minutes to 72 hours, and more preferably to 12 hours to 48 hours. The optimal reaction conditions can be determined through preliminary experiments.

[0060] (4) Recovery step The recovery step is a step for recovering foods and beverages that have undergone a glucose oxidase treatment step and an a-glucosidase treatment step and / or a P-D-fructofuranosidase treatment step. The specific recovery method can be freely combined from one or more general recovery methods used in food and beverage production, depending on the type of foods and beverages being produced.

[0061] Foods and beverages produced via a glucose oxidase treatment step and an a-glucosidase treatment step and / or a P-D-fructofuranosidase treatment step are characterized by a reduced amount of saccharides (monosaccharides + disaccharides) contained. In other words, this technology allows for a recovery step for recovering foods and beverages with a reduced content of saccharides (monosaccharides + disaccharides) compared to foods and beverages produced without performing the glucose oxidase treatment step and the a-glucosidase treatment step and / or the P-D-fructofuranosidase treatment step.

[0062] Some embodiments of the method for producing foods and beverages and the method for reducing saccharides in saccharide-containing foods and beverages according to this technology include the following steps (1), (2), (3) and / or (4), and (5). An enzyme deactivation step may be added as needed after one or more steps selected from steps (2), (3), and (4). Steps (2), (3), and (4) can be performed simultaneously or in any order. (1) A step of preparing saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages. (2) A step of treating the prepared saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with glucose oxidase. (3) A step of treating the prepared saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with a-glucosidase. (4) A step of treating the prepared saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with P-D-fructofuranosidase. (5) A step of recovering foods and beverages produced through steps (1), (2), and (3) and / or (4). EXAMPLES

[0063] The present invention will be described in more detail below based on the following examples. The examples described below are merely representative examples of the present invention and should not be interpreted as narrowing the scope of the invention.

[0064] 1. Raw materials / enzymes used The raw materials and enzymes used in the examples are shown in Table 1 below.

[0065] [Table 1] Name of substance Product name Distributor Fruit juice 1 V8® Fruit & Vegetable Blends Pomegranate Blueberry V8 Fruit juice 2 Ceres Peach Fruit Juice Blend: 100% Natural, Refreshing, and Delicious Ceres Fruit juice 3 Orange Peach Mango Juice TRADER JOE'S Vegetable juice V8® Vegetable Juice Original 100% Vegetable Juice V8 Glucose oxidase 1 Acremonium chrysogenum -derived glucose oxidase Amano Enzyme Inc. Glucose oxidase 2 Aspergillus niger -derived glucose oxidase Amano Enzyme Inc. Catalase Aspergillus niger- derived catalase Amano Enzyme Inc. P-D-fructofuranosidase Saccharomyces cerevisiae- derived P-D-fructofuranosidase Sigma-Aldrich a-glucosidase Aspergillus niger- derived a-glucosidase Amano Enzyme Inc.

[0066] 2. Method for measuring enzyme activity [Method for measuring glucose oxidase activity] An appropriate amount of enzyme was weighed and dissolved or uniformly dispersed in cooled pH 7.0 potassium phosphate / sodium hydroxide buffer (0.1 mol / L) to make 50 mL of the sample solution. 2.50 g of D(+)-glucose was weighed and dissolved in water to make 25 mL of the substrate solution. 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 solution (25 units / mL), and 0.1 mL of 4-aminoantipyrine solution (1^250) were placed in a quartz cell and warmed at 37°C for 10 minutes. 0.1 mL of the sample solution was added to this solution, mixed well, and warmed at 37°C to make the test solution. Separately, a comparison solution was prepared using pH 7.0 potassium phosphate / sodium hydroxide buffer (0.1 mol / L) or water instead of the sample solution, in the same procedure as the test solution. The absorbance of the test solution and the comparison solution at a wavelength of 500 nm was measured 2 minutes and 5 minutes after the addition of the sample solution. The amount of oxidized glucose was quantified from the molar extinction coefficient of the generated quinone imine dye. The amount of enzyme required to oxidize 1 pmol of glucose per minute was defined as 1 unit (1 U).

[0067] [Method for measuring a-glucosidase activity] The activity was measured using Method 2 of a-glucosidase activity test in the 9th edition of the Japan’s Specification and Standards for Food Additives. (1) 2.0 g of a-methyl-D-glucoside was weighed out and dissolved in water to make 100 mL to prepare an a-methyl-D-glucoside solution. 1 mL of the a-methyl-D-glucoside solution and 1 mL of 0.02 mol / L acetic acid / sodium acetate buffer (pH 5.0) were weighed into a test tube, left to stand at 40°C for 10 to 15 minutes, and then 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) was added and mixed well. This was left to stand at 40°C for exactly 60 minutes. After exactly 60 minutes, it was placed in a boiling water bath and heated for exactly 5 minutes, and then cooled under running water. (2) 3 mL of the color-developing solution from the glucose measurement kit (Fujifilm Wako Pure Chemical Corp., hereinafter the same) was measured into a test tube, 0.2 mL of the reaction solution obtained in (1) was added and shaken well, and then left to stand at 40°C for exactly 5 minutes. The absorbance (E60) of this solution at a wavelength of 505 nm was measured with water as a control. Separately, 1 mL of 0.02 mol / L acetic acid / sodium acetate buffer (pH 5.0) and 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) were measured into test tubes as a blank, heated in a boiling water bath for exactly 5 minutes, and then cooled under running water. After cooling, 1 mL of a-methyl-D-glucoside solution was added and the absorbance (E0) was measured in the same manner as above. (3) Either glucose standard solution I or II from the glucose measurement kit was diluted with water to the specified concentration (20 mg / dL, 40 mg / dL). 3 mL of the color development solution from the glucose measurement kit was measured into a test tube, 0.2 mL of the above glucose solution was added to each, shaken well, and then allowed to stand at 40°C for exactly 5 minutes. For this solution, the absorbance (ES) was measured at a wavelength of 505 nm using water as a control. Separately, the absorbance (EB) was measured using 0.2 mL of water instead of the glucose solution as a blank by performing the same procedure as above. A glucose calibration curve was created from the obtained absorbances, and the amount of glucose (pg) (G) when the absorbance difference was 1.000 was determined. (4) The amount of enzyme that produces 1 pg of glucose in 60 minutes was defined as 1 unit (1 U), and was calculated using the following formula. a-glucosidase activity (U / g, U / mL) = (E60 - E0) * G * 2.5 / 0.1 * n / 0.5 E60: Absorbance of the reaction solution E0: Absorbance of the blank solution G: Amount of glucose (pg) when the absorbance difference is 1.000 (calculated from the glucose calibration curve) 2.5: Volume of reaction system (mL) 0.1: Amount of reaction solution collected (mL) 0.5: Amount of enzyme solution collected (mL) n: Dilution factor per 1 g or 1 mL of sample

[0068] [Method for measuring P-D-fructofuranosidase activity] (1) Preparation of reagents A 100 mM sodium acetate solution (prepared by dissolving sodium acetate trihydrate (Sigma-Aldrich “#S8625”) in purified water) adjusted to pH 4.5 with 1 M HCl at 55°C was used as the buffer. Sucrose (Sigma-Aldrich “#S7903”) was dissolved in 100 mM sodium acetate buffer (pH 4.5) to prepare a 1.0% w / v sucrose substrate solution. Immediately before use, a solution containing 0.3 - 0.4 U / mL P—D-fructofuranosidase was dissolved in cold purified water to prepare the enzyme solution. 1N NaOH (Supelco “#S2567”) was diluted 1:2 with purified water to prepare a 0.5M NaOH dilution. p-hydroxybenzhydrazide (PAHBAH) (Sigma-Aldrich “#H9882”) was dissolved in 0.5 M NaOH to prepare a 0.5% w / v PAHBAH reagent. A 5.56 mM glucose standard solution was prepared by dissolving glucose (Sigma-Aldrich “#G8270”) in purified water. (2) Measurement method (2-1) Preparation of glucose standard solution 1.0 mL of purified water was used as a glucose standard solution blank. Purified water (0.95 mL, 0.90 mL, 0.80 mL, 0.70 mL, 0.60 mL, 0.50 mL) was mixed with the glucose standard solutions prepared above (0.05 mL, 0.10 mL, 0.20 mL, 0.30 mL, 0.40 mL, 0.50 mL) to prepare each glucose standard solution, which was then equilibrated at 55°C. (2-2) Preparation of blank test solution A blank test solution was prepared by mixing 0.10 mL of purified water with 0.90 mL of the sucrose substrate solution prepared above, and equilibrated at 55°C. (2-3) Preparation of the test solution A mixture of 0.90 mL of the sucrose substrate solution prepared above and 0.10 mL of the enzyme solution prepared above was used as the test solution and incubated at 55°C for exactly 20 minutes. (2-4) Measurement of absorbance 0.10 mL was measured from each of the glucose standard solutions, blank test solutions, and test solutions prepared above, and added to a tube containing 2.90 mL of the 0.5% w / v PAHBAH reagent prepared above. The mixture was quickly mixed, placed in a boiling water bath, heated for 5 minutes, and allowed to cool to room temperature. After cooling, the mixture was mixed, and the solution was transferred to a suitable cuvette. The absorbance of all reaction mixtures at 410 nm was measured using a suitable spectrophotometer. The amount of enzyme required to hydrolyze 1 pmol of sucrose into invert sugar per minute was defined as 1 unit (1 U).

[0069] 3. Experimental Examples <Experimental Example 1> (1) Experimental method 50 mL of commercially available fruit juice (V8 Pomegranate Blueberry Juice) was pre-incubated at 35°C for 10 minutes with stirring. Next, each enzyme shown in Table 3 below was added, and the mixture was treated with enzymes at 35°C for 24 hours with stirring. After the enzymatic reaction was complete, the enzyme activity was deactivated by treating at 90°C for 20 minutes, and the mixture was cooled to room temperature to obtain fruit juice 1.

[0070] (2) Sugar composition analysis The obtained fruit juice 1 was diluted fivefold with deionized water and then centrifuged at 13300G for 10 minutes. The supernatant was diluted twofold with ethanol (final ethanol concentration of 50% or more) and passed through a membrane filter (0.22 um), and analyzed using high-performance liquid chromatography (differential refractive index detector (RI) “RID-20A” Shimadzu Corporation). Details of the column and each condition are shown in Table 2 below. The saccharide composition was analyzed and quantified using standards (Sigma-made) for each saccharide. In addition, the ratio of glucose and sucrose to the total saccharide content (saccharide composition) was calculated from the obtained saccharide composition.

[0071] [Table 2] Column Asahipak NH2P-50 4E (250x4.6mm, 5um, Shodex®) (Resonac Corp.) Solvent 70% acetonitrile / water (v / v) Column temperature 30r Detector RI (RID-20A : Shimadzu Corp.) Flow rate 1.0mL / min

[0072] (3) Results The results are shown in Table 3 below.

[0073] [Table 3] Control Example Comparative Example Example 1 1 2 3 4 5 6 1 2 Enzyme used Glucose oxidase 1 U / g-total sugar - 158 - - 158 - - - - Glucose oxidase 2 - - - - - 594 - 594 150 Catalase - - 1979 - 1979 1979 - - - P -D-fruc tofUranosidase - - - - - - 3000 - 3000 a-glucosidase - - - 118750 - - - 118750 - Saccharide analysis value Glucose 3.34 3.37 2.78 4.21 2.79 3.06 4.32 1.98 2.94 Glucose (reduction rate) 0% -1% 17% -26% 16% 8% -29% 41% 12% Sucrose 1.88 1.45 1.02 0.10 1.02 0.44 0.02 0.03 0.02 Sucrose (reduction rate) 0% 22% 46% 94% 45% 77% 99% 99% 99% Gluc ose + Sucros e (Total) 5.22 4.82 3.79 4.32 3.81 3.50 4.34 2.01 2.96 Glucose + Sucrose (residual saccharide rate) 100% 92% 73% 83% 73% 67% 83% 38% 57% Glucose + Sucrose (reduction rate) 0% 8% 27% 17% 27% 33% 17% 62% 43%

[0074] (4) Discussion As shown in Comparative Examples 1-3 and 6, when glucose oxidase, catalase, P-D-fructofuranosidase, or a-glucosidase was used alone, the reduction rate of saccharides was less than 30%. Also, as shown in Comparative Examples 4 and 5, when glucose oxidase and catalase were used in combination, the reduction rate of saccharides was less than 35%. On the other hand, Example 1, which used glucose oxidase and a-glucosidase in combination, and Example 2, which used glucose oxidase and P-D-fructofuranosidase in combination, showed a high reduction rate of over 40%.

[0075] <Experimental Example 2> (1) Experimental method 50 mL of commercially available fruit juice (Ceres Peach Juice) was preincubated at 35°C for 10 minutes with stirring. Each enzyme shown in Table 4 below was added, and the mixture was treated at 35°C for 24 hours. After the enzymatic reaction was complete, the enzyme activity was deactivated by treating at 90°C for 20 minutes, and the mixture was cooled to room temperature to obtain fruit juice 2.

[0076] (2) Saccharide composition analysis The obtained fruit juice 2 was subjected to a saccharide composition analysis using the same method as in Experimental Example 1.

[0077] (3) Results The results are shown in Table 4 below.

[0078] [Table 4] Control Example Comparative Example Example 2 7 8 9 10 11 12 13 3 Enzyme used Glucose oxidase 2 U / g-total sugar - 313 625 1875 - 625 625 625 625 Catalase - - - - - 1042 2083 6250 - a-glucosidase - - - - 125000 - - - 125000 Saccharide analysis value Glucose 2.23 1.22 1.84 1.77 2.59 1.14 1.42 1.75 1.01 Glucose (reduction rate) 0% 45% 17% 20% -16% 49% 36% 21% 55% Sucrose 1.84 0.71 0.45 0.09 0.11 0.33 0.27 0.14 0.10 Sucrose (reduction rate) 0% 62% 76% 95% 94% 82% 85% 92% 95% Glucose + Sucrose (Total) 4.07 1.93 2.29 1.86 2.70 1.48 1.69 1.89 1.11 Glucose + Sucrose (residual saccharide rate) 100% 47% 56% 46% 66% 36% 42% 46% 27% Glucose + Sucrose (reduction rate) 0% 53% 44% 54% 34% 64% 58% 54% 73%

[0079] (4) Discussion As shown in Table 4, Example 3, which used glucose oxidase and a-glucosidase in combination, showed a higher reduction rate compared to Comparative Examples 7-10, which used glucose oxidase or a-glucosidase alone, and Comparative Examples 11-13, which used glucose oxidase and catalase in combination.

[0080] <Experimental Example 3> (1) Experimental method 50 mL of commercially available fruit juice (TRADER JOE'S Orange Peach Mango Juice) was pre-incubated at 35°C for 10 minutes with stirring. The enzymes shown in Table 5 below were added and the mixture was treated at 35°C for 24 hours. After the enzymatic reaction was complete, the mixture was treated at 90°C for 20 minutes to deactivate the enzyme activity, and then cooled to room temperature to obtain fruit juice 3.

[0081] (2) Saccharide composition analysis The obtained fruit juice 3 was subjected to a saccharide composition analysis using the same method as in Experimental Example 1.

[0082] (3) Results The results are shown in Table 5 below.

[0083] [Table 5] Control Example Comparative Example Example 3 14 15 4 5 6 Enzyme used Glucose oxidase 2 U / g-total sugar - 391 0 391 781 2344 a-glucosidase - 0 156250 156250 156250 156250 Saccharide analysis value Glucose + Sucrose (Total) 7.64 5.29 4.89 3.62 3.51 4.32 Glucose + Sucrose (residual saccharide rate) 100% 69% 64% 47% 46% 57% Glucose + Sucrose (reduction rate) 0% 31% 36% 53% 54% 43%

[0084] (4) Discussion As shown in Table 5, Examples 4 to 6, which used glucose oxidase and a-glucosidase in combination, showed a higher reduction rate compared to Comparative Examples 14 and 15, which used glucose oxidase or a-glucosidase alone.

[0085] <Experimental Example 4> (1) Experimental method 50 mL of commercially available fruit juice (V8 Vegetable Juice) was preincubated at 35°C for 10 minutes with stirring. Each enzyme shown in Table 6 below was added, and the mixture was treated at 35°C for 24 hours. After the enzymatic reaction was complete, the enzyme activity was deactivated by treating at 90°C for 20 minutes, and the mixture was cooled to room temperature to obtain fruit juice 4.

[0086] (2) Saccharide composition analysis The obtained fruit juice 4 was subjected to a saccharide composition analysis using the same method as in Experimental Example 1.

[0087] (3) Results The results are shown in Table 6 below.

[0088] [Table 6] Control Example Example 4 7 8 9 Enzyme used Glucose oxidase 2 U / g-total sugar - 109 219 656 a-glucosidase - 43750 43750 43750 Saccharide analysis value Glucose 1.30 0.70 0.56 0.00 Glucose (reduction rate) 0% 47% 57% 100% Sucrose 0.28 0.10 0.07 0.07 Sucrose (reduction rate) 0% 65% 75% 74% Glucose + Sucrose (Total) 1.59 0.79 0.63 0.07 Glucose + Sucrose (residual saccharide rate) 100% 50% 40% 5% Glucose + Sucrose (reduction rate) 0% 50% 60% 95%

[0089] (4) Discussion As shown in Table 6, it was found that increasing the amount of glucose oxidase used in combination with a-glucosidase improved the saccharide reduction rate.

Claims

1. A saccharide-reducing agent for saccharide-containing foods and beverages, comprisingglucose oxidase anda-glucosidase and / or P-D-fructofuranosidase.

2. The saccharide-reducing agent according to claim 1, which reduces glucose and sucrose in saccharide-containing foods and beverages.

3. The saccharide-reducing agent according to claim 1, wherein the saccharide-containing foods and beverages are sucrose-containing foods and beverages.

4. The saccharide-reducing agent according to claim 1, wherein the saccharide-containing foods and beverages are beverages made from fruits and / or vegetables as raw materials.

5. Foods and beverages using the saccharide-reducing agent according to any one of claims 1 to 4.

6. A method for producing foods and beverages, comprisinga step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with glucose oxidase, anda step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with a-glucosidase and / or P-D-fructofuranosidase.

7. A method for reducing saccharides in saccharide-containing foods and beverages, comprisinga step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with glucose oxidase, anda step of treating saccharide-containing foods and beverages or raw materials for the saccharide-containing foods and beverages with a-glucosidase and / or P-D-fructofuranosidase.