WO2022138771A1 - Enzyme-treated composition for stevia plants - Google Patents

Enzyme-treated composition for stevia plants Download PDF

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Publication number
WO2022138771A1
WO2022138771A1 PCT/JP2021/047718 JP2021047718W WO2022138771A1 WO 2022138771 A1 WO2022138771 A1 WO 2022138771A1 JP 2021047718 W JP2021047718 W JP 2021047718W WO 2022138771 A1 WO2022138771 A1 WO 2022138771A1
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weight
enzyme
ppm
rebaugioside
composition
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PCT/JP2021/047718
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French (fr)
Japanese (ja)
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聡一郎 浦井
浩二 長尾
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サントリーホールディングス株式会社
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Priority to CN202180086857.8A priority Critical patent/CN116634887A/en
Publication of WO2022138771A1 publication Critical patent/WO2022138771A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof

Definitions

  • the present invention relates to an enzyme-treated composition of Stevia plant and a method for producing the composition.
  • the present invention also relates to a food or drink containing the composition produced by the above-mentioned production method, and a method for enhancing the sweetness of the food or drink.
  • Non-Patent Documents 1 and 2 a method using an enzyme when extracting a stevia plant is also known.
  • the present invention is as shown below.
  • An enzyme-treated composition of Stevia plant A composition in which the weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more.
  • the composition according to [1] which contains 100 to 10000 ppm of amino acids with respect to the total weight of the enzyme-treated composition.
  • the amino acid is one or more selected from the group consisting of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan and cystine.
  • a method for producing an enzyme-treated composition of Stevia plant Extracting Stevia plants with a solvent and A method comprising enzymatically treating the residue after extraction with hemicellulase and / or protease. [10] The method according to [9], wherein the residue is a residue after at least one extraction. [11] The method according to [9] or [10], further comprising solid-liquid separation after the enzymatic treatment. [12] The method according to any one of [9] to [11], wherein the amount of the hemicellulase and / or protease added is 0.5 to 15% by weight based on the dry weight of the Stevia plant.
  • an enzyme-treated composition of a Stevia plant containing a steviol glycoside and an amino acid it is possible to provide a food or drink containing the composition and a method for enhancing the sweetness of the food or drink.
  • stevia plant includes the whole plant and parts of the plant of the stevia plant.
  • plant part includes the leaves, stems, flowers, roots of the Stevia plant and optionally cuts thereof, as well as the cells and tissues of the Stevia plant.
  • enzyme treatment means a composition obtained by subjecting the residue after extracting the stevia plant with a solvent to an enzyme treatment. The enzyme treatment will be described later.
  • the enzyme-treated composition of Stevia plant contains a large amount of amino acids.
  • the enzyme-treated composition of Stevia plants may contain small amounts of steviol glycosides.
  • the enzyme-treated composition of the Stevia plant is preferably substantially free of steviol glycosides, but inevitably contains a small amount of steviol glycosides derived from the Stevia plant that cannot be completely removed. May be.
  • the amino acid preferably contains one or more of the 20 amino acids that form the protein. Specifically, alanine (Ala), arginine (Arg), aspartic acid (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His). , Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionin (Met), Phenylalanine (Phe), Proline (Pro), Serin (Ser), Threonin (Threonine) (Thr), Tryptophan (Trp), Tyrosine (Tyr) and valin (Val).
  • the amino acid is preferably an amino acid derived from Stevia plant.
  • the term "derived from a Stevia plant” means an amino acid obtained from the Stevia plant.
  • the amino acids contained in the enzyme-treated composition can be of Stevia plant origin, unless other components including amino acids are added.
  • the steviol glycosides include rebaugioside A, rebaugioside B, rebaugioside C, rebaugioside D, rebaugioside E, rebaugioside F, rebaugioside G, rebaugioside I, rebaugioside J, rebaugioside M, and rebaugioside N.
  • the enzyme-treated composition comprises rebaugioside A, rebaugioside B, rebaugioside C, rebaugioside D, rebaugioside E, rebaugioside F, rebaugioside G, rebaugioside I, rebaugioside M, rebaugioside N, steviolbioside, steviolbioside.
  • TSG total steviol glycoside
  • steviol glycoside content or "steviol glycoside concentration” means the total amount and total concentration of the total steviol glycosides listed above, unless otherwise specified. Steviol glycosides can be quantified by known methods using a liquid chromatograph mass spectrometer.
  • the weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is, for example, 0.3 or more, 0.5 or more, 0.7 or more, 1 or more, 1.2 or more, 1. 5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more , 18 or more, 19 or more, 20 or more, and the like.
  • the higher the ratio of amino acids the higher the purity of the material, which is preferable.
  • the amino acids are, for example, 100 to 10000 ppm, 150 to 10000 ppm, 200 to 10000 ppm, 250 to 10000 pm, 300 to 10000 ppm, 350 to 10000 ppm, 100 to 9000 ppm with respect to the total weight of the enzyme-treated composition.
  • the steviol glycoside is 3000 ppm or less, 2500 ppm or less, 2300 ppm or less, 2000 ppm or less, 1500 ppm or less, 1000 ppm or less, 750 ppm or less, 720 ppm or less, 700 ppm or less with respect to the total weight of the enzyme-treated composition.
  • the amino acid content contained in the enzyme-treated composition of the Stevia plant is, for example, 0.1 to 50% by weight and 0.5 to 50% by weight with respect to the solid content of the enzyme-treated composition.
  • the protease may be an exopeptidase that cleaves from the end of the peptide chain or an endopeptidase that cleaves from the center of the peptide chain. Both can be used together. Further, the peptidase may be derived from a plant such as papaya, pineapple, ginger, fig or kiwifruit, or may be derived from a bacterium such as jiuqu, mold or natto fungus. Hemicellulase and protease can be purchased and used on the market.
  • the preferred dried leaves of Stevia plants to be used in the production method of the present invention are 100 to 800 mg of arginine, 100 to 400 mg of histidine, and 100 to 600 mg of tyrosine in 100 g of dried leaves when the water content is 10% by weight or less. It may contain 100-800 mg of isoleucine, 100-1000 mg valine, 100-900 mg glycine, 400-2000 mg glutamic acid, 100-800 mg threonine, 50-400 mg tryptophan.
  • the solvent may be heated.
  • the temperature at the time of extraction may be, for example, 10 to 80 ° C, 25 to 80 ° C, 30 to 75 ° C, 35 to 70 ° C, 40 to 65 ° C, 45 to 70 ° C, and preferably 45 to 70 ° C. Is.
  • the lower limit value and the upper limit value of the temperature may be, for example, ⁇ 1 ° C., ⁇ 2 ° C., ⁇ 3 ° C., ⁇ 4 ° C., ⁇ 5 ° C. of each temperature.
  • Extraction may be performed not only once but multiple times. By performing multiple extractions, more steviol glycosides contained in the leaves are extracted.
  • the extraction rate of the Stevia plant can be, for example, 10 to 60% as a guide, preferably 20 to 50%, more preferably 25 to 45%, and 30 to 40%. More preferred.
  • the extraction rate is the ratio (%) of the amount of solid content in the extract to the weight of the raw material Stevia plant.
  • the residue after extraction is treated with hemicellulase and / or protease. Includes enzymatic treatment.
  • hemicellulase and / or protease those described in "1. Enzyme-treated composition of Stevia plant" can be used.
  • the post-extraction residue of the Stevia plant is at least one post-extraction residue.
  • the residue is the residue after at least two extractions.
  • the residue is the residue after at least 3 extractions.
  • the production method according to one aspect of the present invention may further include making a slurry of the residue after extraction of Stevia plant before or after adding the enzyme in the step (B).
  • the solvent used for the slurry include the aqueous solvent described in (A) above.
  • the concentration of the aqueous slurry is not particularly limited, but is preferably 5 to 70%, more preferably 5 to 50%, still more preferably 7 to 30%, in consideration of fluidity and treatment efficiency.
  • hemicellulase and protease are preferably used in combination. This makes it possible to increase the concentration of amino acids contained in the enzyme-treated composition.
  • protease treatment may be performed after hemicellulase treatment, or hemicellulase treatment and protease treatment may be performed at the same time. More specifically, hemicellulase may be added first in the step (A), then a protease may be added in the step (B), or both hemicellulase and the protease may be added in the step (B). You may.
  • the total amount added is 0.5 to 15% by weight, 1 to 14.5% by weight, 1.5 to 14% by weight, and 2 to 13 to the dry weight of the Stevia plant. .5% by weight, 2.5 to 13% by weight, 3 to 12.5% by weight, 3.5 to 12% by weight, 4 to 11.5% by weight, 4.5 to 11% by weight, 5 to 10.5 It may be% by weight, 5 to 10% by weight, or the like.
  • the amount of each enzyme added when hemicellulase and protease are used in combination can be appropriately adjusted based on the above total amount of addition.
  • the amount of hemicellulase added is 0.5 to 15% by weight, 1 to 14% by weight, 1.5 to 13% by weight, 2 to 12% by weight, 2.5 to 25% by weight based on the dry weight of the Stevia plant. It may be 11% by weight, 3 to 10% by weight, or the like.
  • the amount of protease added is 0.5 to 15% by weight, 1 to 14% by weight, 1.5 to 13% by weight, 2 to 12% by weight, 2.5 to 25% by weight based on the dry weight of the Stevia plant. It may be 11% by weight, 3 to 10% by weight, or the like.
  • the addition amount when hemicellulase or protease is used alone can be added based on the total addition amount when these are used in combination.
  • the time of enzyme treatment is, for example, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 18 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 18 hours, 3 to. 48 hours, 3 to 36 hours, 3 to 24 hours, 3 to 18 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to 18 hours, 5 to 48 hours, 5 to 36 hours, 5 to 24 hours, 5-18 hours, 6-48 hours, 6-36 hours, 6-24 hours, 6-18 hours, 7-48 hours, 7-36 hours, 7-24 hours, 7-18 hours, 8- 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 18 hours, 9 to 48 hours, 9 to 36 hours, 9 to 24 hours, 9 to 18 hours, 10 to 48 hours, 10 to 36 hours, 10 to It may be 24 hours, 10 to 18 hours, 10 to 16 hours, or the like.
  • the time of the enzyme reaction can be appropriately adjusted in consideration of the optimum temperature of the enzyme to be used, the amount of the enzyme added, and the like.
  • the temperature of the enzyme reaction can be appropriately changed depending on the type of enzyme used, and is, for example, 10 to 80 ° C, 15 to 80 ° C, 20 to 80 ° C, 25 ° C to 80 ° C, 30 to 80 ° C, 35 to. 80 ° C, 40-80 ° C, 10-75 ° C, 15-75 ° C, 20-75 ° C, 25 ° C-75 ° C, 30-75 ° C, 35-75 ° C, 40-75 ° C, 10-70 ° C, 15 It may be about 70 ° C., 20 to 70 ° C., 25 ° C. to 70 ° C., 30 to 70 ° C., 35 to 70 ° C., 40 to 70 ° C., or the like. Further, the temperature can be adjusted to the above range by using a heating device.
  • the heating device is not particularly limited, and examples thereof include a steam heater, an electric heater, a water jacket heater, and an electromagnetic heater.
  • the pH at the time of the enzyme reaction can be appropriately changed depending on the type of enzyme used, for example, 2 to 10, 2.2 to 10, 2.4 to 10, 2.6 to 10, 2.8 to 10. 3-10, 2-9, 2.2-9, 2.4-9, 2.6-9, 2.8-9, 3-9, 2-8, 2.2-8, 2.4 It may be ⁇ 8, 2.6-8, 2.8-8, 3-8, etc.
  • alkaline agents such as alkali metal hydroxides, alkaline earth metal hydroxides, silicates, carbonates and hydrogen carbonates
  • organic systems such as citric acid, lactic acid and acetic acid.
  • inorganic pH adjusters such as phosphoric acid, hydrochloric acid, sulfuric acid, and carbon dioxide can be used.
  • the Brix (solid content) of the reaction solution during the enzyme treatment is, for example, 0.25 to 30, 0.25 to 25, 0.25 to 20, 0.25 to 18, 0.25 to 16, 0.25 to. 14, 0.25 to 12, 0.25 to 10, 0.25 to 8.0, 0.25 to 6.0, 0.25 to 5.0, 0.25 to 4.0, 0.25 to 3.0, 0.5-30, 0.5-25, 0.5-20, 0.5-18, 0.5-16, 0.5-14, 0.5-12, 0.5- It may be 10, 0.5 to 8.0, 0.5 to 6.0, 0.5 to 5.0, 0.5 to 4.0, 0.5 to 3.0, and the like.
  • the enzyme treatment may be performed while stirring the residue after extraction of the Stevia plant (for example, the slurryed residue) in order to carry out the enzymatic reaction efficiently.
  • the stirring device is not particularly limited, and for example, a vertical axis stirring device, a horizontal axis stirring device, a magnetic stirrer, a shaker, or the like can be used.
  • the residue after extraction of the Stevia plant may be enzymatically treated in a plurality of times.
  • the residue may be enzymatically treated in 2 to 10 times, 2 to 5 times or 2 to 4 times.
  • the production method may include terminating the enzyme reaction by heating the enzyme reaction solution containing the residue to a high temperature.
  • the conditions vary depending on the heat resistance of the enzyme used, but for example, the enzyme can be inactivated by heating the enzyme reaction solution containing the residue to a temperature exceeding 80 ° C, 90 ° C, 95 ° C or 100 ° C. can.
  • the time for inactivating the enzyme may be, for example, 30 seconds to 1 hour, 1 to 30 minutes, 1 to 10 minutes, or the like.
  • a plate heater or a countercurrent contact device can be used for enzyme inactivation.
  • the production method according to one aspect of the present invention may include further concentrating, drying, granulating or the like the enzyme-treated composition which has been subjected to the solid-liquid separation treatment. Treatments such as concentration, drying, and granulation can be carried out by using known methods.
  • Foods include, for example, confectionery, breads, flour, noodles, rice, processed agricultural / forest foods, processed livestock products, processed marine products, milk / dairy products, fats / oils / fat processed products, seasonings or other food materials. And so on.
  • Beverages include, for example, carbonated beverages, sports drinks, flavored water, fruit juice beverages, alcoholic beverages, non-alcoholic beverages, beer-taste beverages such as beer and non-alcoholic beer, coffee beverages, tea beverages, cocoa beverages, nutritional beverages, and functional beverages. And so on.
  • carbonated drinks include sparkling drinks, colas, diet colas, ginger ales, ciders, fruit juice flavored carbonated drinks, and carbonated waters to which fruit juice flavor is imparted.
  • the beverage has a stevia plant-derived amino acid content of, for example, 1-600 ppm, 10-600 ppm, 20-600 ppm, 30-600 ppm, 40-600 ppm, 50-600 ppm, 1-500 ppm.
  • the sweetness of the beverage can be enhanced.
  • the sweetness of sucrose can be enhanced by setting the amino acid content in the above range.
  • the amino acid content may be in the above range to enhance the sweetness of rebaugioside A.
  • the beverage may contain sweeteners other than steviol glycosides.
  • sweeteners are not particularly limited, but are, for example, from sucrose, fructose-glucose liquid sugar, erythritol, mogloside V, corn syrup, aspartame (also referred to as L-phenylalanine compound), sucralose, acesulfame potassium, saccharin and xylitol. It may further contain one or more sweeteners selected from the group. Among them, it is preferable to use a natural sweetener from the viewpoint of refreshing, easy to drink, natural taste, and imparting an appropriate richness, and in particular, fructose-glucose liquid sugar, sucrose, and corn syrup are preferably used.
  • These sweeteners have a Brix equivalent of 5.0 or less, 4.5 or less, 4.0 or less, 3.5 or less, 3.0 or less, 2.5 or less, 2.0 or less in a beverage. It may be contained in an amount corresponding to 1.5 or less, 1.0 or less, 0.5 or less, etc., and the lower limit may be 0.1 or more.
  • the gas content in the effervescent beverage can be defined by the gas pressure.
  • gas pressure refers to carbon dioxide gas in a beverage after the temperature of the effervescent beverage in the container is set to 20 ° C. and then the air in the headspace is once opened to the atmosphere (snift). The gas pressure of.
  • the gas pressure of the effervescent beverage is measured by using the method.
  • the Brix in terms of sucrose in the beverage is not particularly limited, but is preferably 1 to 15, more preferably 3 to 14, still more preferably 5 to 13, and particularly preferably 7 to 11.
  • Brix can be calculated from the known sweetness of each sweetener such as steviol glycoside with respect to sucrose and the content of each sweetener.
  • the relative ratio of the sweetness of various sweeteners to the sweetness 1 of sucrose can be obtained from a known sugar sweetness conversion table (for example, page 11 of "Beverage Glossary", Beverage Japan Co., Ltd.).
  • the relative ratio of sweetness to sweetness 1 of sucrose can be determined by a sensory test.
  • a sample in which sugar is added to pure water from Brix 3.0 to 5.0 in 0.5 increments is prepared, and the sample is equivalent to an aqueous solution of a sweetener having a predetermined concentration.
  • a method of selecting a sugar-added sample having a sweetness intensity can be mentioned.
  • Beverages may contain alcohol.
  • the alcoholic beverage is a beverage containing alcohol, and as described above, the alcohol here means ethyl alcohol (ethanol) unless otherwise specified.
  • the type of alcoholic beverage is not particularly limited as long as it contains alcohol.
  • Alcohol content such as beer, soft drinks, chu-hi and cocktails is 0.05-40v / v%, 1.0-10v / v%, 2.0-9.0v / v% or 3.0-8. It may be a 0v / v% beverage, or may be a beverage having an alcohol content of less than 0.05v / v%, such as non-alcoholic beer, chu-hi taste beverages, and soft drinks.
  • the flavor of the beverage is not particularly limited and can be adjusted to various flavors.
  • the beverage may be an orange-flavored, lemon-flavored, lime-flavored, grape-flavored, ginger-ale-flavored, cassis-flavored, green-tea-flavored, oolong-tea-flavored, black-tea-flavored, coffee-flavored or cola-flavored beverage.
  • the flavor of the beverage of the present invention is an ingredient approved as a food additive such as fruit juice, acidulant, flavoring, plant extract, dairy product, and other flavors, or even if it is not approved, it has a long history of eating. It can be adjusted by adding ingredients that are generally recognized as safe.
  • additives may be added to the beverage as long as the effects of the present invention are not impaired.
  • additives include acidulants, fragrances, vitamins, pigments, antioxidants, emulsifiers, preservatives, seasonings, extracts, pH regulators, quality stabilizers and the like.
  • the energy (total energy amount) of the beverage is not particularly limited, but is 0 to 50 Kcal / 100 ml, 0 to 45 Kcal / 100 ml, 0 to 40 Kcal / 100 ml, 0 to 35 Kcal / 100 ml, 0 to 30 Kcal / 100 ml, 0 to 24 Kcal / 100 ml.
  • the beverage may be prepared as a packaged beverage that has been sterilized by heating and packed in a container.
  • the container is not particularly limited, and examples thereof include PET bottles, aluminum cans, steel cans, paper packs, chilled cups, and bottles.
  • heat sterilization the type is not particularly limited, and for example, it can be performed using ordinary methods such as UHT sterilization and retort sterilization.
  • the temperature of the heat sterilization step is not particularly limited, but is, for example, 65 to 130 ° C., preferably 85 to 120 ° C., for 10 to 40 minutes. However, if a sterilization value equivalent to the above conditions can be obtained, there is no problem in sterilization at an appropriate temperature for several seconds, for example, 5 to 30 seconds.
  • the method for producing food and drink is not particularly limited as long as food and drink containing each of the above components can be obtained.
  • a method for producing a food or drink which comprises obtaining an enzyme-treated composition of a stevia plant and adding the enzyme-treated composition to the food or drink or a raw material thereof. The method is provided.
  • Obtaining the enzyme-treated composition of Stevia plant is as described in "2.
  • the enzyme-treated composition can be added to a food or drink or a raw material thereof at any step in the manufacturing process of the food or drink, for example, when mixing the raw materials of the food or drink or the final taste of the food or drink. It may be done at the time of adjustment.
  • the present invention also relates to a method for enhancing sweetness of food and drink.
  • the present invention relates to a method for enhancing the sweetness of foods and drinks, which comprises blending the composition described in the above "1. Enzyme-treated composition of Stevia plant” into foods and drinks (for example, beverages). The composition is as described in "1. Enzyme-treated composition of Stevia plant” above.
  • blending the composition into foods and drinks is, for example, when mixing raw materials for foods and drinks, as described in "3. Foods and drinks containing enzyme-treated compositions of stevia plants", and foods and drinks.
  • the composition may be added (or blended) to a food or drink or a raw material thereof at the time of final adjustment of the taste quality of.
  • enhancing sweetness means that a food or drink containing an enzyme-treated composition of a stevia plant according to an aspect of the present invention has a sweetness as compared with a food or drink not containing the enzyme-treated composition. It means to have a property that strongly feels or to obtain the property.
  • the sweetness enhancing method in one aspect of the present invention may enhance the sweetness of steviol glycosides and sucrose.
  • the method may enhance the sweetness of sucrose.
  • the method may enhance the sweetness of steviol glycosides.
  • the method may enhance the sweetness of rebaugioside A.
  • the method for enhancing sweetness in one aspect of the present invention may include adding the enzyme-treated composition to foods and drinks so that the content of amino acids derived from Stevia plants is 1 to 600 ppm.
  • the content of the amino acid in foods and drinks is, for example, 10 to 600 ppm, 20 to 600 ppm, 30 to 600 ppm, 40 to 600 ppm, 50 to 600 ppm, 1 to 500 ppm, 10 to 500 ppm, 20 to 500 ppm, 30 to 500 ppm, 40 to 40.
  • the present invention also relates to a food and drink sweetness enhancer. Further, the present invention may be a composition for enhancing the sweetness of food and drink.
  • the term "sweetness enhancer” or “sweetness enhancer composition” means a substance that enhances the sweetness of a food or drink when added to a food or drink (for example, a beverage).
  • the description of the sweetness enhancer also applies to the sweetness enhancer composition.
  • the sweetness enhancer can preferably enhance the sweetness of the food or drink when added to the food or drink without the consumer recognizing the taste of the sweetness enhancer itself.
  • the sweetness enhancer includes the enzyme-treated composition described in "1. Enzyme-treated composition of Stevia plant” above.
  • the active ingredient in the sweetness enhancer may be a Stevia plant-derived amino acid contained in the enzyme-treated composition.
  • the content of the enzyme-treated composition contained in the sweetness enhancer is 30 to 100% by weight, 40 to 99% by weight, 50 to 98% by weight, based on the total weight of the sweetness enhancer. It may be 60 to 97% by weight, 70 to 96% by weight, 80 to 95% by weight, or the like.
  • the sweetness enhancer may consist substantially solely of amino acids derived from Stevia plants. As used herein, the phrase "consisting substantially only of amino acids derived from Steviol plants" means that steviol glycosides and other impurities inevitably contained in the process of producing an enzyme-treated composition may be contained. Means.
  • An enzyme-treated composition of Stevia plant The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
  • a composition containing 100 to 10000 ppm of amino acids and 500 ppm or less of steviol glycoside with respect to the total weight of the enzyme-treated composition is provided.
  • the amino acids are 100-2000 ppm, 150-2000 ppm, 200-2000 ppm, 250-2000 pm, 300-2000 ppm, 350-2000 ppm, 100-1000 ppm, 150-1000 ppm, 200-1000 ppm, 250-1000 pm, 300-1000 ppm.
  • An enzyme-treated composition of Stevia plant The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
  • a composition is provided in which the amino acid content contained in the enzyme-treated composition is 0.1 to 50% by weight based on the solid content of the enzyme-treated composition.
  • the amino acid content is 1.0 to 50% by weight, 2.0 to 40% by weight, 3.0 to 30% by weight, 3.0 to 20% by weight, 3.5 to 20% by weight, 4 It may be 0.0 to 20% by weight, 4.5 to 20% by weight, 5.0 to 20% by weight, 5.5 to 20% by weight, 6.0 to 20% by weight, or the like.
  • a method for producing an enzyme-treated composition of Stevia plant Extracting Stevia plants with a solvent and Provided are methods comprising enzymatically treating the residue after extraction with hemicellulase and / or protease, wherein the reaction solution during the enzymatic treatment has a Brix of 0.25 to 30.
  • the Brix of the reaction solution during the enzyme treatment is 0.25 to 10, 0.25 to 8.0, 0.25 to 6.0, 0.25 to 5.0, 0.5 to 10, It may be 0.5 to 8.0, 0.5 to 6.0, 0.5 to 5.0, or the like.
  • test solution was sealed, hydrolyzed, diluted or concentrated as appropriate after volume determination, and the pH was adjusted to 2.2, and analyzed using an automatic amino acid analyzer.
  • cystine and methionine a performic acid solution is added to the sample, and the oxidized product is concentrated to dryness under reduced pressure, hydrolyzed by adding hydrochloric acid, diluted or concentrated as appropriate after volume determination, and the pH is adjusted to 2.
  • the solution adjusted to 2 was used as a test solution and analyzed using an automatic amino acid analyzer.
  • Example B Confirmation of amino acid amount when various proteases are used
  • Enzymatic treatment is performed using various enzymes by the same method as in the above production example except that the conditions of the enzymatic reaction are set to the conditions shown in Table 4 below.
  • An enzyme-treated composition of Stevia plant was obtained.
  • the amount of amino acids was analyzed under the conditions according to Example A. The results are shown in FIG.
  • the composition of each amino acid shown in FIG. 4 is as shown in Table 5 below.
  • each composition obtained in Example B had an amino acid composition substantially similar to that of the composition obtained in the above-mentioned production example. rice field.
  • Example C Evaluation of sweetness enhancing effect of rebaugioside A Rebaugioside A (manufactured by Morita Chemical Industry Co., Ltd., J-100 (purity 95% or more)) and the stevia plant obtained in the above production example so as to have the concentration shown in Table 6 below.
  • the enzyme-treated composition of No. 1 was added to pure water to prepare an aqueous solution.
  • the enzyme-treated composition was added so as to have the amino acid concentration shown in Table 6.
  • Sample 1 is a control sample that does not contain the enzyme-treated composition. Each sample was evaluated according to the following criteria by panelists who were trained in sensuality (3 persons). The results are shown in Table 7.

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Abstract

A technique for further effectively using stevia plants was required. This composition is an enzyme-treated composition for stevia plants, wherein the weight ratio of amino acid/steviol glycoside in the enzyme-treated composition is at least 0.3.

Description

ステビア植物の酵素処理組成物Enzyme-treated composition of stevia plant
　本発明は、ステビア植物の酵素処理組成物、および該組成物の製造方法に関する。また、本発明は、前記製造方法により製造された組成物を含む飲食品、および飲食品の甘味増強方法にも関する。 The present invention relates to an enzyme-treated composition of Stevia plant and a method for producing the composition. The present invention also relates to a food or drink containing the composition produced by the above-mentioned production method, and a method for enhancing the sweetness of the food or drink.
　キク科ステビア(Stevia rebaudiana)の葉にはジテルペノイドの一種であるステビオール(Steviol)とよばれる二次代謝産物が含まれており、ステビオール配糖体は砂糖の約300倍もの甘味を呈することからカロリーレスの甘味料として食品産業に利用されている。肥満が深刻な社会問題として国際的に発展しており、健康増進および医療費削減の観点からもカロリーレスの甘味料の要望は日々大きくなっている。現在では人工的に合成されたアミノ酸誘導体のアスパルテーム(Aspartame)やアセスルファムカリウム(Acesulfame Potassium)が人工甘味料として利用されているが、ステビオール配糖体のように天然に存在するカロリーレス甘味料はより安全で消費者理解（Public Acceptance）が得られやすいと期待される。 The leaves of Stevia rebaudiana contain a secondary metabolite called Steviol, which is a type of diterpenoid, and steviol glycosides are about 300 times as sweet as sugar, so they are calories. It is used in the food industry as a sweetener for les. Obesity is developing internationally as a serious social problem, and the demand for calorie-less sweeteners is increasing day by day from the viewpoint of health promotion and reduction of medical expenses. Currently, artificially synthesized amino acid derivatives Aspartame and Acesulfame Potassium are used as artificial sweeteners, but naturally occurring calorie-less sweeteners such as steviol glycosides are more common. It is expected that it will be safe and easy to obtain public acceptance.
　近年、ステビア植物を抽出処理した後に発生する残渣の廃棄量を抑える等の目的から、ステビア植物の抽出残渣を有効に活用する取り組みが報告されている（例えば、非特許文献１および２）。また、ステビア植物の抽出時に酵素を利用する手法も知られている（特許文献１）。 In recent years, efforts to effectively utilize the extract residue of Stevia plant have been reported for the purpose of suppressing the amount of waste generated after the extraction process of Stevia plant (for example, Non-Patent Documents 1 and 2). In addition, a method using an enzyme when extracting a stevia plant is also known (Patent Document 1).
米国特許第１０４６３０６５号公報US Pat. No. 10,463065
　このような状況の下、ステビア植物の更なる有効活用を図るための技術が求められていた。 Under such circumstances, a technique for further effective utilization of Stevia plants was required.
　本発明は、下記に示すとおりである。
［１］
　ステビア植物の酵素処理組成物であって、
　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上である、組成物。
［２］
　前記酵素処理組成物の総重量に対して、１００～１００００ｐｐｍのアミノ酸を含む、［１］に記載の組成物。
［３］
　前記アミノ酸は、アルギニン、リジン、ヒスチジン、フェニルアラニン、チロシン、ロイシン、イソロイシン、メチオニン、バリン、アラニン、グリシン、プロリン、グルタミン酸、セリン、スレオニン、アスパラギン酸、トリプトファンおよびシスチンからなる群から選択される１種以上である、［１］または［２］に記載の組成物。
［４］
　前記アミノ酸が、ステビア植物由来のアミノ酸である請求項［１］～［３］のいずれか一項に記載の組成物。
［５］
　前記ステビオール配糖体は、レバウジオシドＡ、レバウジオシドＢ、レバウジオシドＣ、レバウジオシドＤ、レバウジオシドＥ、レバウジオシドＦ、レバウジオシドＧ、レバウジオシドＩ、レバウジオシドＪ、レバウジオシドＫ、レバウジオシドＭ、レバウジオシドＮ、レバウジオシドＯ、レバウジオシドＱ、レバウジオシドＲ、ズルコシドＡ、ズルコシドＣ、ルブソシド、ステビオールモノシド、ステビオールビオシドおよびステビオシドからなる群から選択される１種以上である、［１］～［４］のいずれか一項に記載の組成物。
［６］
　前記酵素処理組成物は、ヘミセルラーゼおよび／またはプロテアーゼにより酵素処理されたものである、［１］～［５］のいずれか一項に記載の組成物。
［７］
　前記ステビア植物は、ステビア植物の葉、茎および／または組織を含む、［１］～［６］のいずれか一項に記載の組成物。
［８］
　［１］～［７］のいずれか一項に記載の組成物を含む、飲食品。
［９］
　ステビア植物の酵素処理組成物の製造方法であって、
　ステビア植物を溶媒を用いて抽出することと、
　ヘミセルラーゼおよび／またはプロテアーゼを用いて前記抽出後の残留物を酵素処理することと、を含む、方法。
［１０］
　前記残留物は、少なくとも１回以上の抽出後の残留物である、［９］に記載の方法。
［１１］
　前記酵素処理後に固液分離することをさらに含む、［９］または［１０］に記載の方法。
［１２］
　前記ヘミセルラーゼおよび／またはプロテアーゼの添加量が、ステビア植物の乾燥重量に対して０．５～１５重量％である、［９］～［１１］のいずれか一項に記載の方法。
［１３］
　酵素処理時間が１～４８時間である、［９］～［１２］のいずれか一項に記載の方法。
［１４］
　酵素処理時のｐＨが２～１０である、［９］～［１３］のいずれか一項に記載の方法。
［１５］
　酵素処理時の温度が１０～８０℃である、［９］～［１４］のいずれか一項に記載の方法。
［１６］
　［９］～［１５］のいずれか一項に記載の方法により得られる、組成物。
［１７］
　［１６］に記載の組成物を含む、飲食品。
［１８］
　［１］～［７］および［１６］のいずれか一項に記載の組成物を飲食品に配合することを含む、飲食品の甘味増強方法。 The present invention is as shown below.
[1]
An enzyme-treated composition of Stevia plant
A composition in which the weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more.
[2]
The composition according to [1], which contains 100 to 10000 ppm of amino acids with respect to the total weight of the enzyme-treated composition.
[3]
The amino acid is one or more selected from the group consisting of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan and cystine. The composition according to [1] or [2].
[4]
The composition according to any one of claims [1] to [3], wherein the amino acid is an amino acid derived from a Stevia plant.
[5]
The steviol glycosides are rebaugioside A, rebaugioside B, rebaugioside C, rebaugioside D, rebaugioside E, rebaugioside F, rebaugioside G, rebaugioside I, rebaugioside J, rebaugioside K, rebaugioside M, rebaugioside N, rebaugioside N, rebaugioside. The composition according to any one of [1] to [4], which is one or more selected from the group consisting of R, zulcoside A, zulcoside C, rubusoside, steviol monoside, steviol bioside and stevioside.
[6]
The composition according to any one of [1] to [5], wherein the enzyme-treated composition is enzymatically treated with hemicellulase and / or protease.
[7]
The composition according to any one of [1] to [6], wherein the stevia plant comprises leaves, stems and / or tissues of the stevia plant.
[8]
A food or drink containing the composition according to any one of [1] to [7].
[9]
A method for producing an enzyme-treated composition of Stevia plant.
Extracting Stevia plants with a solvent and
A method comprising enzymatically treating the residue after extraction with hemicellulase and / or protease.
[10]
The method according to [9], wherein the residue is a residue after at least one extraction.
[11]
The method according to [9] or [10], further comprising solid-liquid separation after the enzymatic treatment.
[12]
The method according to any one of [9] to [11], wherein the amount of the hemicellulase and / or protease added is 0.5 to 15% by weight based on the dry weight of the Stevia plant.
[13]
The method according to any one of [9] to [12], wherein the enzyme treatment time is 1 to 48 hours.
[14]
The method according to any one of [9] to [13], wherein the pH at the time of enzyme treatment is 2 to 10.
[15]
The method according to any one of [9] to [14], wherein the temperature at the time of enzyme treatment is 10 to 80 ° C.
[16]
A composition obtained by the method according to any one of [9] to [15].
[17]
A food or drink containing the composition according to [16].
[18]
A method for enhancing the sweetness of a food or drink, which comprises blending the composition according to any one of [1] to [7] and [16] into the food and drink.
　本発明によれば、ステビオール配糖体およびアミノ酸を含むステビア植物の酵素処理組成物、またその製造方法を提供することができる。また、本発明によれば、前記組成物を含む飲食品、飲食品の甘味増強方法を提供することができる。 According to the present invention, it is possible to provide an enzyme-treated composition of a Stevia plant containing a steviol glycoside and an amino acid, and a method for producing the same. Further, according to the present invention, it is possible to provide a food or drink containing the composition and a method for enhancing the sweetness of the food or drink.
酵素処理中のＢｒｉｘの推移を示す図である。It is a figure which shows the transition of Brix during enzyme treatment. ステビア植物の酵素処理組成物に含まれるアミノ酸量を示す図である。It is a figure which shows the amount of amino acids contained in the enzyme treatment composition of Stevia plant. ステビア植物の酵素処理組成物に含まれるステビオール配糖体量を示す図である。It is a figure which shows the amount of steviol glycoside contained in the enzyme treatment composition of a Stevia plant. 種々の酵素を用いて酵素処理した際の酵素処理組成物に含まれるアミノ酸組成を示す図である。It is a figure which shows the amino acid composition contained in the enzyme treatment composition at the time of enzyme treatment using various enzymes.
　以下、本発明を詳細に説明する。以下の実施の形態は、本発明を説明するための例示であり、本発明をこの実施の形態のみに限定する趣旨ではない。本発明は、その要旨を逸脱しない限り、様々な形態で実施をすることができる。なお、本明細書において引用した全ての文献、および公開公報、特許公報その他の特許文献は、参照として本明細書に組み込むものとする。 Hereinafter, the present invention will be described in detail. The following embodiments are examples for explaining the present invention, and the present invention is not intended to be limited to this embodiment alone. The present invention can be implemented in various forms as long as it does not deviate from the gist thereof. All documents cited in this specification, as well as published publications, patent publications and other patent documents shall be incorporated herein by reference.
　本明細書において、「Reb」および「Reb.」は同じ意味を表すものであり、いずれも「レバウジオシド（rebaudioside）」を意味するものである。 In the present specification, "Reb" and "Reb." Have the same meaning, and both of them mean "rebaudioside".
１．ステビア植物の酵素処理組成物
　本発明は、ステビア植物の酵素処理組成物（以下、単に「酵素処理組成物」または「組成物」とも称する）に関する。具体的には、本発明は、ステビア植物の酵素処理組成物であって、前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が３以上である、組成物に関する。 1. 1. Enzyme-treated composition of Stevia plant The present invention relates to an enzyme-treated composition of Stevia plant (hereinafter, also simply referred to as "enzyme-treated composition" or "composition"). Specifically, the present invention relates to an enzyme-treated composition of a Stevia plant, wherein the weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 3 or more.
　本明細書において、「ステビア植物」には、ステビア植物の植物全体および植物の部分が包含される。「植物の部分」には、ステビア植物の葉、茎、花、根およびこれらを任意に切断したもの、並びにステビア植物の細胞および組織が包含される。
　本明細書において、「ステビア植物の酵素処理組成物」とは、前記ステビア植物を溶媒を用いて抽出した後の残留物（残渣）に酵素処理を施すことで得られる組成物を意味する。酵素処理については後述する。 As used herein, the term "stevia plant" includes the whole plant and parts of the plant of the stevia plant. The "plant part" includes the leaves, stems, flowers, roots of the Stevia plant and optionally cuts thereof, as well as the cells and tissues of the Stevia plant.
As used herein, the term "enzymatically treated composition of stevia plant" means a composition obtained by subjecting the residue after extracting the stevia plant with a solvent to an enzyme treatment. The enzyme treatment will be described later.
　ステビアは、ステビオール配糖体以外にも多種多様な成分を含んでいる。そのような成分としては、水溶性成分と不溶性成分とがある。水溶性成分としては、例えば、水溶性食物繊維などの多糖類、アルカロイドおよびフラボノイドおよびテルペノイドなどの二次代謝産物、メタノール、ポリフェノール、ミネラル、ビタミン、アミノ酸、有機酸、水溶性タンパク質、ならびに様々な他の配糖体が挙げられる。また、不溶性成分としては、例えば、不溶性食物繊維などを含む不溶性多糖類、不溶性タンパク質および脂質が挙げられる。 Stevia contains a wide variety of ingredients in addition to steviol glycosides. Such components include water-soluble components and insoluble components. Water-soluble components include, for example, polysaccharides such as water-soluble dietary fiber, secondary metabolites such as alkaloids and flavonoids and terpenoids, methanol, polyphenols, minerals, vitamins, amino acids, organic acids, water-soluble proteins, and various others. Glycoside of. In addition, examples of the insoluble component include insoluble polysaccharides including insoluble dietary fiber, insoluble proteins and lipids.
　本発明の一態様によれば、ステビア植物の酵素処理組成物は、アミノ酸を多く含む。また、いくつかの態様において、ステビア植物の酵素処理組成物には、少量のステビオール配糖体が含まれ得る。いくつかの態様において、ステビア植物の酵素処理組成物は、ステビオール配糖体を実質的に含まないことが好ましいが、ステビア植物に由来する除去しきれないステビオール配糖体が不可避的に少量含まれていてもよい。 According to one aspect of the present invention, the enzyme-treated composition of Stevia plant contains a large amount of amino acids. Also, in some embodiments, the enzyme-treated composition of Stevia plants may contain small amounts of steviol glycosides. In some embodiments, the enzyme-treated composition of the Stevia plant is preferably substantially free of steviol glycosides, but inevitably contains a small amount of steviol glycosides derived from the Stevia plant that cannot be completely removed. May be.
　アミノ酸は、タンパク質を形成する２０種のアミノ酸の１種以上が含まれることが好ましい。具体的には、アラニン（Ala）、アルギニン（Arg）、アスパラギン（Asn）、アスパラギン酸（Asp）、システイン（Cys）、グルタミン（Gln）、グルタミン酸（Glu）、グリシン（Gly）、ヒスチジン（His）、イソロイシン（Ile）、ロイシン（Leu）、リジン（Lys）、メチオニン（Met）、フェニルアラニン（Phe）、プロリン（Pro）、セリン（Ser）、スレオニン（トレオニン）（Thr）、トリプトファン（Trp）、チロシン（Tyr）およびバリン（Val）が挙げられる。
　また、アミノ酸は、アルギニン、リジン、ヒスチジン、フェニルアラニン、チロシン、ロイシン、イソロイシン、メチオニン、バリン、アラニン、グリシン、プロリン、グルタミン酸、セリン、スレオニン、アスパラギン酸、トリプトファンおよびシスチンからなる群から選択される１種以上であってもよい。
　本明細書において、「アミノ酸含有量」または「アミノ酸濃度」というときは、特に断らない限り、上記１８種のアミノ酸の合計量および合計濃度を意味する。アミノ酸は、その種類に応じて、アミノ酸自動分析機やＨＰＬＣを用いて公知の方法で定量することができる。 The amino acid preferably contains one or more of the 20 amino acids that form the protein. Specifically, alanine (Ala), arginine (Arg), aspartic acid (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His). , Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionin (Met), Phenylalanine (Phe), Proline (Pro), Serin (Ser), Threonin (Threonine) (Thr), Tryptophan (Trp), Tyrosine (Tyr) and valin (Val).
The amino acid is one selected from the group consisting of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan and cystine. It may be the above.
In the present specification, the term "amino acid content" or "amino acid concentration" means the total amount and total concentration of the above 18 kinds of amino acids unless otherwise specified. Amino acids can be quantified by a known method using an automatic amino acid analyzer or HPLC depending on the type.
　ここで、アミノ酸は、ステビア植物由来のアミノ酸であることが好ましい。本明細書において、「ステビア植物由来」とは、前記ステビア植物から得られるアミノ酸を意味する。アミノ酸を含む他の成分を加えていない限り、酵素処理組成物に含まれるアミノ酸はステビア植物由来のものであり得る。 Here, the amino acid is preferably an amino acid derived from Stevia plant. As used herein, the term "derived from a Stevia plant" means an amino acid obtained from the Stevia plant. The amino acids contained in the enzyme-treated composition can be of Stevia plant origin, unless other components including amino acids are added.
　本発明の一態様において、アミノ酸は、Ｄ体であってもよく、Ｌ体であってもよく、Ｄ体とＬ体からなるラセミ体であってもよい。アミノ酸は、好ましくは、Ｄ体と比較してＬ体のものを多く含む。具体的には、酵素処理組成物に含まれるＬ体のアミノ酸は、アミノ酸の含有量に対して５０％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上または９５％以上等であってもよい。 In one aspect of the present invention, the amino acid may be D-form, L-form, or racemic-form consisting of D-form and L-form. Amino acids preferably contain more L-forms as compared to D-forms. Specifically, the L-form amino acids contained in the enzyme-treated composition are 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80 with respect to the amino acid content. % Or more, 85% or more, 90% or more, 95% or more, and the like.
　本発明の一態様において、ステビオール配糖体としては、レバウジオシドＡ、レバウジオシドＢ、レバウジオシドＣ、レバウジオシドＤ、レバウジオシドＥ、レバウジオシドＦ、レバウジオシドＧ、レバウジオシドＩ、レバウジオシドＪ、レバウジオシドＫ、レバウジオシドＭ、レバウジオシドＮ、レバウジオシドＯ、レバウジオシドＱ、レバウジオシドＲ、ズルコシドＡ、ズルコシドＣ、ルブソシド、ステビオール、ステビオールモノシド、ステビオールビオシドおよびステビオシドなどが挙げられる。
　また、いくつかの態様において、酵素処理組成物は、レバウジオシドＡ、レバウジオシドＢ、レバウジオシドＣ、レバウジオシドＤ、レバウジオシドＥ、レバウジオシドＦ、レバウジオシドＧ、レバウジオシドＩ、レバウジオシドＭ、レバウジオシドＮ、ステビオシド、ステビオールビオシドズルコシドＡおよびルブソシドからなる総ステビオール配糖体（ＴＳＧ）を含むものであってもよい。
　本明細書において、「ステビオール配糖体含有量」または「ステビオール配糖体濃度」というときは、特に断らない限り、上記に列挙した総ステビオール配糖体の合計量および合計濃度を意味する。ステビオール配糖体は、液体クロマトグラフ質量分析計を用いて公知の方法で定量することができる。 In one embodiment of the present invention, the steviol glycosides include rebaugioside A, rebaugioside B, rebaugioside C, rebaugioside D, rebaugioside E, rebaugioside F, rebaugioside G, rebaugioside I, rebaugioside J, rebaugioside M, and rebaugioside N. Examples thereof include rebaugioside O, rebaugioside Q, rebaugioside R, zulcoside A, zulcoside C, rubusoside, steviol, steviol monoside, steviolbioside and stevioside.
Further, in some embodiments, the enzyme-treated composition comprises rebaugioside A, rebaugioside B, rebaugioside C, rebaugioside D, rebaugioside E, rebaugioside F, rebaugioside G, rebaugioside I, rebaugioside M, rebaugioside N, steviolbioside, steviolbioside. It may contain a total steviol glycoside (TSG) consisting of cosid A and rubusoside.
In the present specification, the term "steviol glycoside content" or "steviol glycoside concentration" means the total amount and total concentration of the total steviol glycosides listed above, unless otherwise specified. Steviol glycosides can be quantified by known methods using a liquid chromatograph mass spectrometer.
　本発明の一態様において、酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比は、例えば、０．３以上、０．５以上、０．７以上、１以上、１．２以上、１．５以上、２以上、３以上、４以上、５以上、６以上、７以上、８以上、９以上、１０以上、１１以上、１２以上、１３以上、１４以上、１５以上、１６以上、１７以上、１８以上、１９以上または２０以上等であってもよい。アミノ酸の比率が高いほど、素材としての純度が高くなるため好ましい。 In one embodiment of the present invention, the weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is, for example, 0.3 or more, 0.5 or more, 0.7 or more, 1 or more, 1.2 or more, 1. 5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more , 18 or more, 19 or more, 20 or more, and the like. The higher the ratio of amino acids, the higher the purity of the material, which is preferable.
　本発明の一態様において、アミノ酸は、酵素処理組成物の総重量に対して、例えば、１００～１００００ｐｐｍ、１５０～１００００ｐｐｍ、２００～１００００ｐｐｍ、２５０～１００００ｐｍ、３００～１００００ｐｐｍ、３５０～１００００ｐｐｍ、１００～９０００ｐｐｍ、１５０～９０００ｐｐｍ、２００～９０００ｐｐｍ、２５０～９０００ｐｍ、３００～９０００ｐｐｍ、３５０～９０００ｐｐｍ、１００～８０００ｐｐｍ、１５０～８０００ｐｐｍ、２００～８０００ｐｐｍ、２５０～８０００ｐｍ、３００～８０００ｐｐｍ、３５０～８０００ｐｐｍ、１００～７０００ｐｐｍ、１５０～７０００ｐｐｍ、２００～７０００ｐｐｍ、２５０～７０００ｐｍ、３００～７０００ｐｐｍ、３５０～７０００ｐｐｍ、１００～６０００ｐｐｍ、１５０～６０００ｐｐｍ、２００～６０００ｐｐｍ、２５０～６０００ｐｍ、３００～６０００ｐｐｍ、３５０～６０００ｐｐｍ、１００～５０００ｐｐｍ、１５０～５０００ｐｐｍ、２００～５０００ｐｐｍ、２５０～５０００ｐｍ、３００～５０００ｐｐｍ、３５０～５０００ｐｐｍ、１００～４０００ｐｐｍ、１５０～４０００ｐｐｍ、２００～４０００ｐｐｍ、２５０～４０００ｐｍ、３００～４０００ｐｐｍ、３５０～４０００ｐｐｍ、１００～３０００ｐｐｍ、１５０～３０００ｐｐｍ、２００～３０００ｐｐｍ、２５０～３０００ｐｍ、３００～３０００ｐｐｍ、３５０～３０００ｐｐｍ、１００～２０００ｐｐｍ、１５０～２０００ｐｐｍ、２００～２０００ｐｐｍ、２５０～２０００ｐｍ、３００～２０００ｐｐｍ、３５０～２０００ｐｐｍ、１００～１０００ｐｐｍ、１５０～１０００ｐｐｍ、２００～１０００ｐｐｍ、２５０～１０００ｐｍ、３００～１０００ｐｐｍまたは３５０～１０００ｐｐｍ等であってもよい。 In one aspect of the invention, the amino acids are, for example, 100 to 10000 ppm, 150 to 10000 ppm, 200 to 10000 ppm, 250 to 10000 pm, 300 to 10000 ppm, 350 to 10000 ppm, 100 to 9000 ppm with respect to the total weight of the enzyme-treated composition. , 150-9000ppm, 200-9000ppm, 250-9000pm, 300-9000ppm, 350-9000ppm, 100-8000ppm, 150-8000ppm, 200-8000ppm, 250-8000pm, 300-8000ppm, 350-8000ppm, 100-7000ppm, 150 ~ 7000ppm, 200 ~ 7000ppm, 250 ~ 7000pm, 300 ~ 7000ppm, 350 ~ 7000ppm, 100 ~ 6000ppm, 150 ~ 6000ppm, 200 ~ 6000ppm, 250 ~ 6000pm, 300 ~ 6000ppm, 350 ~ 6000ppm, 100 ~ 5000ppm, 150 ~ 5000ppm , 200-5000ppm, 250-5000pm, 300-5000ppm, 350-5000ppm, 100-4000ppm, 150-4000ppm, 200-4000ppm, 250-4000pm, 300-4000ppm, 350-4000ppm, 100-3000ppm, 150-3000ppm, 200 ~ 3000ppm, 250 ~ 3000pm, 300 ~ 3000ppm, 350 ~ 3000ppm, 100 ~ 2000ppm, 150 ~ 2000ppm, 200 ~ 2000ppm, 250 ~ 2000pm, 300 ~ 2000ppm, 350 ~ 2000ppm, 100 ~ 1000ppm, 150 ~ 1000ppm, 200 ~ 1000ppm , 250-1000 pm, 300-1000 ppm, 350-1000 ppm and the like.
　本発明の一態様において、ステビオール配糖体は、酵素処理組成物の総重量に対して、３０００ｐｐｍ以下、２５００ｐｐｍ以下、２３００ｐｐｍ以下、２０００ｐｐｍ以下、１５００ｐｐｍ以下、１０００ｐｐｍ以下、７５０ｐｐｍ以下、７２０ｐｐｍ以下、７００ｐｐｍ以下、５００ｐｐｍ以下、４５０ｐｐｍ以下、４００ｐｐｍ以下、３５０ｐｐｍ以下、３００ｐｐｍ以下、２５０ｐｐｍ以下、２００ｐｐｍ以下、１５０ｐｐｍ以下、１００ｐｐｍ以下、９０ｐｐｍ以下、８５ｐｐｍ以下、８０ｐｐｍ以下、７５ｐｐｍ以下、７０ｐｐｍ以下、６５ｐｐｍ以下、６０ｐｐｍ以下、５５ｐｐｍ以下、５０ｐｐｍ以下、４５ｐｐｍ以下、４０ｐｐｍ以下、３５ｐｐｍ以下、３０ｐｐｍ以下、２５ｐｐｍ以下、２０ｐｐｍ以下、１５ｐｐｍ以下、１０ｐｐｍ以下、５ｐｐｍ以下または１ｐｐｍ以下等であってもよい。なお、ステビオール配糖体濃度の下限は、０である。 In one aspect of the invention, the steviol glycoside is 3000 ppm or less, 2500 ppm or less, 2300 ppm or less, 2000 ppm or less, 1500 ppm or less, 1000 ppm or less, 750 ppm or less, 720 ppm or less, 700 ppm or less with respect to the total weight of the enzyme-treated composition. , 500 ppm or less, 450 ppm or less, 400 ppm or less, 350 ppm or less, 300 ppm or less, 250 ppm or less, 200 ppm or less, 150 ppm or less, 100 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm Below, it may be 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, and the like. The lower limit of the steviol glycoside concentration is 0.
　本発明の一態様において、ステビア植物の酵素処理組成物に含まれるアミノ酸含有量は、酵素処理組成物の固形分量に対して、例えば、０．１～５０重量％、０．５～５０重量％、１．０～５０重量％、１．５～５０重量％、１．０～５０重量％、２．５～５０重量％、３．０～５０重量％、３．５～５０重量％、４．０～５０重量％、４．５～５０重量％、５．０～５０重量％、５．５～５０重量％、６．０～５０重量％、０．１～４０重量％、０．５～４０重量％、１．０～４０重量％、１．５～４０重量％、２．０～４０重量％、２．５～４０重量％、３．０～４０重量％、３．５～４０重量％、４．０～４０重量％、４．５～４０重量％、５．０～４０重量％、５．５～４０重量％、６．０～４０重量％、０．１～３０重量％、０．５～３０重量％、１．０～３０重量％、１．５～３０重量％、２．０～３０重量％、２．５～３０重量％、３．０～３０重量％、３．５～３０重量％、４．０～３０重量％、４．５～３０重量％、５．０～３０重量％、５．５～３０重量％、６．０～３０重量％、０．１～２０重量％、０．５～２０重量％、１．０～２０重量％、１．５～２０重量％、２．０～２０重量％、２．５～２０重量％、３．０～２０重量％、３．５～２０重量％、４．０～２０重量％、４．５～２０重量％、５．０～２０重量％、５．５～２０重量％または６．０～２０重量％等であってもよい。酵素処理組成物のＢｒｉｘ（固形分量）は、組成物が液体である場合は、Ｂｒｉｘ計（例えば、ATAGO製Brix計RX-5000αなど）を用いて求めることができ、ＨＰＬＣ等で測定したアミノ酸の量を用いて、組成物の固形分量に対するアミノ酸の量を算出することができる。 In one embodiment of the present invention, the amino acid content contained in the enzyme-treated composition of the Stevia plant is, for example, 0.1 to 50% by weight and 0.5 to 50% by weight with respect to the solid content of the enzyme-treated composition. , 1.0-50% by weight, 1.5-50% by weight, 1.0-50% by weight, 2.5-50% by weight, 3.0-50% by weight, 3.5-50% by weight, 4 .0-50% by weight, 4.5-50% by weight, 5.0-50% by weight, 5.5-50% by weight, 6.0-50% by weight, 0.1-40% by weight, 0.5 ~ 40% by weight, 1.0 ~ 40% by weight, 1.5 ~ 40% by weight, 2.0 ~ 40% by weight, 2.5 ~ 40% by weight, 3.0 ~ 40% by weight, 3.5 ~ 40 Weight%, 4.0-40% by weight, 4.5-40% by weight, 5.0-40% by weight, 5.5-40% by weight, 6.0-40% by weight, 0.1-30% by weight , 0.5-30% by weight, 1.0-30% by weight, 1.5-30% by weight, 2.0-30% by weight, 2.5-30% by weight, 3.0-30% by weight, 3 .5-30% by weight, 4.0-30% by weight, 4.5-30% by weight, 5.0-30% by weight, 5.5-30% by weight, 6.0-30% by weight, 0.1 ~ 20% by weight, 0.5 ~ 20% by weight, 1.0 ~ 20% by weight, 1.5 ~ 20% by weight, 2.0 ~ 20% by weight, 2.5 ~ 20% by weight, 3.0 ~ 20 %%, 3.5-20% by weight, 4.0-20% by weight, 4.5-20% by weight, 5.0-20% by weight, 5.5-20% by weight or 6.0-20% by weight And so on. The Brix (solid content) of the enzyme-treated composition can be determined using a Brix meter (for example, ATAGO Brix meter RX-5000α) when the composition is a liquid, and the amino acid measured by HPLC or the like. The amount can be used to calculate the amount of amino acids relative to the solid content of the composition.
　本発明の一態様において、ステビア植物の酵素処理組成物に含まれるアミノ酸含有量は、乾燥重量基準で、酵素処理組成物の総重量に対して、例えば、０．１～６０重量％、０．５～６０重量％、１．０～６０重量％、１．５～６０重量％、２．０～６０重量％、２．５～６０重量％、３．０～６０重量％、３．５～６０重量％、４．０～６０重量％、４．５～６０重量％、５．０～６０重量％、５．５～６０重量％、６．０～６０重量％、０．１～５０重量％、０．５～５０重量％、１．０～５０重量％、１．５～５０重量％、２．０～５０重量％、２．５～５０重量％、３．０～５０重量％、３．５～５０重量％、４．０～５０重量％、４．５～５０重量％、５．０～５０重量％、５．５～５０重量％、６．０～５０重量％、０．１～４０重量％、０．５～４０重量％、１．０～４０重量％、１．５～４０重量％、２．０～４０重量％、２．５～４０重量％、３．０～４０重量％、３．５～４０重量％、４．０～４０重量％、４．５～４０重量％、５．０～４０重量％、５．５～４０重量％、６．０～４０重量％、０．１～３０重量％、０．５～３０重量％、１．０～３０重量％、１．５～３０重量％、２．０～３０重量％、２．５～３０重量％、３．０～３０重量％、３．５～３０重量％、４．０～３０重量％、４．５～３０重量％、５．０～３０重量％、５．５～３０重量％、６．０～３０重量％、０．１～２０重量％、０．５～２０重量％、１．０～２０重量％、１．５～２０重量％、２．０～２０重量％、２．５～２０重量％、３．０～２０重量％、３．５～２０重量％、４．０～２０重量％、４．５～２０重量％、５．０～２０重量％、５．５～２０重量％または６．０～２０重量％等であってもよい。 In one embodiment of the present invention, the amino acid content contained in the enzyme-treated composition of the Stevia plant is, for example, 0.1 to 60% by weight, 0. 5-60% by weight, 1.0-60% by weight, 1.5-60% by weight, 2.0-60% by weight, 2.5-60% by weight, 3.0-60% by weight, 3.5- 60% by weight, 4.0-60% by weight, 4.5-60% by weight, 5.0-60% by weight, 5.5-60% by weight, 6.0-60% by weight, 0.1-50% by weight %, 0.5-50% by weight, 1.0-50% by weight, 1.5-50% by weight, 2.0-50% by weight, 2.5-50% by weight, 3.0-50% by weight, 3.5 to 50% by weight, 4.0 to 50% by weight, 4.5 to 50% by weight, 5.0 to 50% by weight, 5.5 to 50% by weight, 6.0 to 50% by weight, 0. 1-40% by weight, 0.5-40% by weight, 1.0-40% by weight, 1.5-40% by weight, 2.0-40% by weight, 2.5-40% by weight, 3.0- 40% by weight, 3.5-40% by weight, 4.0-40% by weight, 4.5-40% by weight, 5.0-40% by weight, 5.5-40% by weight, 6.0-40% by weight %, 0.1-30% by weight, 0.5-30% by weight, 1.0-30% by weight, 1.5-30% by weight, 2.0-30% by weight, 2.5-30% by weight, 3.0 to 30% by weight, 3.5 to 30% by weight, 4.0 to 30% by weight, 4.5 to 30% by weight, 5.0 to 30% by weight, 5.5 to 30% by weight, 6. 0 to 30% by weight, 0.1 to 20% by weight, 0.5 to 20% by weight, 1.0 to 20% by weight, 1.5 to 20% by weight, 2.0 to 20% by weight, 2.5 to 20% by weight, 3.0 to 20% by weight, 3.5 to 20% by weight, 4.0 to 20% by weight, 4.5 to 20% by weight, 5.0 to 20% by weight, 5.5 to 20% by weight. % Or 6.0 to 20% by weight and the like.
　本発明の一態様において、ステビア植物の酵素処理組成物は、ヘミセルラーゼおよび／またはプロテアーゼにより酵素処理されたものである。ヘミセルラーゼは、ヘミセルロースを加水分解する酵素であれば特に制限されず、公知の酵素を使用することができる。ヘミセルラーゼは、例えば、キシラン、アラビノキシラン、キシログルカンおよびグルコマンナンなどの多糖類であるヘミセルロースを分解するものであり、キシラナーゼやガラクタナーゼが知られている。
　プロテアーゼは、ペプチド結合加水分解酵素を意味するものである。プロテアーゼは、ペプチド鎖の末端から切断するエキソペプチダーゼであってもよいし、ペプチド鎖の中央から切断するエンドペプチダーゼであってもよい。両者を併用することもできる。また、ペプチダーゼは、例えば、パパイヤ、パイナップル、ショウガ、イチジク、キウイフルーツなどの植物由来であってもよいし、麹、カビ、納豆菌などの細菌類に由来するものであってもよい。ヘミセルラーゼおよびプロテアーゼは、市販のものを購入して使用することができる。 In one aspect of the invention, the enzyme-treated composition of a stevia plant is enzymatically treated with hemicellulase and / or protease. The hemicellulase is not particularly limited as long as it is an enzyme that hydrolyzes hemicellulose, and a known enzyme can be used. Hemicellulase decomposes hemicellulose, which is a polysaccharide such as xylan, arabinoxylan, xyloglucan and glucomannan, and xylanase and galactanase are known.
Protease means a peptide bond hydrolase. The protease may be an exopeptidase that cleaves from the end of the peptide chain or an endopeptidase that cleaves from the center of the peptide chain. Both can be used together. Further, the peptidase may be derived from a plant such as papaya, pineapple, ginger, fig or kiwifruit, or may be derived from a bacterium such as jiuqu, mold or natto fungus. Hemicellulase and protease can be purchased and used on the market.
　本発明の一態様において、ステビア植物の酵素処理組成物は、液状、ペースト状、粉末状または顆粒状等の形態であってもよい。液状またはペースト状とする場合、溶媒は、水道水、イオン交換水、軟水、蒸留水のほか、これらの水を脱気処理した脱気水などが挙げられる。 In one aspect of the present invention, the enzyme-treated composition of Stevia plant may be in the form of liquid, paste, powder, granule or the like. In the case of liquid or paste, the solvent includes tap water, ion-exchanged water, soft water, distilled water, and degassed water obtained by degassing these waters.
２．ステビア植物の酵素処理組成物の製造方法
　本発明は、ステビア植物の酵素処理組成物の製造方法にも関する。具体的には、本発明は、ステビア植物の酵素処理組成物の製造方法であって、ステビア植物を溶媒を用いて抽出することと、ヘミセルラーゼおよび／またはプロテアーゼを用いて前記抽出後の残留物を酵素処理することと、を含む、方法に関する。 2. 2. Method for Producing Enzyme-Treatment Composition for Stevia Plant The present invention also relates to a method for producing an enzyme-treated composition for Stevia plant. Specifically, the present invention is a method for producing an enzyme-treated composition of a stevia plant, in which the stevia plant is extracted using a solvent and the residue after the extraction using hemicellulase and / or a protease. With respect to methods, including enzymatic treatment.
（Ａ）ステビア植物を溶媒を用いて抽出すること
　本発明の一態様における製造方法には、ステビア植物を溶媒を用いて抽出することが含まれる。例えば、前述したステビア植物を、水性溶媒を用いて抽出することが挙げられる。 (A) Extraction of Stevia Plant Using Solvent The production method according to one aspect of the present invention includes extraction of Stevia plant using a solvent. For example, the above-mentioned Stevia plant can be extracted using an aqueous solvent.
　ステビア植物は、例えば、ステビアの乾燥葉（茎や他の組織が含まれていてもよい）を使用することができる。ステビアの乾燥葉とは、ステビア植物の新鮮葉を乾燥させることにより含水量を減らしたものをいう。ステビア植物の乾燥葉の含水率は、好ましくは１～１０重量％、より好ましくは、２～８重量％、特に好ましくは３～４重量％である。 As the stevia plant, for example, dried leaves of stevia (which may contain stems and other tissues) can be used. The dried leaves of Stevia are those in which the water content is reduced by drying the fresh leaves of the Stevia plant. The water content of the dried leaves of the Stevia plant is preferably 1 to 10% by weight, more preferably 2 to 8% by weight, and particularly preferably 3 to 4% by weight.
　本発明の製造方法に用いるのに好ましいステビア植物の乾燥葉は、含水率が１０重量％以下の場合に乾燥葉１００ｇ中に１００～８００ｍｇのアルギニン、１００～４００ｍｇのヒスチジン、１００～６００ｍｇのチロシン、１００～８００ｍｇのイソロイシン、１００～１０００ｍｇのバリン、１００～９００ｍｇのグリシン、４００～２０００ｍｇのグルタミン酸、１００～８００ｍｇのスレオニン、５０～４００ｍｇのトリプトファンを含むものであってもよい。 The preferred dried leaves of Stevia plants to be used in the production method of the present invention are 100 to 800 mg of arginine, 100 to 400 mg of histidine, and 100 to 600 mg of tyrosine in 100 g of dried leaves when the water content is 10% by weight or less. It may contain 100-800 mg of isoleucine, 100-1000 mg valine, 100-900 mg glycine, 400-2000 mg glutamic acid, 100-800 mg threonine, 50-400 mg tryptophan.
　溶媒としては、水やアルコール、あるいはそれらの混合溶液等が挙げられる。好ましい溶媒としては、イオン交換水、純水（例えば、ミリＱ水）およびエタノール水溶液などが挙げられる。抽出する際には、ステビア植物（例えば、乾燥葉）を破砕してもよく、破砕しなくてもよい。破砕する場合は、ボールミルなどを用いて破砕してもよい。あるいは、カラム抽出機（ＧＥヘルスケア製）やニーダー抽出器（ＳＫＮ－Ｒ１００、三友機器株式会社製）等を用いて抽出処理をしてもよい。 Examples of the solvent include water, alcohol, and a mixed solution thereof. Preferred solvents include ion-exchanged water, pure water (eg, milliQ water), aqueous ethanol and the like. At the time of extraction, Stevia plants (eg, dried leaves) may or may not be crushed. When crushing, it may be crushed using a ball mill or the like. Alternatively, the extraction process may be performed using a column extractor (manufactured by GE Healthcare), a kneader extractor (SKN-R100, manufactured by Sanyu Kikai Co., Ltd.) or the like.
　抽出時には、溶媒を加熱してもよい。抽出する際の温度は、例えば、１０～８０℃、２５～８０℃、３０～７５℃、３５～７０℃、４０～６５℃、４５～７０℃であってもよく、好ましくは４５～７０℃である。なお、本明細書において、温度の下限値及び上限値は、例えば、各温度の±１℃、±２℃、±３℃、±４℃、±５℃の温度であってもよい。 At the time of extraction, the solvent may be heated. The temperature at the time of extraction may be, for example, 10 to 80 ° C, 25 to 80 ° C, 30 to 75 ° C, 35 to 70 ° C, 40 to 65 ° C, 45 to 70 ° C, and preferably 45 to 70 ° C. Is. In the present specification, the lower limit value and the upper limit value of the temperature may be, for example, ± 1 ° C., ± 2 ° C., ± 3 ° C., ± 4 ° C., ± 5 ° C. of each temperature.
　抽出は、１回だけでなく、複数回行ってもよい。複数回抽出を行うことで、葉に含まれているステビオール配糖体がより多く抽出される。 Extraction may be performed not only once but multiple times. By performing multiple extractions, more steviol glycosides contained in the leaves are extracted.
　ステビア植物の抽出率は、例えば、１０～６０％を目安とすることができ、２０～５０％であることが好ましく、２５～４５％であることがより好ましく、３０～４０％であることがさらに好ましい。なお、抽出率は、原料のステビア植物の重量に対する抽出液中の固形分量の比率（％）である。 The extraction rate of the Stevia plant can be, for example, 10 to 60% as a guide, preferably 20 to 50%, more preferably 25 to 45%, and 30 to 40%. More preferred. The extraction rate is the ratio (%) of the amount of solid content in the extract to the weight of the raw material Stevia plant.
（Ｂ）ヘミセルラーゼおよび／またはプロテアーゼを用いて前記抽出後の残留物を酵素処理すること
　本発明の一態様における製造方法には、ヘミセルラーゼおよび／またはプロテアーゼを用いて前記抽出後の残留物を酵素処理することが含まれる。ヘミセルラーゼおよび／またはプロテアーゼは、前記「１．ステビア植物の酵素処理組成物」で述べたものを使用することができる。いくつかの態様では、ステビア植物の抽出後の残留物は、少なくとも１回の抽出後の残留物である。別のいくつかの態様では、前記残留物は、少なくとも２回の抽出後の残留物である。更に別のいくつかの態様では、前記残留物は、少なくとも３回の抽出後の残留物である。 (B) Enzymatic treatment of the residue after extraction with hemicellulase and / or protease In the production method according to one embodiment of the present invention, the residue after extraction is treated with hemicellulase and / or protease. Includes enzymatic treatment. As the hemicellulase and / or protease, those described in "1. Enzyme-treated composition of Stevia plant" can be used. In some embodiments, the post-extraction residue of the Stevia plant is at least one post-extraction residue. In some other embodiments, the residue is the residue after at least two extractions. In yet some other embodiments, the residue is the residue after at least 3 extractions.
　また、本発明の一態様における製造方法は、上記（Ｂ）の工程において酵素を添加する前または後に、ステビア植物の抽出後の残留物をスラリーにすることをさらに含んでもよい。スラリーに使用する溶媒としては、上記（Ａ）で述べた水性溶媒が挙げられる。水性スラリーの濃度は特に制限されないが、流動性や処理効率などを考慮すると、５～７０％であることが好ましく、５～５０％がより好ましく、７～３０％がさらに好ましい。 Further, the production method according to one aspect of the present invention may further include making a slurry of the residue after extraction of Stevia plant before or after adding the enzyme in the step (B). Examples of the solvent used for the slurry include the aqueous solvent described in (A) above. The concentration of the aqueous slurry is not particularly limited, but is preferably 5 to 70%, more preferably 5 to 50%, still more preferably 7 to 30%, in consideration of fluidity and treatment efficiency.
　本発明の一態様において、ヘミセルラーゼおよびプロテアーゼは、併用することが好ましい。これにより、酵素処理組成物に含まれるアミノ酸濃度を増加させることができる。
　また、ヘミセルラーゼおよびプロテアーゼを併用する場合、ヘミセルラーゼ処理後にプロテアーゼ処理を行ってもよいし、ヘミセルラーゼ処理とプロテアーゼ処理を同時に行ってもよい。より具体的には、前記（Ａ）の工程において先ずヘミセルラーゼを添加し、次に（Ｂ）の工程においてプロテアーゼを添加してもよいし、（Ｂ）の工程においてヘミセルラーゼおよびプロテアーゼをともに添加してもよい。 In one aspect of the invention, hemicellulase and protease are preferably used in combination. This makes it possible to increase the concentration of amino acids contained in the enzyme-treated composition.
When hemicellulase and protease are used in combination, protease treatment may be performed after hemicellulase treatment, or hemicellulase treatment and protease treatment may be performed at the same time. More specifically, hemicellulase may be added first in the step (A), then a protease may be added in the step (B), or both hemicellulase and the protease may be added in the step (B). You may.
　ヘミセルラーゼおよびプロテアーゼを併用する場合の合計添加量は、ステビア植物の乾燥重量に対して、０．５～１５重量％、１～１４．５重量％、１．５～１４重量％、２～１３．５重量％、２．５～１３重量％、３～１２．５重量％、３．５～１２重量％、４～１１．５重量％、４．５～１１重量％、５～１０．５重量％または５～１０重量％等であってもよい。
　また、ヘミセルラーゼおよびプロテアーゼを併用する場合のそれぞれの酵素の添加量は、上記の合計添加量を踏まえ適宜調整することができる。例えば、ヘミセルラーゼの添加量は、ステビア植物の乾燥重量に対して、０．５～１５重量％、１～１４重量％、１．５～１３重量％、２～１２重量％、２．５～１１重量％または３～１０重量％等であってもよい。同様に、プロテアーゼの添加量は、ステビア植物の乾燥重量に対して、０．５～１５重量％、１～１４重量％、１．５～１３重量％、２～１２重量％、２．５～１１重量％または３～１０重量％等であってもよい。なお、ヘミセルラーゼまたはプロテアーゼを単独で使用する場合の添加量は、これらを併用する場合の合計添加量を目安に添加することができる。 When hemicellulase and protease are used in combination, the total amount added is 0.5 to 15% by weight, 1 to 14.5% by weight, 1.5 to 14% by weight, and 2 to 13 to the dry weight of the Stevia plant. .5% by weight, 2.5 to 13% by weight, 3 to 12.5% by weight, 3.5 to 12% by weight, 4 to 11.5% by weight, 4.5 to 11% by weight, 5 to 10.5 It may be% by weight, 5 to 10% by weight, or the like.
In addition, the amount of each enzyme added when hemicellulase and protease are used in combination can be appropriately adjusted based on the above total amount of addition. For example, the amount of hemicellulase added is 0.5 to 15% by weight, 1 to 14% by weight, 1.5 to 13% by weight, 2 to 12% by weight, 2.5 to 25% by weight based on the dry weight of the Stevia plant. It may be 11% by weight, 3 to 10% by weight, or the like. Similarly, the amount of protease added is 0.5 to 15% by weight, 1 to 14% by weight, 1.5 to 13% by weight, 2 to 12% by weight, 2.5 to 25% by weight based on the dry weight of the Stevia plant. It may be 11% by weight, 3 to 10% by weight, or the like. The addition amount when hemicellulase or protease is used alone can be added based on the total addition amount when these are used in combination.
　酵素処理の時間は、例えば、１～４８時間、１～３６時間、１～２４時間、１～１８時間、２～４８時間、２～３６時間、２～２４時間、２～１８時間、３～４８時間、３～３６時間、３～２４時間、３～１８時間、４～４８時間、４～３６時間、４～２４時間、４～１８時間、５～４８時間、５～３６時間、５～２４時間、５～１８時間、６～４８時間、６～３６時間、６～２４時間、６～１８時間、７～４８時間、７～３６時間、７～２４時間、７～１８時間、８～４８時間、８～３６時間、８～２４時間、８～１８時間、９～４８時間、９～３６時間、９～２４時間、９～１８時間、１０～４８時間、１０～３６時間、１０～２４時間、１０～１８時間または１０～１６時間等としてもよい。酵素反応の時間は、使用する酵素の至適温度や酵素の添加量等を踏まえ、適宜調整することができる。 The time of enzyme treatment is, for example, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 18 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 18 hours, 3 to. 48 hours, 3 to 36 hours, 3 to 24 hours, 3 to 18 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to 18 hours, 5 to 48 hours, 5 to 36 hours, 5 to 24 hours, 5-18 hours, 6-48 hours, 6-36 hours, 6-24 hours, 6-18 hours, 7-48 hours, 7-36 hours, 7-24 hours, 7-18 hours, 8- 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 18 hours, 9 to 48 hours, 9 to 36 hours, 9 to 24 hours, 9 to 18 hours, 10 to 48 hours, 10 to 36 hours, 10 to It may be 24 hours, 10 to 18 hours, 10 to 16 hours, or the like. The time of the enzyme reaction can be appropriately adjusted in consideration of the optimum temperature of the enzyme to be used, the amount of the enzyme added, and the like.
　酵素反応の温度は、使用する酵素の種類によって適宜変更することができるが、例えば、１０～８０℃、１５～８０℃、２０～８０℃、２５℃～８０℃、３０～８０℃、３５～８０℃、４０～８０℃、１０～７５℃、１５～７５℃、２０～７５℃、２５℃～７５℃、３０～７５℃、３５～７５℃、４０～７５℃、１０～７０℃、１５～７０℃、２０～７０℃、２５℃～７０℃、３０～７０℃、３５～７０℃または４０～７０℃等であってもよい。また、加熱装置を用いて上記範囲の温度に調整することもできる。加熱装置は、特に制限されるものではないが、例えば、スチーム加熱機、電熱加熱機、ウォータージャケット加熱機、電磁加熱機等を挙げることができる。 The temperature of the enzyme reaction can be appropriately changed depending on the type of enzyme used, and is, for example, 10 to 80 ° C, 15 to 80 ° C, 20 to 80 ° C, 25 ° C to 80 ° C, 30 to 80 ° C, 35 to. 80 ° C, 40-80 ° C, 10-75 ° C, 15-75 ° C, 20-75 ° C, 25 ° C-75 ° C, 30-75 ° C, 35-75 ° C, 40-75 ° C, 10-70 ° C, 15 It may be about 70 ° C., 20 to 70 ° C., 25 ° C. to 70 ° C., 30 to 70 ° C., 35 to 70 ° C., 40 to 70 ° C., or the like. Further, the temperature can be adjusted to the above range by using a heating device. The heating device is not particularly limited, and examples thereof include a steam heater, an electric heater, a water jacket heater, and an electromagnetic heater.
　酵素反応時のｐＨは、使用する酵素の種類によって適宜変更することができるが、例えば、２～１０、２．２～１０、２．４～１０、２．６～１０、２．８～１０、３～１０、２～９、２．２～９、２．４～９、２．６～９、２．８～９、３～９、２～８、２．２～８、２．４～８、２．６～８、２．８～８または３～８等であってもよい。
　ｐＨを調整する際には、例えば、アルカリ金属水酸化物、アルカリ土類金属水酸化物、ケイ酸塩、炭酸塩、炭酸水素塩等のアルカリ剤や、クエン酸、乳酸、酢酸等の有機系のｐＨ調整剤、リン酸、塩酸、硫酸、二酸化炭素等の無機系のｐＨ調整剤を使用することができる。 The pH at the time of the enzyme reaction can be appropriately changed depending on the type of enzyme used, for example, 2 to 10, 2.2 to 10, 2.4 to 10, 2.6 to 10, 2.8 to 10. 3-10, 2-9, 2.2-9, 2.4-9, 2.6-9, 2.8-9, 3-9, 2-8, 2.2-8, 2.4 It may be ~ 8, 2.6-8, 2.8-8, 3-8, etc.
When adjusting the pH, for example, alkaline agents such as alkali metal hydroxides, alkaline earth metal hydroxides, silicates, carbonates and hydrogen carbonates, and organic systems such as citric acid, lactic acid and acetic acid. , And inorganic pH adjusters such as phosphoric acid, hydrochloric acid, sulfuric acid, and carbon dioxide can be used.
　酵素処理中の反応液のＢｒｉｘ（固形分量）は、例えば、０．２５～３０、０．２５～２５、０．２５～２０、０．２５～１８、０．２５～１６、０．２５～１４、０．２５～１２、０．２５～１０、０．２５～８．０、０．２５～６．０、０．２５～５．０、０．２５～４．０、０．２５～３．０、０．５～３０、０．５～２５、０．５～２０、０．５～１８、０．５～１６、０．５～１４、０．５～１２、０．５～１０、０．５～８．０、０．５～６．０、０．５～５．０、０．５～４．０または０．５～３．０等であってもよい。 The Brix (solid content) of the reaction solution during the enzyme treatment is, for example, 0.25 to 30, 0.25 to 25, 0.25 to 20, 0.25 to 18, 0.25 to 16, 0.25 to. 14, 0.25 to 12, 0.25 to 10, 0.25 to 8.0, 0.25 to 6.0, 0.25 to 5.0, 0.25 to 4.0, 0.25 to 3.0, 0.5-30, 0.5-25, 0.5-20, 0.5-18, 0.5-16, 0.5-14, 0.5-12, 0.5- It may be 10, 0.5 to 8.0, 0.5 to 6.0, 0.5 to 5.0, 0.5 to 4.0, 0.5 to 3.0, and the like.
　いくつかの態様では、酵素反応を効率的に実施するため、ステビア植物の抽出後の残留物（例えば、スラリー化した残留物）を撹拌しながら酵素処理してもよい。撹拌装置に特に制限はなく、例えば、縦軸撹拌装置、横軸撹拌装置、マグネチックスターラー、振とう機等を用いることができる。 In some embodiments, the enzyme treatment may be performed while stirring the residue after extraction of the Stevia plant (for example, the slurryed residue) in order to carry out the enzymatic reaction efficiently. The stirring device is not particularly limited, and for example, a vertical axis stirring device, a horizontal axis stirring device, a magnetic stirrer, a shaker, or the like can be used.
　また、いくつかの態様において、ステビア植物の抽出後の残留物（例えば、スラリー化した残留物）は、複数回に分けて酵素処理してもよい。例えば、前記残留物は、２～１０回、２～５回または２～４回に分けて酵素処理してもよい。 Further, in some embodiments, the residue after extraction of the Stevia plant (for example, the slurryed residue) may be enzymatically treated in a plurality of times. For example, the residue may be enzymatically treated in 2 to 10 times, 2 to 5 times or 2 to 4 times.
（Ｃ）その他の工程
　本発明の一態様における製造方法は、前記残留物を含む酵素反応液を高温に加熱することで酵素反応を終了することを含んでいてもよい。使用する酵素の耐熱性によって条件は異なるが、例えば、前記残留物を含む酵素反応液を８０℃、９０℃、９５℃または１００℃を超える温度に加熱することで、酵素を失活させることができる。酵素を失活させるための時間は、例えば、３０秒～１時間、１～３０分間または１～１０分間等であってもよい。酵素失活にはプレートヒーターや向流接触装置を使用することができる。 (C) Other Steps The production method according to one aspect of the present invention may include terminating the enzyme reaction by heating the enzyme reaction solution containing the residue to a high temperature. The conditions vary depending on the heat resistance of the enzyme used, but for example, the enzyme can be inactivated by heating the enzyme reaction solution containing the residue to a temperature exceeding 80 ° C, 90 ° C, 95 ° C or 100 ° C. can. The time for inactivating the enzyme may be, for example, 30 seconds to 1 hour, 1 to 30 minutes, 1 to 10 minutes, or the like. A plate heater or a countercurrent contact device can be used for enzyme inactivation.
　本発明の一態様における製造方法は、上記（Ｂ）の工程後に固液分離処理することを含んでいてもよい。固液分離処理としては、固体と液体が十分に分離されれば特に限定されないが、例えば、遠心分離器やフィルタープレスを用いた処理や、フィルターやメッシュを用いた重力ろ過が挙げられる。 The production method in one aspect of the present invention may include solid-liquid separation treatment after the step (B) above. The solid-liquid separation treatment is not particularly limited as long as the solid and the liquid are sufficiently separated, and examples thereof include treatment using a centrifuge or a filter press, and gravity filtration using a filter or a mesh.
　本発明の一態様における製造方法は、固液分離処理された酵素処理組成物をさらに濃縮、乾燥、造粒等の処理を施すことを含んでいてもよい。濃縮、乾燥、造粒等の処理は、公知の方法を用いて実施することができる。 The production method according to one aspect of the present invention may include further concentrating, drying, granulating or the like the enzyme-treated composition which has been subjected to the solid-liquid separation treatment. Treatments such as concentration, drying, and granulation can be carried out by using known methods.
　以上の各工程により、ステビア植物の酵素処理組成物を製造することができる。得られる酵素処理組成物の特徴は、上記「１．ステビア植物の酵素処理組成物」で述べた特徴と同じである。また、本発明の一態様における製造方法によって得られるステビア植物の酵素処理組成物を含む飲食品の特徴および当該飲食品の製造方法は、後述するとおりである。 By each of the above steps, an enzyme-treated composition of Stevia plant can be produced. The characteristics of the obtained enzyme-treated composition are the same as those described in "1. Enzyme-treated composition of Stevia plant" above. Further, the characteristics of the food and drink containing the enzyme-treated composition of the stevia plant obtained by the production method in one aspect of the present invention and the production method of the food and drink are as described later.
３．ステビア植物の酵素処理組成物を含む飲食品
　本発明は、ステビア植物の酵素処理組成物を含む飲食品にも関する。本発明の飲食品は、ステビア植物の酵素処理組成物を含んでいれば特に限定されない。飲食品とは、飲料および食品を意味する。好ましくは、飲食品は飲料である。 3. 3. Foods and Beverages Containing Enzyme-treated Compositions of Stevia Plants The present invention also relates to foods and drinks containing enzyme-treated compositions of Stevia plants. The food or drink of the present invention is not particularly limited as long as it contains an enzyme-treated composition of Stevia plant. Food and beverage means beverages and food. Preferably, the food or drink is a beverage.
　飲食品に対する酵素処理組成物の配合量は、飲食品の種類に応じて異なるが、飲食品中のステビア植物由来のアミノ酸量に換算して設定することができ、例えば、前記アミノ酸量が０．０００１～１重量％、０．００１～０．１重量％、０．００５～０．１重量％または０．００５～０．０５重量％等となるように設定することができる。 The blending amount of the enzyme-treated composition with respect to the food or drink varies depending on the type of the food or drink, but can be set by converting it into the amount of amino acids derived from the Stevia plant in the food or drink. For example, the amount of the amino acid is 0. It can be set to be 0001 to 1% by weight, 0.001 to 0.1% by weight, 0.005 to 0.1% by weight, 0.005 to 0.05% by weight, or the like.
　食品は、例えば、製菓、製パン類、穀粉、麺類、飯類、農産・林産加工食品、畜産加工品、水産加工品、乳・乳製品、油脂・油脂加工品、調味料またはその他の食品素材等が挙げられる。 Foods include, for example, confectionery, breads, flour, noodles, rice, processed agricultural / forest foods, processed livestock products, processed marine products, milk / dairy products, fats / oils / fat processed products, seasonings or other food materials. And so on.
　飲料は、例えば、炭酸飲料、スポーツドリンク、フレーバーウォーター、果汁飲料、アルコール飲料、非アルコール飲料、ビールやノンアルコールビール等のビールテイスト飲料、コーヒー飲料、茶飲料、ココア飲料、栄養飲料、機能性飲料等が挙げられる。また、炭酸飲料としては、スパークリング飲料、コーラ、ダイエットコーラ、ジンジャーエール、サイダー、果汁フレーバー炭酸飲料および果汁風味が付与された炭酸水等が挙げられる。 Beverages include, for example, carbonated beverages, sports drinks, flavored water, fruit juice beverages, alcoholic beverages, non-alcoholic beverages, beer-taste beverages such as beer and non-alcoholic beer, coffee beverages, tea beverages, cocoa beverages, nutritional beverages, and functional beverages. And so on. Examples of carbonated drinks include sparkling drinks, colas, diet colas, ginger ales, ciders, fruit juice flavored carbonated drinks, and carbonated waters to which fruit juice flavor is imparted.
　本発明の一態様において、飲料は、ステビア植物由来のアミノ酸の含有量が、例えば、１～６００ｐｐｍ、１０～６００ｐｐｍ、２０～６００ｐｐｍ、３０～６００ｐｐｍ、４０～６００ｐｐｍ、５０～６００ｐｐｍ、１～５００ｐｐｍ、１０～５００ｐｐｍ、２０～５００ｐｐｍ、３０～５００ｐｐｍ、４０～５００ｐｐｍ、５０～５００ｐｐｍ、１～４００ｐｐｍ、１０～４００ｐｐｍ、２０～４００ｐｐｍ、３０～４００ｐｐｍ、４０～４００ｐｐｍ、５０～４００ｐｐｍ、１～３５０ｐｐｍ、１０～３５０ｐｐｍ、２０～３５０ｐｐｍ、３０～３５０ｐｐｍ、４０～３５０ｐｐｍ、５０～３５０ｐｐｍ、１～３００ｐｐｍ、１０～３００ｐｐｍ、２０～３００ｐｐｍ、３０～３００ｐｐｍ、４０～３００ｐｐｍ、５０～３００ｐｐｍ、１～２５００ｐｐｍ、１０～２５０ｐｐｍ、２０～２５０ｐｐｍ、３０～２５０ｐｐｍ、４０～２５０ｐｐｍ、５０～２５０ｐｐｍ、１～２００ｐｐｍ、１０～２００ｐｐｍ、２０～２００ｐｐｍ、３０～２００ｐｐｍ、４０～２００ｐｐｍまたは５０～２００ｐｐｍ等であってもよい。アミノ酸の含有量を上記範囲とすることで、飲料の甘味を増強することができる。いくつかの態様では、アミノ酸の含有量を上記範囲とすることで、スクロースの甘味を増強することができる。別のいくつかの態様では、アミノ酸の含有量を上記範囲とすることで、レバウジオシドＡの甘味を増強することができる。 In one aspect of the invention, the beverage has a stevia plant-derived amino acid content of, for example, 1-600 ppm, 10-600 ppm, 20-600 ppm, 30-600 ppm, 40-600 ppm, 50-600 ppm, 1-500 ppm. 10-500ppm, 20-500ppm, 30-500ppm, 40-500ppm, 50-500ppm, 1-400ppm, 10-400ppm, 20-400ppm, 30-400ppm, 40-400ppm, 50-400ppm, 1-350ppm, 10- 350ppm, 20-350ppm, 30-350ppm, 40-350ppm, 50-350ppm, 1-300ppm, 10-300ppm, 20-300ppm, 30-300ppm, 40-300ppm, 50-300ppm, 1-2500ppm, 10-250ppm, It may be 20 to 250 ppm, 30 to 250 ppm, 40 to 250 ppm, 50 to 250 ppm, 1 to 200 ppm, 10 to 200 ppm, 20 to 200 ppm, 30 to 200 ppm, 40 to 200 ppm, 50 to 200 ppm, or the like. By setting the amino acid content in the above range, the sweetness of the beverage can be enhanced. In some embodiments, the sweetness of sucrose can be enhanced by setting the amino acid content in the above range. In some other embodiments, the amino acid content may be in the above range to enhance the sweetness of rebaugioside A.
　また、いくつかの態様において、飲料は、ステビオール配糖体を含んでいてもよい。飲料におけるステビオール配糖体の含有量は、１～８００ｐｐｍであるのが好ましく、例えば、２０～７５０ｐｐｍ、２０～７００ｐｐｍ、２０～６５０ｐｐｍ、２０～６００ｐｐｍ、２０～５５０ｐｐｍ、２５～５５０ｐｐｍ、３０～５５０ｐｐｍ、３５～５５０ｐｐｍ、４０～５５０ｐｐｍ、４５～５５０ｐｐｍ、５０～５５０ｐｐｍ、５５～５５０ｐｐｍ、２０～５４０ｐｐｍ、２５～５４０ｐｐｍ、３０～５４０ｐｐｍ、３５～５４０ｐｐｍ、４０～５４０ｐｐｍ、４５～５４０ｐｐｍ、５０～５４０ｐｐｍ、５５～５４０ｐｐｍ、２０～５３０ｐｐｍ、２５～５３０ｐｐｍ、３０～５３０ｐｐｍ、３５～５３０ｐｐｍ、４０～５３０ｐｐｍ、４５～５３０ｐｐｍ、５０～５３０ｐｐｍ、５５～５３０ｐｐｍ、２０～５２０ｐｐｍ、２５～５２０ｐｐｍ、３０～５２０ｐｐｍ、３５～５２０ｐｐｍ、４０～５２０ｐｐｍ、４５～５２０ｐｐｍ、５０～５２０ｐｐｍ、５５～５２０ｐｐｍ、２０～５１０ｐｐｍ、２５～５１０ｐｐｍ、３０～５１０ｐｐｍ、３５～５１０ｐｐｍ、４０～５１０ｐｐｍ、４５～５１０ｐｐｍ、５０～５１０ｐｐｍ、５５～５１０ｐｐｍ、２０～５０５ｐｐｍ、２５～５０５ｐｐｍ、３０～５０５ｐｐｍ、３５～５０５ｐｐｍ、４０～５０５ｐｐｍ、４５～５０５ｐｐｍ、５０～５０５ｐｐｍ、５５～５０５ｐｐｍ、２０～５００ｐｐｍ、２５～５００ｐｐｍ、３０～５００ｐｐｍ、３５～５００ｐｐｍ、４０～５００ｐｐｍ、４５～５００ｐｐｍ、５０～５００ｐｐｍ、５５～５００ｐｐｍ、２０～４９５ｐｐｍ、２５～４９５ｐｐｍ、３０～４９５ｐｐｍ、３５～４９５ｐｐｍ、４０～４９５ｐｐｍ、４５～４９５ｐｐｍ、５０～４９５ｐｐｍ、５５～４９５ｐｐｍ、２０～４９０ｐｐｍ、２５～４９０ｐｐｍ、３０～４９０ｐｐｍ、３５～４９０ｐｐｍ、４０～４９０ｐｐｍ、４５～４９０ｐｐｍ、５０～４９０ｐｐｍ、５５～４９０ｐｐｍ、１００～４００ｐｐｍ、１５０～４００ｐｐｍ、２００～４００ｐｐｍ、２５０～４００ｐｐｍ、３００～４００ｐｐｍ、１００～１５０ｐｐｍ、１００～２００ｐｐｍ、１００～２５０ｐｐｍまたは１００～３００ｐｐｍ等であってもよい。ステビオール配糖体の含有量をこの範囲とすることで、飲料に適度な甘みを付与することができる。 Also, in some embodiments, the beverage may contain steviol glycosides. The content of steviol glycoside in the beverage is preferably 1 to 800 ppm, for example, 20 to 750 ppm, 20 to 700 ppm, 20 to 650 ppm, 20 to 600 ppm, 20 to 550 ppm, 25 to 550 ppm, 30 to 550 ppm, 35-550 ppm, 40-550 ppm, 45-550 ppm, 50-550 ppm, 55-550 ppm, 20-540 ppm, 25-540 ppm, 30-540 ppm, 35-540 ppm, 40-540 ppm, 45-540 ppm, 50-540 ppm, 55- 540ppm, 20-530ppm, 25-530ppm, 30-530ppm, 35-530ppm, 40-530ppm, 45-530ppm, 50-530ppm, 55-530ppm, 20-520ppm, 25-520ppm, 30-520ppm, 35-520ppm, 40-520 ppm, 45-520 ppm, 50-520 ppm, 55-520 ppm, 20-510 ppm, 25-510 ppm, 30-510 ppm, 35-510 ppm, 40-510 ppm, 45-510 ppm, 50-510 ppm, 55-510 ppm, 20- 505 ppm, 25-505 ppm, 30-505 ppm, 35-505 ppm, 40-505 ppm, 45-505 ppm, 50-505 ppm, 55-505 ppm, 20-500 ppm, 25-500 ppm, 30-500 ppm, 35-500 ppm, 40-500 ppm, 45-500ppm, 50-500ppm, 55-500ppm, 20-495ppm, 25-495ppm, 30-495ppm, 35-495ppm, 40-495ppm, 45-495ppm, 50-495ppm, 55-495ppm, 20-490ppm, 25- 490ppm, 30-490ppm, 35-490ppm, 40-490ppm, 45-490ppm, 50-490ppm, 55-490ppm, 100-400ppm, 150-400ppm, 200-400ppm, 250-400ppm, 300-400ppm, 100-150ppm, It may be 100 to 200 ppm, 100 to 250 ppm, 100 to 300 ppm, or the like. By setting the content of the steviol glycoside in this range, it is possible to impart an appropriate sweetness to the beverage.
　本発明の一態様において、飲料は、ステビオール配糖体以外の甘味料を含んでいてもよい。そのような甘味料としては特に限定されないが、例えば、スクロース、果糖ぶどう糖液糖
、エリスリトール、モグロシドＶ、コーンシロップ、アスパルテーム（Ｌ－フェニルアラニン化合物とも称される）、スクラロース、アセスルファムカリウム、サッカリンおよびキシリトールからなる群から選択される１種以上の甘味料をさらに含んでいてもよい。その中でも、すっきりさ、飲みやすさ、自然な味わい、適度なコク味の付与の観点から、天然甘味料を用いることが好ましく、特に、果糖ぶどう糖液糖、スクロース、コーンシロップが好適に用いられる。これら甘味成分は１種類のみ用いてもよく、また複数種類を用いてもよい。これらの甘味料は、飲料中にショ糖換算のＢｒｉｘで５．０以下、４．５以下、４．０以下、３．５以下、３．０以下、２．５以下、２．０以下、１．５以下、１．０以下または０．５以下等に相当する量で含まれていてもよく、下限値は０．１以上であってもよい。 In one aspect of the invention, the beverage may contain sweeteners other than steviol glycosides. Such sweeteners are not particularly limited, but are, for example, from sucrose, fructose-glucose liquid sugar, erythritol, mogloside V, corn syrup, aspartame (also referred to as L-phenylalanine compound), sucralose, acesulfame potassium, saccharin and xylitol. It may further contain one or more sweeteners selected from the group. Among them, it is preferable to use a natural sweetener from the viewpoint of refreshing, easy to drink, natural taste, and imparting an appropriate richness, and in particular, fructose-glucose liquid sugar, sucrose, and corn syrup are preferably used. Only one kind of these sweetness components may be used, or a plurality of kinds may be used. These sweeteners have a Brix equivalent of 5.0 or less, 4.5 or less, 4.0 or less, 3.5 or less, 3.0 or less, 2.5 or less, 2.0 or less in a beverage. It may be contained in an amount corresponding to 1.5 or less, 1.0 or less, 0.5 or less, etc., and the lower limit may be 0.1 or more.
　飲料が発泡性飲料である場合、ガス圧は、２．２ｋｇｆ／ｃｍ２～５．０ｋｇｆ／ｃｍ２であってもよい。いくつかの態様において、発泡性飲料のガス圧は、２．２ｋｇｆ／ｃｍ２～４．５ｋｇｆ／ｃｍ２、２．２ｋｇｆ／ｃｍ２～４．０ｋｇｆ／ｃｍ２、２．２ｋｇｆ／ｃｍ２～３．５ｋｇｆ／ｃｍ２、２．２ｋｇｆ／ｃｍ２～３．３ｋｇｆ／ｃｍ２、２．２ｋｇｆ／ｃｍ２～３．２ｋｇｆ／ｃｍ２、２．３ｋｇｆ／ｃｍ２～４．０ｋｇｆ／ｃｍ２、２．３ｋｇｆ／ｃｍ２～３．５ｋｇｆ／ｃｍ２、２．３ｋｇｆ／ｃｍ２～３．２ｋｇｆ／ｃｍ２、３．０ｋｇｆ／ｃｍ２～４．０ｋｇｆ／ｃｍ２または３．０ｋｇｆ／ｃｍ２～３．５ｋｇｆ／ｃｍ２等であってもよい。発泡性飲料におけるガスの含量は、ガス圧で規定することができる。本明細書において「ガス圧」とは、特に記載がなければ、容器内の発泡性飲料の液温を２０℃にし、その後一度ヘッドスペースのエアを大気開放（スニフト）した後の飲料における炭酸ガスのガス圧をいう。ガス圧の測定は、液温２０℃にした飲料をガス内圧計に固定し、一度ガス内圧計活栓を開いて大気開放することでヘッドスペース内の炭酸ガスを抜いた後、再度活栓を閉じ、ガス内圧計を振り動かして指針が一定の位置に達した時の値を読み取ることにより行うことができる。本明細書においては、特に記載がなければ、当該方法を用いて発泡性飲料のガス圧を測定する。 When the beverage is a sparkling beverage, the gas pressure may be 2.2 kgf / cm 2 to 5.0 kgf / cm 2 . In some embodiments, the gas pressure of the effervescent beverage is 2.2 kgf / cm 2 to 4.5 kgf / cm 2 , 2.2 kgf / cm 2 to 4.0 kgf / cm 2 , 2.2 kgf / cm 2 to 3. .5kgf / cm 2 , 2.2kgf / cm 2 to 3.3kgf / cm 2 , 2.2kgf / cm 2 to 3.2kgf / cm 2 , 2.3kgf / cm 2 to 4.0kgf / cm 2 , 2. 3 kgf / cm 2 to 3.5 kgf / cm 2 , 2.3 kgf / cm 2 to 3.2 kgf / cm 2 , 3.0 kgf / cm 2 to 4.0 kgf / cm 2 or 3.0 kgf / cm 2 to 3.5 kgf It may be / cm 2 or the like. The gas content in the effervescent beverage can be defined by the gas pressure. Unless otherwise specified, the term "gas pressure" as used herein refers to carbon dioxide gas in a beverage after the temperature of the effervescent beverage in the container is set to 20 ° C. and then the air in the headspace is once opened to the atmosphere (snift). The gas pressure of. To measure the gas pressure, fix the beverage at a liquid temperature of 20 ° C to the gas internal pressure gauge, open the stopcock of the gas internal pressure gauge once and open it to the atmosphere to remove the carbon dioxide gas in the headspace, and then close the stopcock again. This can be done by swinging the gas pressure gauge and reading the value when the pointer reaches a certain position. In the present specification, unless otherwise specified, the gas pressure of the effervescent beverage is measured by using the method.
　飲料のショ糖換算のＢｒｉｘは、特に限定されないが、好ましくは１～１５、より好ましくは３～１４、さらに好ましくは５～１３、特に好ましくは７～１１である。Ｂｒｉｘは、ショ糖（スクロース）に対するステビオール配糖体等の各甘味料の公知の甘味度と、各甘味料の含有量から計算することができる。なお、ショ糖の甘味１に対する各種甘味料の甘味の相対比は、公知の砂糖甘味換算表（例えば、ビバレッジジャパン社「飲料用語辞典」資料１１頁）等から求めることができる。甘味の値が数値範囲で記載されているものや、文献によって値が異なる甘味料については、ショ糖の甘味１に対する甘味の相対比を官能試験によって定めることができる。そのような官能試験としては、例えば、Brix３．０から５．０まで０．５刻となるよう砂糖を純水に添加したサンプルを調製し、その中から所定濃度の甘味料の水溶液と同等の甘味強度を持つ砂糖添加サンプルを選択する方法が挙げられる。 The Brix in terms of sucrose in the beverage is not particularly limited, but is preferably 1 to 15, more preferably 3 to 14, still more preferably 5 to 13, and particularly preferably 7 to 11. Brix can be calculated from the known sweetness of each sweetener such as steviol glycoside with respect to sucrose and the content of each sweetener. The relative ratio of the sweetness of various sweeteners to the sweetness 1 of sucrose can be obtained from a known sugar sweetness conversion table (for example, page 11 of "Beverage Glossary", Beverage Japan Co., Ltd.). For sweeteners whose sweetness values are described in a numerical range or whose values differ depending on the literature, the relative ratio of sweetness to sweetness 1 of sucrose can be determined by a sensory test. As such a sensory test, for example, a sample in which sugar is added to pure water from Brix 3.0 to 5.0 in 0.5 increments is prepared, and the sample is equivalent to an aqueous solution of a sweetener having a predetermined concentration. A method of selecting a sugar-added sample having a sweetness intensity can be mentioned.
　飲料は、アルコールを含有してもよい。アルコール飲料とは、アルコールを含有する飲料のことであるが、ここでいうアルコールとは、上述のとおり、特に断らない限り、エチルアルコール（エタノール）を意味する。アルコール飲料は、アルコールを含有していれば特に種類は問わない。ビール、発泡酒、チューハイやカクテルのようなアルコール含有量が０．０５～４０ｖ／ｖ％、１．０～１０ｖ／ｖ％、２．０～９．０ｖ／ｖ％または３．０～８．０ｖ／ｖ％の飲料であってもよく、ノンアルコールビール、チューハイテイスト飲料や清涼飲料水のようなアルコール含有量が０．０５ｖ／ｖ％未満の飲料であってもよい。いくつかの態様では、飲料はアルコール含有量が０．０５ｖ／ｖ％未満であることが好ましく、さらに０．００ｖ／ｖ％であることが好ましい。なお、本明細書において、アルコール含有量は体積／体積基準の百分率（ｖ／ｖ％）で示されるものとする。また、飲料のアルコール含有量は、公知のいずれの方法によっても測定することができるが、例えば、振動式密度計によって測定することができる。 Beverages may contain alcohol. The alcoholic beverage is a beverage containing alcohol, and as described above, the alcohol here means ethyl alcohol (ethanol) unless otherwise specified. The type of alcoholic beverage is not particularly limited as long as it contains alcohol. Alcohol content such as beer, soft drinks, chu-hi and cocktails is 0.05-40v / v%, 1.0-10v / v%, 2.0-9.0v / v% or 3.0-8. It may be a 0v / v% beverage, or may be a beverage having an alcohol content of less than 0.05v / v%, such as non-alcoholic beer, chu-hi taste beverages, and soft drinks. In some embodiments, the beverage preferably has an alcohol content of less than 0.05 v / v%, more preferably 0.00 v / v%. In this specification, the alcohol content is expressed as a volume / volume-based percentage (v / v%). The alcohol content of the beverage can be measured by any known method, for example, by a vibration densitometer.
　飲料の風味（フレーバー）は特に限定されず、種々の風味に調整することができる。例えば、飲料は、オレンジ風味、レモン風味、ライム風味、グレープ風味、ジンジャーエール風味、カシス風味、緑茶風味、ウーロン茶風味、紅茶風味、コーヒー風味またはコーラ風味の飲料であってもよい。本発明の飲料の風味は、果汁、酸味料、香料、植物の抽出物、乳製品、その他のフレーバー等、食品添加物として認可されている成分、または認可されていなくても古くから食経験があり、一般的に安全であると認識されている成分を添加することで調整することができる。 The flavor of the beverage is not particularly limited and can be adjusted to various flavors. For example, the beverage may be an orange-flavored, lemon-flavored, lime-flavored, grape-flavored, ginger-ale-flavored, cassis-flavored, green-tea-flavored, oolong-tea-flavored, black-tea-flavored, coffee-flavored or cola-flavored beverage. The flavor of the beverage of the present invention is an ingredient approved as a food additive such as fruit juice, acidulant, flavoring, plant extract, dairy product, and other flavors, or even if it is not approved, it has a long history of eating. It can be adjusted by adding ingredients that are generally recognized as safe.
  飲料には、本発明の効果を妨げない範囲で、各種の添加剤を配合してもよい。そのような添加剤としては、例えば、酸味料、香料、ビタミン類、色素類、酸化防止剤、乳化剤、保存料、調味料、エキス類、ｐＨ調整剤、品質安定剤等を挙げることができる。 Various additives may be added to the beverage as long as the effects of the present invention are not impaired. Examples of such additives include acidulants, fragrances, vitamins, pigments, antioxidants, emulsifiers, preservatives, seasonings, extracts, pH regulators, quality stabilizers and the like.
　飲料のエネルギー（総エネルギー量）は、特に限定されないが、０～５０Ｋｃａｌ／１００ｍｌ、０～４５Ｋｃａｌ／１００ｍｌ、０～４０Ｋｃａｌ／１００ｍｌ、０～３５Ｋｃａｌ／１００ｍｌ、０～３０Ｋｃａｌ／１００ｍｌ、０～２４Ｋｃａｌ／１００ｍｌ、０～２２Ｋｃａｌ／１００ｍｌ、０～２０Ｋｃａｌ／１００ｍｌ、０～１５Ｋｃａｌ／１００ｍｌ、０～１０Ｋｃａｌ／１００ｍｌ、０～５Ｋｃａｌ／１００ｍｌ、０．１～５０Ｋｃａｌ／１００ｍｌ、０．１～４５Ｋｃａｌ／１００ｍｌ、０．１～４０Ｋｃａｌ／１００ｍｌ、０．１～３５Ｋｃａｌ／１００ｍｌ、０．１～３０Ｋｃａｌ／１００ｍｌ、０．１～２４Ｋｃａｌ／１００ｍｌ、０．１～２２Ｋｃａｌ／１００ｍｌ、０．１～２０Ｋｃａｌ／１００ｍｌ、０．１～１５Ｋｃａｌ／１００ｍｌ、０．１～１０Ｋｃａｌ／１００ｍｌ、０．１～５Ｋｃａｌ／１００ｍｌ、１～５０Ｋｃａｌ／１００ｍｌ、１～４５Ｋｃａｌ／１００ｍｌ、１～４０Ｋｃａｌ／１００ｍｌ、１～３５Ｋｃａｌ／１００ｍｌ、１～３０Ｋｃａｌ／１００ｍｌ、１～２４Ｋｃａｌ／１００ｍｌ、１～２２Ｋｃａｌ／１００ｍｌ、１～２０Ｋｃａｌ／１００ｍｌ、１～１５Ｋｃａｌ／１００ｍｌ、１～１０Ｋｃａｌ／１００ｍｌ、１～５Ｋｃａｌ／１００ｍｌ、５～５０Ｋｃａｌ／１００ｍｌ、５～４５Ｋｃａｌ／１００ｍｌ、５～４０Ｋｃａｌ／１００ｍｌ、５～３５Ｋｃａｌ／１００ｍｌ、５～３０Ｋｃａｌ／１００ｍｌ、５～２４Ｋｃａｌ／１００ｍｌ、５～２０Ｋｃａｌ／１００ｍｌ、５～１５Ｋｃａｌ／１００ｍｌ、５～１０Ｋｃａｌ／１００ｍｌ、１０～５０Ｋｃａｌ／１００ｍｌ、１０～４５Ｋｃａｌ／１００ｍｌ、１０～４０Ｋｃａｌ／１００ｍｌ、１０～３５Ｋｃａｌ／１００ｍｌ、１０～３０Ｋｃａｌ／１００ｍｌ、１０～２４Ｋｃａｌ／１００ｍｌ、１０～２０Ｋｃａｌ／１００ｍｌ、１０～１５Ｋｃａｌ／１００ｍｌ、１５～５０Ｋｃａｌ／１００ｍｌ、１５～４５Ｋｃａｌ／１００ｍｌ、１５～４０Ｋｃａｌ／１００ｍｌ、１５～３５Ｋｃａｌ／１００ｍｌ、１５～３０Ｋｃａｌ／１００ｍｌ、１５～２４Ｋｃａｌ／１００ｍｌ、１５～２０Ｋｃａｌ／１００ｍｌ、２０～５０Ｋｃａｌ／１００ｍｌ、２０～４５Ｋｃａｌ／１００ｍｌ、２０～４０Ｋｃａｌ／１００ｍｌ、２０～３５Ｋｃａｌ／１００ｍｌ、２０～３０Ｋｃａｌ／１００ｍｌ、２０～２４Ｋｃａｌ／１００ｍｌ、２４～５０Ｋｃａｌ／１００ｍｌ、２４～４５Ｋｃａｌ／１００ｍｌ、２４～４０Ｋｃａｌ／１００ｍｌ、２４～３５Ｋｃａｌ／１００ｍｌまたは２４～３０Ｋｃａｌ／１００ｍｌ等であってもよい。 The energy (total energy amount) of the beverage is not particularly limited, but is 0 to 50 Kcal / 100 ml, 0 to 45 Kcal / 100 ml, 0 to 40 Kcal / 100 ml, 0 to 35 Kcal / 100 ml, 0 to 30 Kcal / 100 ml, 0 to 24 Kcal / 100 ml. , 0-22 Kcal / 100 ml, 0-20 Kcal / 100 ml, 0-15 Kcal / 100 ml, 0-10 Kcal / 100 ml, 0-5 Kcal / 100 ml, 0.1-50 Kcal / 100 ml, 0.1-45 Kcal / 100 ml, 0.1 ~ 40Kcal / 100ml, 0.1 ~ 35Kcal / 100ml, 0.1 ~ 30Kcal / 100ml, 0.1 ~ 24Kcal / 100ml, 0.1 ~ 22Kcal / 100ml, 0.1 ~ 20Kcal / 100ml, 0.1 ~ 15Kcal / 100 ml, 0.1-10 Kcal / 100 ml, 0.1-5 Kcal / 100 ml, 1-50 Kcal / 100 ml, 1-45 Kcal / 100 ml, 1-40 Kcal / 100 ml, 1-35 Kcal / 100 ml, 1-30 Kcal / 100 ml, 1 ~ 24Kcal / 100ml, 1 ~ 22Kcal / 100ml, 1 ~ 20Kcal / 100ml, 1 ~ 15Kcal / 100ml, 1 ~ 10Kcal / 100ml, 1 ~ 5Kcal / 100ml, 5 ~ 50Kcal / 100ml, 5 ~ 45Kcal / 100ml, 5 ~ 40Kcal / 100 ml, 5 to 35 Kcal / 100 ml, 5 to 30 Kcal / 100 ml, 5 to 24 Kcal / 100 ml, 5 to 20 Kcal / 100 ml, 5 to 15 Kcal / 100 ml, 5 to 10 Kcal / 100 ml, 10 to 50 Kcal / 100 ml, 10 to 45 Kcal / 100 ml. 10-40 Kcal / 100 ml, 10-35 Kcal / 100 ml, 10-30 Kcal / 100 ml, 10-24 Kcal / 100 ml, 10-20 Kcal / 100 ml, 10-15 Kcal / 100 ml, 15-50 Kcal / 100 ml, 15-45 Kcal / 100 ml, 15 ~ 40Kcal / 100ml, 15 ~ 35Kcal / 100ml, 15 ~ 30Kcal / 100ml, 15 ~ 24Kcal / 100ml, 15 ~ 20Kcal / 100ml, 20 ~ 50Kcal / 100ml, 20 ~ 45Kcal / 100ml, 20 ~ 40Kcal / 100ml, 20 ~ 35Kcal / 100ml, 20-30Kcal / 100ml, 2 It may be 0 to 24 Kcal / 100 ml, 24 to 50 Kcal / 100 ml, 24 to 45 Kcal / 100 ml, 24 to 40 Kcal / 100 ml, 24-35 Kcal / 100 ml, 24 to 30 Kcal / 100 ml, or the like.
　飲料は、加熱殺菌をされ、容器に詰められた状態の容器詰飲料として調製してもよい。容器としては、特に限定されず、例えば、ＰＥＴボトル、アルミ缶、スチール缶、紙パック、チルドカップ、瓶などを挙げることができる。加熱殺菌を行う場合、その種類は特に限定されず、例えば、ＵＨＴ殺菌およびレトルト殺菌等の通常の手法を用いて行うことができる。加熱殺菌工程の温度は特に限定されないが、例えば、６５～１３０℃、好ましくは８５～１２０℃で、１０～４０分である。ただし、上記の条件と同等の殺菌価が得られれば適当な温度で数秒、例えば５～３０秒での殺菌でも問題はない。 The beverage may be prepared as a packaged beverage that has been sterilized by heating and packed in a container. The container is not particularly limited, and examples thereof include PET bottles, aluminum cans, steel cans, paper packs, chilled cups, and bottles. When heat sterilization is performed, the type is not particularly limited, and for example, it can be performed using ordinary methods such as UHT sterilization and retort sterilization. The temperature of the heat sterilization step is not particularly limited, but is, for example, 65 to 130 ° C., preferably 85 to 120 ° C., for 10 to 40 minutes. However, if a sterilization value equivalent to the above conditions can be obtained, there is no problem in sterilization at an appropriate temperature for several seconds, for example, 5 to 30 seconds.
　飲食品の製造方法は、上記の各成分を含む飲食品が得られれば特に限定されない。本発明の一態様によれば、飲食品の製造方法であって、ステビア植物の酵素処理組成物を得ることと、前記酵素処理組成物を飲食品またはその原料に添加することと、を含む製造方法が提供される。ステビア植物の酵素処理組成物を得ることは、上記「２．ステビア植物の酵素処理組成物の製造方法」に記載のとおりである。酵素処理組成物を飲食品またはその原料に添加することは、飲食品の製造工程における任意の工程で行うことができ、例えば、飲食品の原料を混合する際や、飲食品の味質の最終調整の際に行ってもよい。 The method for producing food and drink is not particularly limited as long as food and drink containing each of the above components can be obtained. According to one aspect of the present invention, a method for producing a food or drink, which comprises obtaining an enzyme-treated composition of a stevia plant and adding the enzyme-treated composition to the food or drink or a raw material thereof. The method is provided. Obtaining the enzyme-treated composition of Stevia plant is as described in "2. Method for producing enzyme-treated composition of Stevia plant" above. The enzyme-treated composition can be added to a food or drink or a raw material thereof at any step in the manufacturing process of the food or drink, for example, when mixing the raw materials of the food or drink or the final taste of the food or drink. It may be done at the time of adjustment.
４．飲食品の甘味増強方法
　本発明は、飲食品の甘味増強方法にも関する。具体的には、本発明は、上記「１．ステビア植物の酵素処理組成物」で述べた組成物を飲食品（例えば、飲料）に配合することを含む、飲食品の甘味増強方法に関する。前記組成物については、上記「１．ステビア植物の酵素処理組成物」で述べたとおりである。また、前記組成物を飲食品に配合することは、上記「３．ステビア植物の酵素処理組成物を含む飲食品」で述べたように、例えば、飲食品の原料を混合する際や、飲食品の味質の最終調整の際に、前記組成物を飲食品またはその原料に添加（または配合）してもよい。 4. Method for enhancing sweetness of food and drink The present invention also relates to a method for enhancing sweetness of food and drink. Specifically, the present invention relates to a method for enhancing the sweetness of foods and drinks, which comprises blending the composition described in the above "1. Enzyme-treated composition of Stevia plant" into foods and drinks (for example, beverages). The composition is as described in "1. Enzyme-treated composition of Stevia plant" above. In addition, blending the composition into foods and drinks is, for example, when mixing raw materials for foods and drinks, as described in "3. Foods and drinks containing enzyme-treated compositions of stevia plants", and foods and drinks. The composition may be added (or blended) to a food or drink or a raw material thereof at the time of final adjustment of the taste quality of.
　本明細書において、「甘味増強」または「甘味を増強する」とは、本発明の一態様に係るステビア植物の酵素処理組成物を含む飲食品が、これを含まない飲食品と比較して甘味を強く感じる性質、または当該性質を得ることを意味する。 As used herein, "enhancing sweetness" or "enhancing sweetness" means that a food or drink containing an enzyme-treated composition of a stevia plant according to an aspect of the present invention has a sweetness as compared with a food or drink not containing the enzyme-treated composition. It means to have a property that strongly feels or to obtain the property.
　本発明の一態様における甘味増強方法は、ステビオール配糖体およびスクロースの甘味を増強するものであってもよい。いくつかの態様では、前記方法は、スクロースの甘味を増強するものであってもよい。別のいくつかの態様では、前記方法は、ステビオール配糖体の甘味を増強するものであってもよい。更に別のいくつかの態様では、前記方法は、レバウジオシドＡの甘味を増強するものであってもよい。 The sweetness enhancing method in one aspect of the present invention may enhance the sweetness of steviol glycosides and sucrose. In some embodiments, the method may enhance the sweetness of sucrose. In some other embodiments, the method may enhance the sweetness of steviol glycosides. In yet some other embodiments, the method may enhance the sweetness of rebaugioside A.
　本発明の一態様における甘味増強方法は、前記酵素処理組成物を、ステビア植物由来のアミノ酸の含有量が１～６００ｐｐｍとなるように飲食品に添加することを含むものであってもよい。飲食品における前記アミノ酸の含有量は、例えば、１０～６００ｐｐｍ、２０～６００ｐｐｍ、３０～６００ｐｐｍ、４０～６００ｐｐｍ、５０～６００ｐｐｍ、１～５００ｐｐｍ、１０～５００ｐｐｍ、２０～５００ｐｐｍ、３０～５００ｐｐｍ、４０～５００ｐｐｍ、５０～５００ｐｐｍ、１～４００ｐｐｍ、１０～４００ｐｐｍ、２０～４００ｐｐｍ、３０～４００ｐｐｍ、４０～４００ｐｐｍ、５０～４００ｐｐｍ、１～３５０ｐｐｍ、１０～３５０ｐｐｍ、２０～３５０ｐｐｍ、３０～３５０ｐｐｍ、４０～３５０ｐｐｍ、５０～３５０ｐｐｍ、１～３００ｐｐｍ、１０～３００ｐｐｍ、２０～３００ｐｐｍ、３０～３００ｐｐｍ、４０～３００ｐｐｍ、５０～３００ｐｐｍ、１～２５００ｐｐｍ、１０～２５０ｐｐｍ、２０～２５０ｐｐｍ、３０～２５０ｐｐｍ、４０～２５０ｐｐｍ、５０～２５０ｐｐｍ、１～２００ｐｐｍ、１０～２００ｐｐｍ、２０～２００ｐｐｍ、３０～２００ｐｐｍ、４０～２００ｐｐｍまたは５０～２００ｐｐｍ等であってもよい。 The method for enhancing sweetness in one aspect of the present invention may include adding the enzyme-treated composition to foods and drinks so that the content of amino acids derived from Stevia plants is 1 to 600 ppm. The content of the amino acid in foods and drinks is, for example, 10 to 600 ppm, 20 to 600 ppm, 30 to 600 ppm, 40 to 600 ppm, 50 to 600 ppm, 1 to 500 ppm, 10 to 500 ppm, 20 to 500 ppm, 30 to 500 ppm, 40 to 40. 500ppm, 50-500ppm, 1-400ppm, 10-400ppm, 20-400ppm, 30-400ppm, 40-400ppm, 50-400ppm, 1-350ppm, 10-350ppm, 20-350ppm, 30-350ppm, 40-350ppm, 50-350ppm, 1-300ppm, 10-300ppm, 20-300ppm, 30-300ppm, 40-300ppm, 50-300ppm, 1-2500ppm, 10-250ppm, 20-250ppm, 30-250ppm, 40-250ppm, 50- It may be 250 ppm, 1 to 200 ppm, 10 to 200 ppm, 20 to 200 ppm, 30 to 200 ppm, 40 to 200 ppm, 50 to 200 ppm, or the like.
５．飲食品の甘味増強剤
　本発明は、飲食品の甘味増強剤にも関する。また、本発明は、飲食品の甘味増強用組成物であってもよい。本明細書において、「甘味増強剤」または「甘味増強用組成物」とは、飲食品（例えば、飲料）に添加された場合に、当該飲食品の甘味を増強する物質のことを意味する。本明細書において、甘味増強剤についての説明は、甘味増強用組成物についても当てはまる。甘味増強剤は、好ましくは、飲食品に添加された際に、甘味増強剤自体の味を消費者が認識することなく、その飲食品の甘味を増強することができる。 5. Food and Beverage Sweetness Enhancer The present invention also relates to a food and drink sweetness enhancer. Further, the present invention may be a composition for enhancing the sweetness of food and drink. As used herein, the term "sweetness enhancer" or "sweetness enhancer composition" means a substance that enhances the sweetness of a food or drink when added to a food or drink (for example, a beverage). As used herein, the description of the sweetness enhancer also applies to the sweetness enhancer composition. The sweetness enhancer can preferably enhance the sweetness of the food or drink when added to the food or drink without the consumer recognizing the taste of the sweetness enhancer itself.
　本発明の一態様において、甘味増強剤は、上記「１．ステビア植物の酵素処理組成物」で述べた酵素処理組成物を含む。甘味増強剤における有効成分は、前記酵素処理組成物に含まれるステビア植物由来のアミノ酸であり得る。 In one aspect of the present invention, the sweetness enhancer includes the enzyme-treated composition described in "1. Enzyme-treated composition of Stevia plant" above. The active ingredient in the sweetness enhancer may be a Stevia plant-derived amino acid contained in the enzyme-treated composition.
　本発明の一態様において、甘味増強剤は、液状、ペースト状、粉末状または顆粒状等の形態であってもよい。 In one aspect of the present invention, the sweetness enhancer may be in the form of a liquid, a paste, a powder, a granule, or the like.
　本発明の一態様において、甘味増強剤に含まれる酵素処理組成物の含有量は、甘味増強剤の全重量に対して、３０～１００重量％、４０～９９重量％、５０～９８重量％、６０～９７重量％、７０～９６重量％または８０～９５重量％等であってもよい。いくつかの態様において、甘味増強剤は、実質的にステビア植物由来のアミノ酸のみからなっていてもよい。本明細書において、「実質的にステビア植物由来のアミノ酸のみからなる」とは、酵素処理組成物の製造過程において不可避的に含まれるステビオール配糖体や他の不純物は含まれていてもよいことを意味する。例えば、甘味増強剤の全重量に対して、５重量％以下、４重量％以下、３重量％以下、２重量％以下、１．５重量％以下、１．０重量％以下または０．５重量％以下等の前記アミノ酸以外のステビオール配糖体や他の不純物を含んでいてもよい。 In one embodiment of the present invention, the content of the enzyme-treated composition contained in the sweetness enhancer is 30 to 100% by weight, 40 to 99% by weight, 50 to 98% by weight, based on the total weight of the sweetness enhancer. It may be 60 to 97% by weight, 70 to 96% by weight, 80 to 95% by weight, or the like. In some embodiments, the sweetness enhancer may consist substantially solely of amino acids derived from Stevia plants. As used herein, the phrase "consisting substantially only of amino acids derived from Steviol plants" means that steviol glycosides and other impurities inevitably contained in the process of producing an enzyme-treated composition may be contained. Means. For example, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1.5% by weight or less, 1.0% by weight or less or 0.5% by weight with respect to the total weight of the sweetness enhancer. It may contain steviol glycosides other than the above amino acids such as% or less and other impurities.
　いくつかの態様において、甘味増強剤は、酵素処理組成物以外の成分を含んでいてもよい。そのような成分として、例えば、甘味料が挙げられる。甘味料としては、例えば、果糖、砂糖、果糖ぶどう糖液糖、ぶどう糖、麦芽糖、高果糖液糖、糖アルコール、オリゴ糖、はちみつ、サトウキビ搾汁液（黒糖蜜）、水飴、羅漢果末、羅漢果抽出物、甘草末、甘草抽出物、ソーマトコッカスダニエリ種子末、ソーマトコッカスダニエリ種子抽出物などの天然甘味料や、アセスルファムカリウム、スクラロース、ネオテーム、アスパルテーム、サッカリンなどの人工甘味料などが挙げられる。その中でも、すっきりさ、飲みやすさ、自然な味わい、適度なコク味の付与の観点から、天然甘味料を用いることが好ましく、特に、果糖、ぶどう糖、麦芽糖、ショ糖、砂糖が好適に用いられる。これら甘味料は、１種のみを含んでもよく、複数を含んでもよい。 In some embodiments, the sweetness enhancer may contain components other than the enzyme-treated composition. Examples of such ingredients include sweeteners. Examples of sweeteners include fructose, sugar, high fructose corn syrup, high fructose corn syrup, maltose, high fructose corn syrup, sugar alcohol, oligosaccharide, honey, sugar cane juice (brown sugar honey), water candy, Rakan fruit powder, Rakan fruit extract, etc. Examples include natural sweeteners such as licorice powder, licorice extract, somatococcus daneri seed powder, somatococcus daneri seed extract, and artificial sweeteners such as acesulfam potassium, sucralose, neotame, aspartame, and saccharin. Among them, it is preferable to use a natural sweetener from the viewpoint of refreshing, easy to drink, natural taste, and imparting an appropriate richness, and in particular, fructose, glucose, maltose, sucrose, and sugar are preferably used. .. These sweeteners may contain only one kind or may contain a plurality of kinds.
６．微生物の発酵促進剤
　本発明は、微生物の発酵促進剤にも関する。また、本発明は、微生物の発酵促進用組成物であってもよい。本明細書において、「発酵促進剤」または「発酵促進用組成物」とは、微生物を培養する培地や微生物を含む飲食品（例えば、飲料）に添加された場合に、微生物の発酵や増殖を促進するものを意味する。本明細書において、発酵促進剤についての説明は、発酵促進用組成物についても当てはまる。 6. Microbial Fermentation Accelerator The present invention also relates to a microbial fermentation accelerator. Further, the present invention may be a composition for promoting fermentation of microorganisms. As used herein, the term "fermentation-promoting agent" or "fermentation-promoting composition" refers to the fermentation or proliferation of microorganisms when added to a medium for culturing microorganisms or a food or drink containing microorganisms (for example, a beverage). Means what promotes. As used herein, the description of the fermentation accelerator also applies to the fermentation promoting composition.
　本発明の一態様において、発酵促進剤は、上記「１．ステビア植物の酵素処理組成物」で述べた酵素処理組成物を含む。発酵促進剤における有効成分は、前記酵素処理組成物に含まれるステビア植物由来のアミノ酸であり得る。 In one aspect of the present invention, the fermentation accelerator includes the enzyme-treated composition described in "1. Enzyme-treated composition of Stevia plant" above. The active ingredient in the fermentation accelerator may be a Stevia plant-derived amino acid contained in the enzyme-treated composition.
　本発明の一態様において、発酵促進剤は、液状、ペースト状、粉末状または顆粒状等の形態であってもよい。 In one aspect of the present invention, the fermentation accelerator may be in the form of liquid, paste, powder, granules or the like.
　本発明の一態様において、発酵促進剤に含まれる酵素処理組成物の含有量は、発酵促進剤の全重量に対して、３０～１００重量％、４０～９９重量％、５０～９８重量％、６０～９７重量％、７０～９６重量％または８０～９５重量％等であってもよい。いくつかの態様において、発酵促進剤は、実質的にステビア植物由来のアミノ酸のみからなっていてもよい。また、別のいくつかの態様において、発酵促進剤は、糖、ビタミンおよびミネラル等の他の成分を任意に含んでいてもよい。 In one embodiment of the present invention, the content of the enzyme-treated composition contained in the fermentation accelerator is 30 to 100% by weight, 40 to 99% by weight, 50 to 98% by weight, based on the total weight of the fermentation accelerator. It may be 60 to 97% by weight, 70 to 96% by weight, 80 to 95% by weight, or the like. In some embodiments, the fermentation accelerator may consist solely of amino acids derived from the Stevia plant. Further, in some other embodiments, the fermentation accelerator may optionally contain other components such as sugars, vitamins and minerals.
　本発明の一態様における発酵促進剤を適用し得る微生物は、窒素を資化できるものであれば特に制限されず、例えば、細菌や酵母が挙げられる。 The microorganism to which the fermentation accelerator in one aspect of the present invention can be applied is not particularly limited as long as it can assimilate nitrogen, and examples thereof include bacteria and yeast.
［本発明の例示的態様］
　本発明の一態様において、
　ステビア植物の酵素処理組成物であって、
　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上であり、
　前記酵素処理組成物の総重量に対して、１００～１００００ｐｐｍのアミノ酸を含む、組成物が提供される。本態様において、アミノ酸は、１００～２０００ｐｐｍ、１５０～２０００ｐｐｍ、２００～２０００ｐｐｍ、２５０～２０００ｐｍ、３００～２０００ｐｐｍ、３５０～２０００ｐｐｍ、１００～１０００ｐｐｍ、１５０～１０００ｐｐｍ、２００～１０００ｐｐｍ、２５０～１０００ｐｍ、３００～１０００ｐｐｍまたは３５０～１０００ｐｐｍ等であってもよい。 [Exemplary aspect of the present invention]
In one aspect of the invention
An enzyme-treated composition of Stevia plant
The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
A composition containing 100 to 10000 ppm of amino acids with respect to the total weight of the enzyme-treated composition is provided. In this embodiment, the amino acids are 100-2000 ppm, 150-2000 ppm, 200-2000 ppm, 250-2000 pm, 300-2000 ppm, 350-2000 ppm, 100-1000 ppm, 150-1000 ppm, 200-1000 ppm, 250-1000 pm, 300-1000 ppm. Alternatively, it may be 350 to 1000 ppm or the like.
　本発明の一態様において、
　ステビア植物の酵素処理組成物であって、
　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上であり、
　前記酵素処理組成物の総重量に対して、５００ｐｐｍ以下のステビオール配糖体を含む、組成物が提供される。本態様において、ステビオール配糖体は、４００ｐｐｍ以下、３００ｐｐｍ以下、２００ｐｐｍ以下、１００ｐｐｍ以下、９０ｐｐｍ以下、８０ｐｐｍ以下、７０ｐｐｍ以下、６０ｐｐｍ以下または５０ｐｐｍ以下等であってもよい。 In one aspect of the invention
An enzyme-treated composition of Stevia plant
The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
A composition containing 500 ppm or less of steviol glycoside with respect to the total weight of the enzyme-treated composition is provided. In this embodiment, the steviol glycoside may be 400 ppm or less, 300 ppm or less, 200 ppm or less, 100 ppm or less, 90 ppm or less, 80 ppm or less, 70 ppm or less, 60 ppm or less, 50 ppm or less, and the like.
　本発明の一態様において、
　ステビア植物の酵素処理組成物であって、
　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上であり、
　前記酵素処理組成物の総重量に対して、１００～１００００ｐｐｍのアミノ酸を含み、
　前記アミノ酸は、アルギニン、リジン、ヒスチジン、フェニルアラニン、チロシン、ロイシン、イソロイシン、メチオニン、バリン、アラニン、グリシン、プロリン、グルタミン酸、セリン、スレオニン、アスパラギン酸、トリプトファンおよびシスチンからなる群から選択される１種以上である、組成物が提供される。本態様において、アミノ酸は、１００～２０００ｐｐｍ、１５０～２０００ｐｐｍ、２００～２０００ｐｐｍ、２５０～２０００ｐｍ、３００～２０００ｐｐｍ、３５０～２０００ｐｐｍ、１００～１０００ｐｐｍ、１５０～１０００ｐｐｍ、２００～１０００ｐｐｍ、２５０～１０００ｐｍ、３００～１０００ｐｐｍまたは３５０～１０００ｐｐｍ等であってもよい。 In one aspect of the invention
An enzyme-treated composition of Stevia plant
The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
It contains 100 to 10000 ppm of amino acids with respect to the total weight of the enzyme-treated composition.
The amino acid is one or more selected from the group consisting of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan and cystine. The composition is provided. In this embodiment, the amino acids are 100-2000 ppm, 150-2000 ppm, 200-2000 ppm, 250-2000 pm, 300-2000 ppm, 350-2000 ppm, 100-1000 ppm, 150-1000 ppm, 200-1000 ppm, 250-1000 pm, 300-1000 ppm. Alternatively, it may be 350 to 1000 ppm or the like.
　本発明の一態様において、
　ステビア植物の酵素処理組成物であって、
　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上であり、
　前記酵素処理組成物の総重量に対して、１００～１００００ｐｐｍのアミノ酸を含み、かつ５００ｐｐｍ以下のステビオール配糖体を含む、組成物が提供される。本態様において、アミノ酸は、１００～２０００ｐｐｍ、１５０～２０００ｐｐｍ、２００～２０００ｐｐｍ、２５０～２０００ｐｍ、３００～２０００ｐｐｍ、３５０～２０００ｐｐｍ、１００～１０００ｐｐｍ、１５０～１０００ｐｐｍ、２００～１０００ｐｐｍ、２５０～１０００ｐｍ、３００～１０００ｐｐｍまたは３５０～１０００ｐｐｍ等であってもよい。また、本態様において、ステビオール配糖体は、４００ｐｐｍ以下、３００ｐｐｍ以下、２００ｐｐｍ以下、１００ｐｐｍ以下、９０ｐｐｍ以下、８０ｐｐｍ以下、７０ｐｐｍ以下、６０ｐｐｍ以下または５０ｐｐｍ以下等であってもよい。 In one aspect of the invention
An enzyme-treated composition of Stevia plant
The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
A composition containing 100 to 10000 ppm of amino acids and 500 ppm or less of steviol glycoside with respect to the total weight of the enzyme-treated composition is provided. In this embodiment, the amino acids are 100-2000 ppm, 150-2000 ppm, 200-2000 ppm, 250-2000 pm, 300-2000 ppm, 350-2000 ppm, 100-1000 ppm, 150-1000 ppm, 200-1000 ppm, 250-1000 pm, 300-1000 ppm. Alternatively, it may be 350 to 1000 ppm or the like. Further, in this embodiment, the steviol glycoside may be 400 ppm or less, 300 ppm or less, 200 ppm or less, 100 ppm or less, 90 ppm or less, 80 ppm or less, 70 ppm or less, 60 ppm or less, 50 ppm or less, or the like.
　本発明の一態様において、
　ステビア植物の酵素処理組成物であって、
　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上であり、
　前記酵素処理組成物に含まれるアミノ酸含有量は、酵素処理組成物の固形分量に対して、０．１～５０重量％である、組成物が提供される。本態様において、アミノ酸含有量は、１．０～５０重量％、２．０～４０重量％、３．０～３０重量％、３．０～２０重量％、３．５～２０重量％、４．０～２０重量％、４．５～２０重量％、５．０～２０重量％、５．５～２０重量％または６．０～２０重量等であってもよい。 In one aspect of the invention
An enzyme-treated composition of Stevia plant
The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
A composition is provided in which the amino acid content contained in the enzyme-treated composition is 0.1 to 50% by weight based on the solid content of the enzyme-treated composition. In this embodiment, the amino acid content is 1.0 to 50% by weight, 2.0 to 40% by weight, 3.0 to 30% by weight, 3.0 to 20% by weight, 3.5 to 20% by weight, 4 It may be 0.0 to 20% by weight, 4.5 to 20% by weight, 5.0 to 20% by weight, 5.5 to 20% by weight, 6.0 to 20% by weight, or the like.
　本発明の一態様において、
　ステビア植物の酵素処理組成物であって、
　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上であり、
　前記酵素処理組成物に含まれるアミノ酸含有量は、乾燥重量基準で、酵素処理組成物の総重量に対して、０．１～６０重量％である、組成物が提供される。本態様において、アミノ酸含有量は、１．０～５０重量％、２．０～４０重量％、３．０～３０重量％、３．０～２０重量％、３．５～２０重量％、４．０～２０重量％、４．５～２０重量％、５．０～２０重量％、５．５～２０重量％または６．０～２０重量等であってもよい。 In one aspect of the invention
An enzyme-treated composition of Stevia plant
The weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more, and the weight ratio is 0.3 or more.
A composition is provided in which the amino acid content contained in the enzyme-treated composition is 0.1 to 60% by weight based on the total weight of the enzyme-treated composition on a dry weight basis. In this embodiment, the amino acid content is 1.0 to 50% by weight, 2.0 to 40% by weight, 3.0 to 30% by weight, 3.0 to 20% by weight, 3.5 to 20% by weight, 4 It may be 0.0 to 20% by weight, 4.5 to 20% by weight, 5.0 to 20% by weight, 5.5 to 20% by weight, 6.0 to 20% by weight, or the like.
　本発明の一態様において、
　ステビア植物の酵素処理組成物の製造方法であって、
　ステビア植物を溶媒を用いて抽出することと、
　ステビア植物の抽出後の残留物をスラリーにすることと、
　ヘミセルラーゼおよび／またはプロテアーゼを用いて前記スラリーを酵素処理することと、を含み、前記スラリーの濃度が５～７０％である、方法が提供される。本態様において、スラリー濃度は５～５０％または７～３０％等であってもよい。 In one aspect of the invention
A method for producing an enzyme-treated composition of Stevia plant.
Extracting Stevia plants with a solvent and
Making the residue after extraction of Stevia plants into a slurry and
Provided are methods comprising enzymatically treating the slurry with hemicellulase and / or protease, wherein the concentration of the slurry is 5 to 70%. In this embodiment, the slurry concentration may be 5 to 50%, 7 to 30%, or the like.
　本発明の一態様において、
　ステビア植物の酵素処理組成物の製造方法であって、
　ステビア植物を溶媒を用いて抽出することと、
　ヘミセルラーゼおよび／またはプロテアーゼを用いて前記抽出後の残留物を酵素処理することと、を含み、前記酵素処理中の反応液のＢｒｉｘが０．２５～３０である、方法が提供される。本態様において、酵素処理中の反応液のＢｒｉｘは、０．２５～１０、０．２５～８．０、０．２５～６．０、０．２５～５．０、０．５～１０、０．５～８．０、０．５～６．０または０．５～５．０等であってもよい。 In one aspect of the invention
A method for producing an enzyme-treated composition of Stevia plant.
Extracting Stevia plants with a solvent and
Provided are methods comprising enzymatically treating the residue after extraction with hemicellulase and / or protease, wherein the reaction solution during the enzymatic treatment has a Brix of 0.25 to 30. In this embodiment, the Brix of the reaction solution during the enzyme treatment is 0.25 to 10, 0.25 to 8.0, 0.25 to 6.0, 0.25 to 5.0, 0.5 to 10, It may be 0.5 to 8.0, 0.5 to 6.0, 0.5 to 5.0, or the like.
　以下、本発明を実施例により詳細に説明するが、本発明の内容がこれにより限定されるものではない。 Hereinafter, the present invention will be described in detail by way of examples, but the content of the present invention is not limited thereto.
［製造例］
１．ステビア植物の抽出
　ステビア乾燥葉（含水率：１０重量％以下）を６０℃±５℃に加熱したイオン交換水に浸し、２０分静置した。その後、カラム抽出機（カラム容量：１Ｌ、ＧＥヘルスケア製）にて下部から加熱したイオン交換水を通液し、乾燥葉の３０倍量の抽出液量を採取した（１ｓｔ）。次に９０℃±５℃のイオン交換水を下部から通液し、ステビア乾燥葉の４５倍量の抽出液を採取した（２ｎｄ）。抽出後、カラム中のイオン交換水及びステビア葉を酵素処理用の容器に移し、次工程にて使用した。なお、原料として使用したステビア乾燥葉１００ｇ当たりに含まれるアミノ酸量を酸分解法により事前に分析した結果を表１に示す。アミノ酸量は、アルギニン、リジン、ヒスチジン、フェニルアラニン、チロシン、ロイシン、イソロイシン、バリン、アラニン、グリシン、プロリン、グルタミン酸、セリン、スレオニンおよびアスパラギン酸の１５種については、サンプルに塩酸を添加し、脱気および封管して加水分解し、定容後に適宜希釈または濃縮し、ｐＨを２．２に調整したものを試験溶液とし、アミノ酸自動分析計を用いて分析した。シスチンおよびメチオニンについては、サンプルに過ギ酸溶液を添加し、酸化処理したものを減圧下で濃縮乾固し、塩酸を添加して加水分解し、定容後に適宜希釈または濃縮し、ｐＨを２．２に調整したものを試験溶液とし、アミノ酸自動分析計を用いて分析した。トリプトファンについては、サンプルに水酸化バリウム八水和物、チオジエチレングリコールおよび水を添加して加熱・溶解し、塩酸を加え脱気および封管して加水分解した後に微アルカリに調整し、定容して適宜希釈したものを試験溶液とし、高速液体クロマトグラフィーを用いて分析した。 [Manufacturing example]
1. 1. Extraction of Stevia plants Stevia dried leaves (moisture content: 10% by weight or less) were immersed in ion-exchanged water heated to 60 ° C. ± 5 ° C. and allowed to stand for 20 minutes. Then, ion-exchanged water heated from the bottom was passed through a column extractor (column capacity: 1 L, manufactured by GE Healthcare), and an amount of extract 30 times as much as that of dried leaves was collected (1st). Next, ion-exchanged water at 90 ° C. ± 5 ° C. was passed from the bottom, and 45 times the amount of the extract of dried Stevia leaves was collected (2nd). After extraction, the ion-exchanged water and stevia leaves in the column were transferred to a container for enzyme treatment and used in the next step. Table 1 shows the results of prior analysis of the amount of amino acids contained in 100 g of dried Stevia leaves used as a raw material by an acid decomposition method. For 15 amino acid amounts of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, valine, alanine, glycine, proline, glutamic acid, serine, threonine and aspartic acid, hydrochloric acid was added to the sample for degassing and degassing. The test solution was sealed, hydrolyzed, diluted or concentrated as appropriate after volume determination, and the pH was adjusted to 2.2, and analyzed using an automatic amino acid analyzer. For cystine and methionine, a performic acid solution is added to the sample, and the oxidized product is concentrated to dryness under reduced pressure, hydrolyzed by adding hydrochloric acid, diluted or concentrated as appropriate after volume determination, and the pH is adjusted to 2. The solution adjusted to 2 was used as a test solution and analyzed using an automatic amino acid analyzer. For tryptophan, add barium hydroxide octahydrate, thiodiethylene glycol and water to the sample, heat and dissolve it, add hydrochloric acid, degas and seal it, hydrolyze it, and then adjust it to a slight alkali and set the volume. The test solution was diluted appropriately and analyzed using high-speed liquid chromatography.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
２．抽出残留物の酵素処理
　上記「１．ステビア植物の抽出」で得られた残留物（スラリー）に、ヘミセルラーゼ（天野エンザイム製、ヘミセルラーゼ「アマノ」９０）およびプロテアーゼ（天野エンザイム製、プロテアーゼＡアマノ）を添加し、ｐＨ５．８４、４２℃の条件で１６時間、撹拌しながら酵素処理した。この際、スラリー濃度は約１１．２％とした。酵素の添加量は、ステビア植物の乾燥重量に対してそれぞれ４重量％となるように添加した。また、酵素処理中のＢｒｉｘは、図１に示すように推移した。その後、反応液を温浴で加熱して酵素を失活させた。さらに、遠心分離により固液分離を行い、ステビア植物の酵素処理組成物をエキスとして得た。 2. 2. Enzyme treatment of extraction residue Hemicellulase (manufactured by Amano Enzyme, hemicellulase "Amano" 90) and protease (manufactured by Amano Enzyme, Protease A Amano) were added to the residue (slurry) obtained in the above "1. Extraction of Stevia plant". ) Was added, and the enzyme was treated with stirring under the conditions of pH 5.84 and 42 ° C. for 16 hours. At this time, the slurry concentration was set to about 11.2%. The amount of the enzyme added was 4% by weight based on the dry weight of the Stevia plant. In addition, Brix during the enzyme treatment changed as shown in FIG. Then, the reaction solution was heated in a warm bath to inactivate the enzyme. Further, solid-liquid separation was carried out by centrifugation to obtain an enzyme-treated composition of Stevia plant as an extract.
［実施例Ａ］ステビア植物の酵素処理組成物におけるアミノ酸量およびステビオール配糖体量の確認
　上記製造例に記載の方法で得たステビア植物の酵素処理組成物について、アミノ酸量およびステビオール配糖体量を分析した。結果を図２および図３にそれぞれ示す。なお、アミノ酸量は、アルギニン、リジン、ヒスチジン、フェニルアラニン、チロシン、ロイシン、イソロイシン、メチオニン、シスチン、バリン、アラニン、グリシン、プロリン、グルタミン酸、セリン、スレオニンおよびアスパラギン酸の１７種については、サンプルにスルホサリチル酸溶液を添加して抽出を行い、次いで、ｐＨを２．２に調整し、定容およびろ過を行い、適宜希釈して試験溶液を調整し、アミノ酸自動分析計を用いて分析した。トリプトファンについては、上記１７種と同様にして試験溶液を調整し、微アルカリに調整した後に定容して高速液体クロマトグラフィーを用いて分析した。ステビオール配糖体量は、液体クロマトグラフ質量分析計を用いて分析した（カラム：Shim-pack XR-ODS II (2.0 mm (i.d.) x 150 mm (L)（島津製作所製）、移動相：３０％アセトニトリル／７０％ミリＱ水（０．１％ギ酸を含む）、流量：０．３４ｍｌ／ｍｉｎ)。また、図２および図３において、「１ｓｔ」および「２ｎｄ」のサンプルは、上記製造例における「１．ステビア植物の抽出」で得た１回目の抽出液および２回目の抽出液を分析した際の結果である（以下同様）。また、「３ｒｄ（前）」および「３ｒｄ（後）」は、それぞれ酵素処理前および酵素処理後のサンプルである（以下同様）。 [Example A] Confirmation of amino acid amount and steviol glycoside amount in the enzyme-treated composition of Stevia plant The amino acid amount and steviol glycoside amount of the enzyme-treated composition of Stevia plant obtained by the method described in the above production example. Was analyzed. The results are shown in FIGS. 2 and 3, respectively. The amount of amino acids is arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, cystine, valine, alanine, glycine, proline, glutamic acid, serine, threonine and aspartic acid. The solution was added for extraction, then the pH was adjusted to 2.2, volume and filtration were performed, diluted appropriately to prepare the test solution, and analyzed using an automatic amino acid analyzer. For tryptophan, the test solution was prepared in the same manner as in the above 17 types, adjusted to a slight alkali, and then the volume was adjusted and analyzed using high performance liquid chromatography. The amount of steviol glycoside was analyzed using a liquid chromatograph mass spectrometer (column: Shim-pack XR-ODS II (2.0 mm (id) x 150 mm (L) (manufactured by Shimadzu Corporation), mobile phase: 30). % Acetonitrile / 70% milliQ water (including 0.1% formic acid), flow rate: 0.34 ml / min). Further, in FIGS. 2 and 3, the "1st" and "2nd" samples are the above-mentioned production examples. It is the result when the first extract and the second extract obtained in "1. Steviol plant extraction" were analyzed (the same applies hereinafter), and "3rd (before)" and "3rd (after)". "Is a sample before and after the enzyme treatment, respectively (the same applies hereinafter).
　図２に示されるように、ヘミセルラーゼおよびプロテアーゼによる酵素処理により、得られた組成物のアミノ酸濃度が酵素添加前と比較して著しく増加したことが確認された。なお、図２に示すアミノ酸量における各アミノ酸の組成は、下記表２のとおりであった。 As shown in FIG. 2, it was confirmed that the amino acid concentration of the obtained composition was significantly increased by the enzyme treatment with hemicellulase and protease as compared with that before the enzyme addition. The composition of each amino acid in the amount of amino acid shown in FIG. 2 is as shown in Table 2 below.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　また、図３に示されるように、得られた組成物には、ステビオール配糖体が少量含まれていた（１ｓｔ：２２３１．４２ｐｐｍ、２ｎｄ：７１９．７８ｐｐｍ、３ｒｄ（前）：２８．５１ｐｐｍ、３ｒｄ（後）：５５．５３ｐｐｍ）。ここで、図３に示すステビオール配糖体量は、レバウジオシドＡ、レバウジオシドＢ、レバウジオシドＣ、レバウジオシドＤ、レバウジオシドＥ、レバウジオシドＦ、レバウジオシドＧ、レバウジオシドＩ、レバウジオシドＭ、レバウジオシドＮ、ステビオシド、ステビオールビオシド、ズルコシドＡおよびルブソシドからなる総ステビオール配糖体の含有量である。１回目および２回目の抽出液、並びに酵素処理後のサンプルにおける、総ステビオール配糖体に占める各ステビオール配糖体の割合は、下記表３のとおりであった。 Further, as shown in FIG. 3, the obtained composition contained a small amount of steviol glycoside (1st: 2231.42 ppm, 2nd: 719.78 ppm, 3rd (previous): 28.51 ppm, 3rd (after): 55.53 ppm). Here, the amounts of steviol glycosides shown in FIG. 3 are rebaugioside A, rebaugioside B, rebaugioside C, rebaugioside D, rebaugioside E, rebaugioside F, rebaugioside G, rebaugioside I, rebaugioside M, rebaugioside N, stevioside, steviolbioside. The content of the total steviol glycoside consisting of zulcoside A and rubusoside. The ratio of each steviol glycoside to the total steviol glycosides in the first and second extracts and the sample after the enzyme treatment is shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
［実施例Ｂ］種々のプロテアーゼを使用した際のアミノ酸量の確認
　酵素反応の条件を下記表４に示す条件とした以外は上記製造例と同様の方法で、種々の酵素を用いて酵素処理されたステビア植物の酵素処理組成物を得た。各組成物について、実施例Ａに準じる条件でアミノ酸量を分析した。結果を図４に示す。なお、図４に示す各アミノ酸の組成は、下記表５のとおりであった。 [Example B] Confirmation of amino acid amount when various proteases are used Enzymatic treatment is performed using various enzymes by the same method as in the above production example except that the conditions of the enzymatic reaction are set to the conditions shown in Table 4 below. An enzyme-treated composition of Stevia plant was obtained. For each composition, the amount of amino acids was analyzed under the conditions according to Example A. The results are shown in FIG. The composition of each amino acid shown in FIG. 4 is as shown in Table 5 below.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　図４に示されるように、使用する酵素を変更した場合でも、実施例Ｂで得られた各組成物は、上記製造例で得られた組成物とおおむね同様のアミノ酸組成を有することが確認された。 As shown in FIG. 4, it was confirmed that even when the enzyme used was changed, each composition obtained in Example B had an amino acid composition substantially similar to that of the composition obtained in the above-mentioned production example. rice field.
［実施例Ｃ］レバウジオシドＡの甘味増強効果の評価
　下記表６の濃度となるようにレバウジオシドＡ（守田化学工業製、Ｊ－１００（純度９５％以上））と上記製造例で得られたステビア植物の酵素処理組成物を純水に添加して水溶液を調製した。酵素処理組成物については、表６のアミノ酸濃度となるように添加した。なお、サンプル１は酵素処理組成物を含まない対照サンプルである。各サンプルを、官能に関して訓練を受けた者（３名）がパネラーとなって下記の基準で評価した。結果を表７に示す。 [Example C] Evaluation of sweetness enhancing effect of rebaugioside A Rebaugioside A (manufactured by Morita Chemical Industry Co., Ltd., J-100 (purity 95% or more)) and the stevia plant obtained in the above production example so as to have the concentration shown in Table 6 below. The enzyme-treated composition of No. 1 was added to pure water to prepare an aqueous solution. The enzyme-treated composition was added so as to have the amino acid concentration shown in Table 6. Sample 1 is a control sample that does not contain the enzyme-treated composition. Each sample was evaluated according to the following criteria by panelists who were trained in sensuality (3 persons). The results are shown in Table 7.
Figure JPOXMLDOC01-appb-T000006
 評価基準
　○：対照サンプルと比較して、甘味を強く感じる
　△：対照サンプルと比較して、甘味をやや強く感じる
　×：対照サンプルと比較して、甘味が弱い
Figure JPOXMLDOC01-appb-T000006
 Evaluation Criteria ○: Sweetness is stronger than the control sample Δ: Sweetness is slightly stronger than the control sample ×: Sweetness is weaker than the control sample
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
［実施例Ｄ］スクロースの甘味増強効果の評価
　下記表８の濃度となるようにスクロース（三井製糖製）と上記製造例で得られたステビア植物の酵素処理組成物を純水に添加して水溶液を調製した。酵素処理組成物については、表８のアミノ酸濃度となるように添加した。なお、サンプル１は酵素処理組成物を含まない対照サンプルである。各サンプルを、官能に関して訓練を受けた者（３名）がパネラーとなって下記の基準で評価した。結果を表９に示す。 [Example D] Evaluation of sweetness enhancing effect of sucrose Add sucrose (manufactured by Mitsui Sugar Co., Ltd.) and the enzyme-treated composition of Stevia plant obtained in the above production example to pure water so as to have the concentration shown in Table 8 below, and add an aqueous solution. Was prepared. The enzyme-treated composition was added so as to have the amino acid concentration shown in Table 8. Sample 1 is a control sample that does not contain the enzyme-treated composition. Each sample was evaluated according to the following criteria by panelists who were trained in sensuality (3 persons). The results are shown in Table 9.
Figure JPOXMLDOC01-appb-T000008
 評価基準
　○：対照サンプルと比較して、甘味を強く感じる
　△：対照サンプルと比較して、甘味をやや強く感じる
　×：対照サンプルと比較して、甘味が弱い
Figure JPOXMLDOC01-appb-T000008
 Evaluation Criteria ○: Sweetness is stronger than the control sample Δ: Sweetness is slightly stronger than the control sample ×: Sweetness is weaker than the control sample
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009

Claims (18)

  1. 　ステビア植物の酵素処理組成物であって、
    　前記酵素処理組成物におけるアミノ酸／ステビオール配糖体の重量比が０．３以上である、組成物。     An enzyme-treated composition of Stevia plant
    A composition in which the weight ratio of amino acid / steviol glycoside in the enzyme-treated composition is 0.3 or more.
  2. 　前記酵素処理組成物の総重量に対して、１００～１００００ｐｐｍのアミノ酸を含む、請求項１に記載の組成物。 The composition according to claim 1, which contains 100 to 10000 ppm of amino acids with respect to the total weight of the enzyme-treated composition.
  3. 　前記アミノ酸は、アルギニン、リジン、ヒスチジン、フェニルアラニン、チロシン、ロイシン、イソロイシン、メチオニン、バリン、アラニン、グリシン、プロリン、グルタミン酸、セリン、スレオニン、アスパラギン酸、トリプトファンおよびシスチンからなる群から選択される１種以上である、請求項１または２に記載の組成物。 The amino acid is one or more selected from the group consisting of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan and cystine. The composition according to claim 1 or 2.
  4. 　前記アミノ酸が、ステビア植物由来のアミノ酸である請求項１～３のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 3, wherein the amino acid is an amino acid derived from a Stevia plant.
  5. 　前記ステビオール配糖体は、レバウジオシドＡ、レバウジオシドＢ、レバウジオシドＣ、レバウジオシドＤ、レバウジオシドＥ、レバウジオシドＦ、レバウジオシドＧ、レバウジオシドＩ、レバウジオシドＪ、レバウジオシドＫ、レバウジオシドＭ、レバウジオシドＮ、レバウジオシドＯ、レバウジオシドＱ、レバウジオシドＲ、ズルコシドＡ、ズルコシドＣ、ルブソシド、ステビオールモノシド、ステビオールビオシドおよびステビオシドからなる群から選択される１種以上である、請求項１～４のいずれか一項に記載の組成物。 The steviol glycosides are rebaugioside A, rebaugioside B, rebaugioside C, rebaugioside D, rebaugioside E, rebaugioside F, rebaugioside G, rebaugioside I, rebaugioside J, rebaugioside K, rebaugioside M, rebaugioside N, rebaugioside N, rebaugioside. The composition according to any one of claims 1 to 4, which is one or more selected from the group consisting of R, zulcoside A, zulcoside C, rubusoside, steviol monoside, steviol bioside and stevioside.
  6. 　前記酵素処理組成物は、ヘミセルラーゼおよび／またはプロテアーゼにより酵素処理されたものである、請求項１～５のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 5, wherein the enzyme-treated composition is enzymatically treated with hemicellulase and / or protease.
  7. 　前記ステビア植物は、ステビア植物の葉、茎および／または組織を含む、請求項１～６のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 6, wherein the stevia plant contains leaves, stems and / or tissues of the stevia plant.
  8. 　請求項１～７のいずれか一項に記載の組成物を含む、飲食品。 A food or drink containing the composition according to any one of claims 1 to 7.
  9. 　ステビア植物の酵素処理組成物の製造方法であって、
    　ステビア植物を溶媒を用いて抽出することと、
    　ヘミセルラーゼおよび／またはプロテアーゼを用いて前記抽出後の残留物を酵素処理することと、を含む、方法。     A method for producing an enzyme-treated composition of Stevia plant.
    Extracting Stevia plants with a solvent and
    A method comprising enzymatically treating the residue after extraction with hemicellulase and / or protease.
  10. 　前記残留物は、少なくとも１回以上の抽出後の残留物である、請求項９に記載の方法。 The method according to claim 9, wherein the residue is a residue after extraction at least once.
  11. 　前記酵素処理後に固液分離することをさらに含む、請求項９または１０に記載の方法。 The method according to claim 9 or 10, further comprising solid-liquid separation after the enzyme treatment.
  12. 　前記ヘミセルラーゼおよび／またはプロテアーゼの添加量が、ステビア植物の乾燥重量に対して０．５～１５重量％である、請求項９～１１のいずれか一項に記載の方法。 The method according to any one of claims 9 to 11, wherein the amount of the hemicellulase and / or protease added is 0.5 to 15% by weight based on the dry weight of the Stevia plant.
  13. 　酵素処理時間が１～４８時間である、請求項９～１２のいずれか一項に記載の方法。 The method according to any one of claims 9 to 12, wherein the enzyme treatment time is 1 to 48 hours.
  14. 　酵素処理時のｐＨが２～１０である、請求項９～１３のいずれか一項に記載の方法。 The method according to any one of claims 9 to 13, wherein the pH at the time of enzyme treatment is 2 to 10.
  15. 　酵素処理時の温度が１０～８０℃である、請求項９～１４のいずれか一項に記載の方法。 The method according to any one of claims 9 to 14, wherein the temperature at the time of enzyme treatment is 10 to 80 ° C.
  16. 　請求項９～１５のいずれか一項に記載の方法により得られる、組成物。 A composition obtained by the method according to any one of claims 9 to 15.
  17. 　請求項１６に記載の組成物を含む、飲食品。 A food or drink containing the composition according to claim 16.
  18. 　請求項１～７および１６のいずれか一項に記載の組成物を飲食品に配合することを含む、飲食品の甘味増強方法。  A method for enhancing the sweetness of foods and drinks, which comprises blending the composition according to any one of claims 1 to 7 and 16 into foods and drinks. The
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