CN116634887A

CN116634887A - Enzyme treatment composition for stevia plants

Info

Publication number: CN116634887A
Application number: CN202180086857.8A
Authority: CN
Inventors: 浦井聡一郎; 长尾浩二
Original assignee: Suntory Holdings Ltd
Current assignee: Suntory Holdings Ltd
Priority date: 2020-12-24
Filing date: 2021-12-22
Publication date: 2023-08-22
Also published as: WO2022138771A1; JP2022100883A

Abstract

The present invention pursues a technique for further effective use of stevia plants. The present invention provides a composition which is an enzyme-treated composition of stevia plants, wherein the weight ratio of amino acid/steviol glycoside in the enzyme-treated composition is 0.3 or more.

Description

Enzyme treatment composition for stevia plants

Technical Field

The present invention relates to an enzyme-treated composition of stevia plant and a method for producing the same. The present invention also relates to a food or beverage containing the composition produced by the above-mentioned production method and a method for enhancing sweetness of the food or beverage.

Background

The inclusion of a secondary metabolite called Steviol (Steviol), which is one of diterpenes, in the leaves of stevia (Stevia rebaudiana) belonging to the family Compositae is utilized in the food industry as a low-calorie sweetener because Steviol glycosides exhibit about 300 times the sweetness of granulated sugar. Obesity is becoming a serious internationally serious social problem, and there is an increasing demand for low-calorie sweeteners from the standpoint of health improvement and medical cost reduction. Currently, the artificially synthesized amino acid derivatives Aspartame (Aspartame) and acesulfame potassium (Acesulfame Potassium) are used as artificial sweeteners, but naturally occurring low-calorie sweeteners such as steviol glycosides are safer, and are expected to be easily understood by consumers (Public Acceptance).

In recent years, efforts for effectively utilizing the extracted residues of stevia plants have been reported for the purpose of suppressing the waste amount of the residues generated after the extraction treatment of stevia plants (for example, non-patent documents 1 and 2). In addition, a method using enzymes at the time of extraction of stevia plants is also known (patent document 1).

Patent literature

Patent document 1: U.S. patent No. 10463065

Non-patent literature

Non-patent document 1: journal of Functional Foods, vol.72, september 2020,103983

Non-patent document 2: ecotoxicology and EnvironmentalSafety, vol.172, may 2019,403-410

Disclosure of Invention

Under such circumstances, a technique for further effective use of stevia plants has been sought.

The present invention is as follows.

[1] A composition which is an enzyme-treated composition of stevia plant, characterized in that,

the weight ratio of amino acid/steviol glycoside in the enzyme treatment composition is more than 0.3.

[2] The composition according to [1], wherein the amino acid content is 100 to 10000ppm based on the total weight of the enzyme treatment composition.

[3] The composition according to [1] or [2], wherein the amino acid is at least 1 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.

[4] The composition according to any one of [1] to [3], wherein the amino acid is an amino acid derived from a stevia plant.

[5] The composition according to any one of [1] to [4], wherein the steviol glycoside is 1 or more selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside M, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudioside R, du Kegan A, dulcoside C, rubusoside, steviolmonoside, steviolbioside and stevioside.

[6] The composition according to any one of [1] to [5], wherein the enzyme-treated composition is a composition which is enzymatically treated with a hemicellulase and/or a 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 beverage comprising the composition of any one of [1] to [7 ].

[9] A method for producing an enzyme-treated composition of stevia plant, characterized by comprising the steps of,

Comprises the step of extracting stevia plant with solvent,

and (c) a step of subjecting the extracted residue to an enzyme treatment using a hemicellulase and/or a protease.

[10] The method according to [9], wherein the residue is a residue after at least 1 or more extractions.

[11] The method according to [9] or [10], further comprising a step of performing solid-liquid separation after the enzyme treatment.

[12] The method according to any one of [9] to [11], wherein the amount of the hemicellulase and/or the protease added is 0.5 to 15% by weight relative to 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 the enzyme treatment is 2 to 10.

[15] The method according to any one of [9] to [14], wherein the temperature at the time of the enzyme treatment is 10 to 80 ℃.

[16] A composition obtained by the method of any one of [9] to [15 ].

[17] A food or beverage comprising the composition of [16 ].

[18] A method for enhancing the sweetness of a food or beverage, comprising the step of blending the composition of any one of [1] to [7] and [16] into a food or beverage.

According to the present invention, there can be provided an enzyme-treated composition of stevia plant containing steviol glycoside and amino acid or a method for producing the same. Further, the present invention provides a food or beverage containing the composition and a method for enhancing sweetness of the food or beverage.

Drawings

FIG. 1 is a diagram showing the transition of Brix in enzyme treatment.

FIG. 2 is a graph showing the amounts of amino acids contained in an enzyme-treated composition of stevia plants.

FIG. 3 is a graph showing the amount of steviol glycoside contained in an enzyme-treated composition of stevia plant.

FIG. 4 is a diagram showing the amino acid composition of an enzyme treatment composition when enzyme treatment is performed using various enzymes.

Detailed Description

The present invention will be described in detail below. The following embodiments are illustrative examples for explaining the present invention, and the present invention is not limited to the embodiments. The present invention can be implemented in various ways without departing from the gist thereof. All documents, publications, patent publications, and other patent documents cited in the present specification are incorporated herein by reference.

In the present specification, "Reb" and "Reb" mean the same meaning, and both "rebaudioside (rebaudiosides)".

1. Enzyme treatment composition for stevia plants

The present invention relates to an enzyme treatment composition (hereinafter, also simply referred to as "enzyme treatment composition" or "composition") for stevia plants. Specifically, the present invention relates to a composition which is an enzyme-treated composition of stevia plants, wherein the weight ratio of amino acid/steviol glycoside in the enzyme-treated composition is 3 or more.

In the present specification, "stevia plant" includes the whole plant and a part of the plant of the stevia plant. "part of a plant" includes leaves, stems, flowers, roots, and any substances that cut off the leaves, stems, flowers, roots, and any cells and tissues of a stevia plant.

In the present specification, the term "enzyme-treated composition of stevia plant" refers to a composition obtained by subjecting a residue (residue) obtained by extracting the stevia plant with a solvent to an enzyme treatment. The enzyme treatment will be described later.

Stevia contains various components in addition to steviol glycoside. As such components, there are water-soluble components and insoluble components. Examples of the water-soluble component include polysaccharides such as water-soluble dietary fibers, secondary metabolites such as alkaloids and flavonoids and terpenoids, methanol, polyphenols, minerals, vitamins, amino acids, organic acids, water-soluble proteins, and various other glycosides. Examples of the insoluble component include insoluble polysaccharides, insoluble proteins, and lipids containing insoluble dietary fibers and the like.

According to one mode of the invention, the enzyme treatment composition of stevia plants is rich in amino acids. In addition, in several ways, the enzyme treatment composition of the stevia plant may contain a small amount of steviol glycosides. In several ways, the enzyme treatment composition of the stevia plant is preferably substantially free of steviol glycosides, however, it is inevitable that the enzyme treatment composition contains a small amount of steviol glycosides from the stevia plant that cannot be completely removed.

The amino acid preferably contains 1 or more of 20 amino acids forming a protein. Specifically, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (threonine) (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val) may be mentioned.

The amino acid may be 1 or more selected from arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan, and cystine.

In the present specification, the term "amino acid content" or "amino acid concentration" refers to the total amount and total concentration of the 18 amino acids unless otherwise specified. The amino acid can be quantified by a known method using an amino acid automatic analyzer or HPLC according to the kind thereof.

Here, the amino acid is preferably an amino acid derived from stevia plant. In the present specification, the term "derived from stevia plant" means an amino acid obtained from the stevia plant. The amino acid contained in the enzyme treatment composition may be a substance derived from stevia plant, as long as no other component containing the amino acid is added.

In one embodiment of the present invention, the amino acid may be in the form of D, L, or a racemate comprising D and L. The amino acid preferably contains a larger amount of L form than D form. Specifically, the content of the L-form amino acid in the enzyme-treated composition may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or 95% or more, or the like.

In one embodiment of the present invention, as steviol glycoside, there may be mentioned rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside M, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudioside R, du Kegan a, dulcoside C, rubusoside, steviol, steviolmonoside, steviolbioside, etc.

In addition, in several modes, the enzyme treatment composition may be a substance containing Total Steviol Glycosides (TSG) consisting of rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside I, rebaudioside M, rebaudioside N, stevioside, steviolbioside dulcoside a, and rubusoside.

In the present specification, the term "steviol glycoside content" or "steviol glycoside concentration" refers to the total amount and total concentration of the total steviol glycosides listed above unless otherwise specified. The steviol glycoside can be quantified by a known method using a liquid chromatography mass spectrometer.

In one embodiment of the present invention, the weight ratio of amino acid/steviol glycoside in the enzyme-treated composition may be, 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 20 or more. The higher the ratio of amino acids, the higher the purity as a material is and thus preferable.

In one mode of the invention, the amino acid is present in an amount relative to the total weight of the enzyme treatment composition, for example, it may be 100 to 10000ppm, 150 to 10000ppm, 200 to 10000ppm, 250 to 10000pm, 300 to 10000ppm, 350 to 10000ppm, 100 to 9000ppm, 150 to 9000ppm, 200 to 9000ppm, 250 to 9000pm, 300 to 9000ppm, 350 to 9000ppm, 100 to 8000ppm, 150 to 8000ppm, 200 to 8000ppm, 250 to 8000pm, 300 to 8000ppm, 350 to 8000ppm, 100 to 7000ppm, 150 to 7000ppm, 200 to 7000ppm, 250 to 7000pm, 300 to 7000ppm, 350 to 7000ppm, 100 to 6000ppm, 150 to 6000ppm, 200 to 6000ppm, 250 to 6000pm, 300 to 6000ppm, 350 to 6000ppm 100 to 5000ppm, 150 to 5000ppm, 200 to 5000ppm, 250 to 5000ppm, 300 to 5000ppm, 350 to 5000ppm, 100 to 4000ppm, 150 to 4000ppm, 200 to 4000ppm, 250 to 4000pm, 300 to 4000ppm, 350 to 4000ppm, 100 to 3000ppm, 150 to 3000ppm, 200 to 3000ppm, 250 to 3000pm, 300 to 3000ppm, 350 to 3000ppm, 100 to 2000ppm, 150 to 2000ppm, 200 to 2000ppm, 250 to 2000pm, 300 to 2000ppm, 350 to 2000ppm, 100 to 1000ppm, 150 to 1000ppm, 200 to 1000ppm, 250 to 1000pm, 300 to 1000ppm, or 350 to 1000ppm, etc.

In one embodiment of the present invention, steviol glycoside may be 3000ppm or less, 2500ppm or less, 2300ppm or less, 2000ppm or less, 1500ppm or less, 1000ppm or less, 750ppm or less, 720ppm or less, 700ppm or less, 500ppm or less, 450ppm or less, 400ppm or less, 350ppm or less, 300ppm or less, 250ppm or less, 200ppm or less, 150ppm or less, 100ppm or less, 90ppm or less, 85ppm or less, 80ppm or less, 75ppm or less, 70ppm or less, 65ppm or less, 60ppm or less, 55ppm or less, 50ppm or less, 45ppm or less, 40ppm or less, 35ppm or less, 30ppm or less, 25ppm or less, 20ppm or less, 15ppm or 10ppm or 5ppm or 1ppm or less, etc. based on the total weight of the enzyme treatment composition. In addition, the lower limit of steviol glycoside concentration is 0.

In one embodiment of the present invention, the amino acid content of the enzyme treatment composition for stevia rebaudiana plant may be, for example, 0.1 to 50 wt%, 0.5 to 50 wt%, 1.0 to 50 wt%, 1.5 to 50 wt%, 1.0 to 50 wt%, 2.5 to 50 wt%, 3.0 to 50 wt%, 3.5 to 50 wt%, 4.0 to 50 wt%, 4.5 to 50 wt%, 5.0 to 50 wt%, 5.5 to 50 wt%, 6.0 to 50 wt%, 0.1 to 40 wt%, 0.5 to 40 wt%, 1.0 to 40 wt%, 1.5 to 40 wt%, 2.0 to 40 wt%, 3.0 to 40 wt%, 3.5 to 40 wt%, 4.0 to 40 wt%, 4.5 to 40 wt%, 5.5 to 40 wt%, 6.0 to 40 wt%, 4.0 to 40 wt%, 4.1 to 20 wt%, 0 to 20 wt%, 0.1 to 20 wt%, 0 to 20 wt%, 0.5 to 20 wt%, 0 to 30 wt%, 0.0 to 20 wt%, 0 to 20 wt%, 0.3 to 20 wt%, 0.5 to 20 wt%, 0 wt% to 30 wt%, 0.3 to 20 wt%, 0 wt% to 20 wt% to 30 wt% based on the solid content of the enzyme treatment composition. When the enzyme-treated composition is liquid, brix (solid content) can be obtained by using a Brix meter (for example, brix meter RX-5000. Alpha. Manufactured by ATAGO), and the amount of amino acid relative to the solid content of the composition can be calculated by using the amount of amino acid measured by HPLC or the like.

In one mode of the present invention, the amino acid content contained in the enzyme-treating composition of stevia plant is determined based on the dry weight, relative to the total weight of the enzyme-treating composition, for example, it may be 0.1 to 60 wt%, 0.5 to 60 wt%, 1.0 to 60 wt%, 1.5 to 60 wt%, 2.0 to 60 wt%, 2.5 to 60 wt%, 3.0 to 60 wt%, 3.5 to 60 wt%, 4.0 to 60 wt%, 4.5 to 60 wt%, 5.0 to 60 wt%, 5.5 to 60 wt%, 6.0 to 60 wt%, 0.1 to 50 wt%, 0.5 to 50 wt%, 1.0 to 50 wt%, 1.5 to 50 wt%, 2.0 to 50 wt%, 2.5 to 50 wt%, 3.0 to 50 wt%, 3.5 to 50 wt%, 4.0 to 50 wt%, 4.5 to 50 wt%, 5.0 to 50 wt%, 6.0 to 50 wt%, 0.1 to 40 wt%, 1.0 to 40 wt%, 1.5 to 40 wt%, 2.0 to 40 wt%, etc 2.5 to 40 wt%, 3.0 to 40 wt%, 3.5 to 40 wt%, 4.0 to 40 wt%, 4.5 to 40 wt%, 5.0 to 40 wt%, 5.5 to 40 wt%, 6.0 to 40 wt%, 0.1 to 30 wt%, 0.5 to 30 wt%, 1.0 to 30 wt%, 1.5 to 30 wt%, 2.0 to 30 wt%, 2.5 to 30 wt%, 3.0 to 30 wt%, 3.5 to 30 wt%, 4.0 to 30 wt%, 4.5 to 30 wt%, 5.0 to 30 wt%, 5.5 to 30 wt%, 6.0 to 30 wt%, 0.1 to 20 wt%, 0.5 to 20 wt%, 1.0 to 20 wt%, 2.0 to 20 wt%, 2.5 to 20 wt%, 3.0 to 20 wt%, 4.0 to 20 wt%, 4.5 to 20 wt%, 5 to 20 wt%, 5.0 to 20 wt% 5.5 to 20 wt% or 6.0 to 20 wt% or the like.

In one embodiment of the present invention, the enzyme-treated composition of stevia plant is a composition that is enzymatically treated with hemicellulase and/or protease. The hemicellulase is not particularly limited as long as it is an enzyme capable of hydrolyzing hemicellulose, and known enzymes can be used. Hemicellulases are enzymes that decompose hemicellulose of polysaccharides such as xylan, arabinoxylan, xyloglucan and glucomannan, and xylanases and galactanases are known.

Protease refers to peptide bond hydrolase. The protease may be a peptide chain-end hydrolase that cleaves from the end of a peptide chain, or an endopeptidase that cleaves from the center of a peptide chain. Both may be used. The peptidase may be derived from plants such as papaya, pineapple, ginger, strawberry, kiwi, etc., or bacteria such as koji, mold, bacillus natto, etc. Hemicellulases and proteases are commercially available enzymes.

In one embodiment of the present invention, the enzyme-treated composition of stevia plant may be in the form of liquid, paste, powder or granule. When the liquid or paste is produced, the solvent may be tap water, ion-exchanged water, soft water, distilled water, or deaerated water obtained by deaerating these water.

2. Method for producing enzyme-treated composition of stevia plant

The invention also relates to a method for manufacturing the enzyme treatment composition of stevia plants. Specifically, the present invention relates to a method for producing an enzyme-treated composition for stevia plants, which is characterized by comprising a step of extracting stevia plants with a solvent and a step of subjecting the extracted residue to an enzyme treatment with hemicellulase and/or protease.

(A) Extracting stevia plant with solvent

The method according to one embodiment of the present invention includes a step of extracting stevia plants with a solvent. For example, the extraction of the stevia plant using an aqueous solvent may be mentioned.

For example, stevia plants can use dry leaves (which may also contain stems or other tissues) of stevia. The dry leaf of stevia is a leaf in which the water content is reduced by drying fresh leaves of stevia plant. The moisture content of the dried leaf of stevia plant is preferably 1 to 10% by weight, more preferably 2 to 8% by weight, particularly preferably 3 to 4% by weight.

When the moisture content of the dry leaf of the stevia plant used in the production method of the present invention is 10% by weight or less, the leaf may be a leaf containing 100g of arginine, 100 to 400mg of histidine, 100 to 600mg of tyrosine, 100 to 800mg of isoleucine, 100 to 1000mg of valine, 100 to 900mg of glycine, 400 to 2000mg of glutamic acid, 100 to 800mg of threonine, and 50 to 400mg of tryptophan per 100g of dry leaf.

The solvent may be water, alcohol, or a mixed solution thereof. Preferable solvents include ion-exchanged water, pure water (e.g., milli-Q water), and an aqueous ethanol solution. During extraction, stevia plants (e.g., dried leaves) may or may not be crushed. In the case of pulverization, the pulverization may be performed by using a ball mill or the like. Alternatively, the extraction treatment may be performed by using a column extractor (manufactured by GE Health Care) or a kneader extractor (SKN-R100 manufactured by Sanyou machines Co., ltd.).

The solvent may also be heated during extraction. The temperature at the time of extraction may be, for example, 10 to 80 ℃, 25 to 80 ℃, 30 to 75 ℃, 35 to 70 ℃, 40 to 65 ℃, 45 to 70 ℃, preferably 45 to 70 ℃. In the present specification, the lower limit and the upper limit of the temperature may be, for example, temperatures of ±1℃, ±2℃, ±3℃, ±4℃, and ±5℃, for each temperature.

The extraction may be carried out only 1 time or several times. By performing the extraction a plurality of times, steviol glycosides contained in the leaves can be extracted more.

The extraction rate of stevia plant is, for example, 10 to 60% as a standard, preferably 20 to 50%, more preferably 25 to 45%, still more preferably 30 to 40%. The extraction ratio is the ratio (%) of the amount of solid components in the extract to the weight of stevia plant as a raw material.

(B) A step of enzymatically treating the residue obtained after the extraction with a hemicellulase and/or a protease

In one embodiment of the present invention, the method for producing the extract comprises a step of subjecting the extracted residue to an enzyme treatment using a hemicellulase and/or a protease. As the hemicellulase and/or protease, those described in "enzyme treatment composition for stevia plant" above can be used. In several ways, the extracted residue of the stevia plant is at least 1 time of the extracted residue. In other ways, the residue is at least 2 post-extraction residue. In still other ways, the residue is at least 3 times post-extraction residue.

In the method according to one embodiment of the present invention, the step (B) may further include a step of preparing a slurry from the residue after extraction of the stevia plant, before or after the enzyme is added. The solvent used in the slurry may be 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%, and even more preferably 7 to 30% in view of fluidity, treatment efficiency, and the like.

In one embodiment of the present invention, the hemicellulase and the protease are preferably used in combination. By doing so, the concentration of the amino acid contained in the enzyme treatment composition can be increased.

In the case of using both hemicellulase and protease, the protease treatment may be performed after the hemicellulase treatment, or both the hemicellulase treatment and the protease treatment may be performed at the same time. More specifically, the hemicellulase may be added first in the step (a), and then the protease may be added in the step (B), or both the hemicellulase and the protease may be added simultaneously in the step (B).

The total amount of hemicellulase and protease added may be 0.5 to 15 wt%, 1 to 14.5 wt%, 1.5 to 14 wt%, 2 to 13.5 wt%, 2.5 to 13 wt%, 3 to 12.5 wt%, 3.5 to 12 wt%, 4 to 11.5 wt%, 4.5 to 11 wt%, 5 to 10.5 wt%, or 5 to 10 wt% based on the dry weight of the stevia plant.

In addition, when the hemicellulase and the protease are used in combination, the amount of each enzyme to be added may be appropriately adjusted according to the total amount to be added. For example, the amount of hemicellulase added may be 0.5 to 15 wt%, 1 to 14 wt%, 1.5 to 13 wt%, 2 to 12 wt%, 2.5 to 11 wt%, 3 to 10 wt%, or the like, based on the dry weight of the stevia plant. Similarly, the amount of protease to be added may be 0.5 to 15 wt%, 1 to 14 wt%, 1.5 to 13 wt%, 2 to 12 wt%, 2.5 to 11 wt%, 3 to 10 wt%, or the like based on the dry weight of stevia plant. The amount of hemicellulase or protease added may be added in combination with the total amount of the hemicellulase or protease added.

The time for the enzyme treatment may be, 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 to 18 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 6 to 18 hours, 7 to 48 hours, 7 to 36 hours, 7 to 18 hours, 8 to 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 24 hours, 10 to 18 hours, or 10 to 18 hours. The time of the enzyme reaction may be appropriately adjusted depending on the optimum temperature of the enzyme to be used, the amount of enzyme to be added, and the like.

The temperature of the enzyme reaction may be appropriately changed depending on the kind of the enzyme used, and may be, for example, 10 to 80 ℃, 15 to 80 ℃, 20 to 80 ℃, 25 to 80 ℃, 30 to 80 ℃, 35 to 80 ℃, 40 to 80 ℃, 10 to 75 ℃, 15 to 75 ℃, 20 to 75 ℃, 25 to 75 ℃, 30 to 75 ℃, 35 to 75 ℃, 40 to 75 ℃, 10 to 70 ℃, 15 to 70 ℃, 20 to 70 ℃, 25 to 70 ℃, 30 to 70 ℃, 35 to 70 ℃, 40 to 70 ℃, or the like. In addition, the temperature of the above range can also be adjusted 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, an electromagnetic heater, and the like.

The pH at the time of the enzymatic reaction may be appropriately changed depending on the kind of enzyme used, and may be, for example, 2 to 10, 2.2 to 10, 2.4 to 10, 2.6 to 10, 2.8 to 10, 3 to 10, 2 to 9, 2.2 to 9, 2.4 to 9, 2.6 to 9, 2.8 to 9, 3 to 9, 2 to 8, 2.2 to 8, 2.4 to 8, 2.6 to 8, 2.8 to 8, or 3 to 8.

For adjusting the pH, for example, alkali agents such as alkali metal hydroxides, alkaline earth metal hydroxides, silicates, carbonates, and hydrogencarbonates, organic pH adjusters 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.

Brix (solid content) of the reaction solution in the enzyme treatment may be, 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 to 30, 0.5 to 25, 0.5 to 20, 0.5 to 18, 0.5 to 16, 0.5 to 14, 0.5 to 12, 0.5 to 10, 0.5 to 8.0, 0.5 to 6.0, 0.5 to 5.0, 0.5 to 4.0 or 0.5 to 3.0.

In several modes, in order to effectively perform the enzyme reaction, the enzyme treatment may be performed while stirring the residue after extraction of the stevia plant (for example, the slurried residue). 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 may be used.

Furthermore, in several ways, the post-extraction residue of stevia plants (e.g., slurried residue) may be subjected to enzyme treatment in several passes. For example, the residue may be subjected to an enzyme treatment 2 to 10 times, 2 to 5 times or 2 to 4 times.

(C) Other procedures

The method of the present invention may further comprise ending the enzyme reaction by heating the enzyme reaction solution containing the residue to a high temperature. Depending on the heat resistance of the enzyme used, the conditions may vary, and for example, the enzyme may be inactivated by heating the enzyme reaction solution containing the residue to a temperature exceeding 80 ℃, 90 ℃, 95 ℃, or 100 ℃. 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. Enzyme deactivation may use a chip heater or a countercurrent contacting device.

In the production method according to one aspect of the present invention, the step (B) may include a step of performing a solid-liquid separation treatment. The solid-liquid separation treatment is not particularly limited as long as the solid and the liquid are sufficiently separated, and examples thereof include a treatment using a centrifugal separator or a filter press, and a gravity filtration using a filter or a screen.

The production method according to one embodiment of the present invention may further comprise concentrating, drying, granulating, and the like the enzyme-treated composition subjected to the solid-liquid separation treatment. The treatments such as concentration, drying and granulation can be carried out by a known method.

Through the above steps, an enzyme-treated composition of stevia plant can be produced. The characteristics of the resulting enzyme-treated composition were the same as those described in the above "enzyme-treated composition for stevia plant" described above. The characteristics of the food or drink containing the enzyme-treated composition of stevia plant obtained by the production method according to one embodiment of the present invention and the production method of the food or drink are described below.

3. Food or drink containing enzyme-treated composition of stevia plant

The present invention also relates to a food or beverage containing the enzyme-treated composition of stevia plant. The food or beverage of the present invention is not particularly limited as long as it contains the enzyme-treated composition of stevia plant. The term "food and drink" means a food and drink. The food or beverage is preferably a beverage.

The amount of the enzyme-treated composition to be blended with respect to the food and beverage may be set in accordance with the type of the food and beverage, and may be set in terms of the amount of the stevia-derived amino acid in the food and beverage, for example, such that the amount of the amino acid is 0.0001 to 1% by weight, 0.001 to 0.1% by weight, 0.005 to 0.05% by weight, or the like.

Examples of the food include snack foods, breads, cereal powders, flour, rice, agricultural or forestry processed foods, animal husbandry processed products, aquatic processed products, milk or dairy products, oils or fat processed products, seasonings, and other food materials.

Examples of the beverage include carbonated beverages, sports beverages, flavored waters, fruit juice beverages, alcoholic beverages, nonalcoholic beverages, beer-flavored beverages such as beer and nonalcoholic beer, coffee beverages, tea beverages, cocoa beverages, nutritional beverages, and functional beverages. Examples of the carbonated beverage include a sparkling beverage, cola, sugarless cola, ginger juice soda, lemon soda, a fruit juice-flavored carbonated beverage, and carbonated water to which a fruit juice flavor is imparted.

In one embodiment of the present invention, the content of the amino acid derived from stevia plant in the beverage may be, for example, 1 to 600ppm, 10 to 600ppm, 20 to 600ppm, 30 to 600ppm, 40 to 600ppm, 50 to 600ppm, 1 to 500ppm, 10 to 500ppm, 20 to 500ppm, 30 to 500ppm, 40 to 500ppm, 50 to 500ppm, 1 to 400ppm, 10 to 400ppm, 20 to 400ppm, 30 to 400ppm, 40 to 400ppm, 50 to 400ppm, 1 to 350ppm, 10 to 350ppm, 20 to 350ppm, 30 to 350ppm, 40 to 350ppm, 50 to 350ppm, 1 to 300ppm, 10 to 300ppm, 20 to 300ppm, 30 to 300ppm, 50 to 300ppm, 1 to 2500ppm, 10 to 250ppm, 20 to 250ppm, 30 to 250ppm, 40 to 250ppm, 50 to 250ppm, 1 to 200ppm, 10 to 200ppm, 20 to 200ppm, 30 to 200ppm, 40 to 200ppm, or 50 to 200 ppm. By setting the amino acid content within the above range, the sweetness of the beverage can be enhanced. In several modes, by setting the content of the amino acid within the above-described range, the sweetness of sucrose can be enhanced. In other modes, the sweetness of rebaudioside a can be enhanced by setting the content of amino acids within the above-described range.

In addition, in several ways, the beverage may also contain steviol glycosides. The steviol glycoside content in the beverage is preferably 1 to 800ppm, for example, it may be 20 to 750ppm, 20 to 700ppm, 20 to 650ppm, 20 to 600ppm, 20 to 550ppm, 25 to 550ppm, 30 to 550ppm, 35 to 550ppm, 40 to 550ppm, 45 to 550ppm, 50 to 550ppm, 55 to 550ppm, 20 to 540ppm, 25 to 540ppm, 30 to 540ppm, 35 to 540ppm, 40 to 540ppm, 45 to 540ppm, 50 to 540ppm, 55 to 540ppm, 20 to 530ppm, 25 to 530ppm, 30 to 530ppm, 35 to 530ppm, 40 to 530ppm, 45 to 530ppm, 50 to 530ppm, 55 to 530ppm, 20 to 520ppm, 25 to 520ppm, 30 to 520ppm, 40 to 520ppm, 45 to 520ppm, 55 to 520ppm, 20 to 510ppm, 30 to 510ppm, 35 to 510ppm, 40 to 510ppm, 45 to 510ppm, 50 to 510ppm, 35 to 510ppm 55 to 510ppm, 20 to 505ppm, 25 to 505ppm, 30 to 505ppm, 35 to 505ppm, 40 to 505ppm, 45 to 505ppm, 50 to 505ppm, 55 to 505ppm, 20 to 500ppm, 25 to 500ppm, 30 to 500ppm, 35 to 500ppm, 40 to 500ppm, 45 to 500ppm, 50 to 500ppm, 55 to 500ppm, 20 to 495ppm, 25 to 495ppm, 30 to 495ppm, 35 to 495ppm, 40 to 495ppm, 45 to 495ppm, 50 to 495ppm, 55 to 495ppm, 20 to 490ppm, 25 to 490ppm, 30 to 490ppm, 35 to 490ppm, 40 to 490ppm, 45 to 490ppm, 50 to 490ppm, 55 to 490ppm, 100 to 400ppm, 150 to 400ppm, 200 to 400ppm, 250 to 400ppm, 300 to 400ppm, 100 to 150ppm, 100 to 200ppm, 100 to 250ppm, or 100 to 300ppm, etc. By setting the steviol glycoside content within this range, the beverage can be given a moderate sweetness.

In one embodiment of the present invention, the beverage may contain a sweetener other than steviol glycoside. Such a sweetener is not particularly limited, and may further contain at least 1 sweetener selected from sucrose, fructose-Glucose syrup (Glucose-fructose-syrup), erythritol, mogroside V, corn syrup, aspartame (also referred to as an L-phenylalanine compound), sucralose, acesulfame potassium, saccharin, and xylitol, for example. Among them, natural sweeteners are preferably used from the viewpoints of freshness, ease of drinking, natural taste, and moderate alcohol flavor, and fructose syrup, sucrose, and corn syrup are particularly preferably used. These sweeteners may be used in an amount of 1 or more. These sweeteners may be contained in an amount 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, 1.5 or less, 1.0 or 0.5 or less, etc., in terms of Brix in the beverage, and the lower limit may be 0.1 or more.

When the beverage is a foaming beverage, the air pressure may be 2.2kgf/cm ² ～5.0kgf/cm ² . In several ways, the air pressure of the foamable beverage may be 2.2kgf/cm ² ～4.5kgf/cm ² 、2.2kgf/cm ² ～4.0kgf/cm ² 、2.2kgf/cm ² ～3.5kgf/cm ² 、2.2kgf/cm ² ～3.3kgf/cm ² 、2.2kgf/cm ² ～3.2kgf/cm ² 、2.3kgf/cm ² ～4.0kgf/cm ² 、2.3kgf/cm ² ～3.5kgf/cm ² 、2.3kgf/cm ² ～3.2kgf/cm ² 、3.0kgf/cm ² ～4.0kgf/cm ² Or 3.0kgf/cm ² ～3.5kgf/cm ² Etc. The gas content of the foamable beverage can be regulated by the gas pressure. In the present specification, unless otherwise specified, "air pressure" refers to the air pressure of carbon dioxide in a beverage after the liquid temperature of a foamable beverage in a container is set to 20 ℃, and then the air in the headspace is once opened to the atmosphere (sucked). The measurement of the air pressure can be performed by fixing a beverage having a liquid temperature of 20 ℃ to the gas internal pressure gauge, temporarily opening a stopcock of the gas internal pressure gauge, releasing carbon dioxide in the headspace to the atmosphere, closing the stopcock again, vibrating the gas internal pressure gauge, and reading the value when the pointer reaches a certain position. In the present specification, unless otherwise specified, the air pressure of the foamable beverage is measured by using this method.

The Brix of the beverage in terms of sucrose 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 relative to Sucrose (Sucrose) and the content of each sweetener. The relative ratio of the sweet tastes of the various sweeteners to the sweet taste 1 of sucrose can be obtained from a known sugar sweet taste conversion table (for example, page 11 of "dictionary of Beverage" information by Beverage Japan corporation) or the like. The sweetness values are described in numerical ranges, or relative comparison of sweetness with respect to sweetness 1 of sucrose can be determined by sensory tests for sweeteners having different values from document to document. Examples of such sensory tests include the following methods: a sample was prepared by adding granulated sugar to pure water starting from brix3.0 in a scale of 0.5 to 5.0, and a granulated sugar added sample having the same sweetness intensity as that of an aqueous solution of a sweetener of a predetermined concentration was selected therefrom.

The beverage may also contain alcohol. The alcoholic beverage is a beverage containing alcohol, and here, the term "alcohol" refers to ethyl alcohol (ethanol) as described above. The alcoholic beverage is not particularly limited as long as it contains alcohol. The beverage can be beer, sparkling wine, carbonic acid distilled liquor or cocktail with general alcohol content of 0.05-40 v/v%, 1.0-10 v/v%, 2.0-9.0 v/v% or 3.0-8.0 v/v%, or beverage with general alcohol content lower than 0.05v/v% of non-alcoholic beer, carbonic acid distilled liquor flavor beverage or cool beverage water. In several ways, the beverage preferably has an alcohol content of less than 0.05v/v%, more preferably 0.00v/v%. In the present specification, the alcohol degree refers to an index expressed as a percentage (v/v%) of a volume/volume standard. The alcohol content of the beverage may be measured by any known method, for example, by a vibrating densitometer.

The Flavor (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 juice soda-flavored, blackcurrant-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 can be adjusted by adding the following ingredients: fruit juices, souring agents, spices, extracts of plants, dairy products, other flavors, and the like, components that are approved as food additives, or components that are not approved but have been generally recognized as safe from ancient times of eating experience.

Various additives may be formulated into the beverage within a range that does not hinder the effects of the present invention. Examples of such additives include acidulants, perfumes, vitamins, pigments, antioxidants, emulsifiers, preservatives, flavors, extracts, pH adjusters, and quality stabilizers.

The energy (total energy) of the beverage is not particularly limited, can be 0 to 50Kcal/100ml, 0 to 45Kcal/100ml, 0 to 40Kcal/100ml, 0 to 35Kcal/100ml, 0 to 30Kcal/100ml, 0 to 24Kcal/100ml, 0 to 22Kcal/100ml, 0 to 20Kcal/100ml, 0 to 15Kcal/100ml, 0 to 10Kcal/100ml, 0 to 5Kcal/100ml, 0.1 to 50Kcal/100ml, 0.1 to 45Kcal/100ml, 0.1 to 40Kcal/100ml, 0.1 to 35Kcal/100ml, 0.1 to 30Kcal/100ml, 0.1 to 24Kcal/100ml, 0.1 to 22Kcal/100ml, 0.1 to 20Kcal/100ml, 0.1 to 15Kcal/100ml, 0.1 to 10Kcal/100ml, 0.1 to 5Kcal/100ml, 1 to 50Kcal/100ml, 1 to 35Kcal/100ml, 0.1 to 30Kcal/100ml, 0.1 to 24Kcal/100ml, 0 to 30Kcal/100ml 1 to 10Kcal/100ml, 1 to 5Kcal/100ml, 5 to 50Kcal/100ml, 5 to 45Kcal/100ml, 5 to 40Kcal/100ml, 5 to 35Kcal/100ml, 5 to 30Kcal/100ml, 5 to 24Kcal/100ml, 5 to 20Kcal/100ml, 5 to 15Kcal/100ml, 5 to 10Kcal/100ml, 10 to 50Kcal/100ml, 10 to 45Kcal/100ml, 10 to 40Kcal/100ml, 10 to 35Kcal/100ml, 10 to 30Kcal/100ml, 10 to 24Kcal/100ml, 10 to 20Kcal/100ml, 10 to 15Kcal/100ml, 15 to 50Kcal/100ml, 15 to 45Kcal/100ml, 15 to 40Kcal/100ml, 15 to 35Kcal/100ml, 15 to 30Kcal/100ml, 15 to 24Kcal/100ml, 15 to 20Kcal/100ml, 20 to 20Kcal/100ml, 20Kcal/100ml, 20-24 Kcal/100ml, 24-50 Kcal/100ml, 24-45 Kcal/100ml, 24-40 Kcal/100ml, 24-35 Kcal/100ml or 24-30 Kcal/100ml, etc.

The beverage can be prepared as a packaged beverage in a state of being sterilized by heating and filled in a container. The container is not particularly limited, and examples thereof include PET bottles, aluminum cans, steel cans, cartons, cold cups, bottles, and the like. When the heat sterilization is performed, the type thereof is not particularly limited, and for example, it can be performed by a usual method such as UHT sterilization and sterilization in a sterilization tank. The temperature of the heat sterilization step is not particularly limited, and may be, for example, 65 to 130 ℃, preferably 85 to 120 ℃, for 10 to 40 minutes. However, there is no problem in performing sterilization at an appropriate temperature for several seconds, for example, 5 to 30 seconds, as long as the sterilization effect equivalent to the above conditions is obtained.

The method for producing the food or beverage is not particularly limited as long as the food or beverage containing the above components can be obtained. According to one aspect of the present invention, there is provided a method for producing a food or beverage, comprising a step of obtaining an enzyme-treated composition of a stevia plant, and a step of adding the enzyme-treated composition to the food or beverage or a raw material thereof. The enzyme-treated composition for stevia plant obtained is as described in "2. Method for producing enzyme-treated composition for stevia plant" above. The enzyme-treated composition may be added to the food or beverage or a raw material thereof in any step of the production process of the food or beverage, for example, when the raw material of the food or beverage is mixed or when the taste quality of the food or beverage is finally adjusted.

4. Method for enhancing sweetness of food and drink

The invention also relates to a method for enhancing the sweetness of food and drink. Specifically, the present invention relates to a method for enhancing the sweetness of a food or beverage, which comprises a step of blending the composition described in the "enzyme-treated composition of a stevia plant" described in the above-mentioned "1" into a food or beverage (for example, a beverage). The composition is as described in "enzyme treatment composition of stevia plant" above. The composition may be added (or blended) to a food or a beverage containing the enzyme-treated composition of stevia plant as shown in the above "3", for example, when the raw materials of the food or beverage are mixed or when the taste quality of the food or beverage is finally adjusted.

In the present specification, the term "sweetness-enhanced" or "sweetness-enhanced" means that the food or beverage containing the enzyme-treated composition of a stevia plant according to one embodiment of the present invention has a property of being strongly perceived as sweet or a property of being obtained as compared with the food or beverage not containing the same.

The sweet taste enhancing method of one embodiment of the present invention can enhance the sweet taste of steviol glycoside and sucrose. In several ways, the method may also enhance the sweetness of sucrose. In several other ways, the method may enhance the sweetness of steviol glycosides. In still other ways, the methods may enhance the sweetness of rebaudioside a.

The method for enhancing sweetness of one embodiment of the present invention comprises a step of adding the enzyme-treated composition to a food or beverage such that the content of amino acids derived from stevia plants is 1 to 600 ppm. The content of the amino acid in the food and beverage may be, for example, 10 to 600ppm, 20 to 600ppm, 30 to 600ppm, 40 to 600ppm, 50 to 600ppm, 1 to 500ppm, 10 to 500ppm, 20 to 500ppm, 30 to 500ppm, 40 to 500ppm, 50 to 500ppm, 1 to 400ppm, 10 to 400ppm, 20 to 400ppm, 30 to 400ppm, 40 to 400ppm, 50 to 400ppm, 1 to 350ppm, 10 to 350ppm, 20 to 350ppm, 30 to 350ppm, 40 to 350ppm, 50 to 350ppm, 1 to 300ppm, 10 to 300ppm, 20 to 300ppm, 30 to 300ppm, 40 to 300ppm, 50 to 300ppm, 1 to 2500ppm, 10 to 250ppm, 20 to 250ppm, 30 to 250ppm, 40 to 250ppm, 50 to 250ppm, 1 to 200ppm, 10 to 200ppm, 20 to 200ppm, 30 to 200ppm, 40 to 200ppm, or 50 to 200 ppm.

5. Sweetness enhancer for food and drink

The present invention also relates to a sweetness enhancer for food or beverage. The present invention may also be a composition for enhancing sweetness of a food or beverage. In the present specification, the term "sweetness enhancer" or "sweetness enhancing composition" refers to a substance that enhances the sweetness of a food or beverage (for example, a beverage) when added to the food or beverage. In the present specification, the description of the sweetness enhancer is also applicable to a composition for enhancing sweetness. The sweetness enhancer is preferably added to a food or beverage, and the sweetness enhancer itself does not recognize the taste of the food or beverage by the consumer and enhances the sweetness of the food or beverage.

In one embodiment of the present invention, the sweetness enhancer comprises the enzyme-treated composition described in "enzyme-treated composition of stevia plant" above. The active ingredient in the sweetness enhancer may be an amino acid from stevia plant contained in the enzyme-treated composition.

In one embodiment of the present invention, the sweetness enhancer may be in the form of a liquid, paste, powder, granule, or the like.

In one embodiment of the present invention, the content of the enzyme-treated composition contained in the sweetness enhancer may be 30 to 100 wt%, 40 to 99 wt%, 50 to 98 wt%, 60 to 97 wt%, 70 to 96 wt%, 80 to 95 wt%, or the like, based on the total weight of the sweetness enhancer. In several ways, the sweetness enhancer may consist essentially of only amino acids from stevia plants. In the present specification, "substantially consisting of amino acids derived from stevia plants" means that steviol glycosides or other impurities which are inevitably contained in the enzyme treatment composition during the production process may be contained. For example, steviol glycosides or other impurities other than the amino acids may be contained in an amount of 5 wt% or less, 4 wt% or less, 3 wt% or less, 2 wt% or less, 1.5 wt% or less, 1.0 wt% or 0.5 wt% or less, or the like, based on the total weight of the sweetness enhancer.

In several ways, the sweetness enhancer may also contain ingredients other than the enzyme treatment composition. Examples of such components include sweeteners. Examples of the sweetener include natural sweeteners such as fructose, granulated sugar, fructose syrup (Glucose-fructose syrup), glucose, maltose, high fructose syrup (High Fructose Syrup), sugar alcohols, oligosaccharides, honey, sugarcane juice (brown sugar honey), maltose, lo Han Guo powder, lo Han Guo extract, glycyrrhiza root powder, glycyrrhiza root extract, sasa veitchii seed powder, sasa veitchii seed extract, artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, saccharin, and the like. Among them, natural sweeteners are preferably used from the viewpoints of freshness, ease of drinking, natural taste, and moderate alcohol flavor, and fructose, glucose, maltose, sucrose, and granulated sugar are particularly preferably used. These sweeteners may be used in an amount of 1 or more.

6. Microbial fermentation promoter

The invention also relates to a microbial fermentation promoter. The present invention may also be a composition for promoting fermentation of microorganisms. In the present specification, the term "fermentation accelerator" or "composition for promoting fermentation" refers to a composition for promoting fermentation or proliferation of a microorganism when added to a medium for culturing the microorganism or a food or beverage (for example, beverage) containing the microorganism. In the present specification, the description of the fermentation accelerator is also applicable to the fermentation-promoting composition.

In one embodiment of the present invention, the fermentation accelerator contains the enzyme-treating composition described in "1. Enzyme-treating composition of stevia plant" above. The active ingredient in the fermentation promoter may be an amino acid from stevia plant contained in the enzyme treatment composition.

In one embodiment of the present invention, the fermentation accelerator may be in the form of liquid, paste, powder, granule, or the like.

In one embodiment of the present invention, the content of the enzyme-treating composition contained in the fermentation accelerator may be 30 to 100 wt%, 40 to 99 wt%, 50 to 98 wt%, 60 to 97 wt%, 70 to 96 wt%, 80 to 95 wt%, or the like, based on the total weight of the fermentation accelerator. In several ways, the fermentation promoters may consist essentially of only amino acids from stevia plants. In other modes, the fermentation accelerator may optionally contain other components such as sugar, vitamins, minerals, and the like.

The microorganism which can be used as a fermentation promoter in one embodiment of the present invention is not particularly limited as long as it can assimilate nitrogen, and examples thereof include bacteria and yeasts.

Exemplary embodiments of the invention

In one aspect of the present invention, there is provided a composition which is an enzyme-treated composition of stevia plants, characterized in that,

The weight ratio of amino acid/steviol glycoside in the enzyme treatment composition is more than 0.3,

contains 100 to 10000ppm of amino acids relative to the total weight of the enzyme treatment composition. In this embodiment, the amino acid may be 100 to 2000ppm, 150 to 2000ppm, 200 to 2000ppm, 250 to 2000pm, 300 to 2000ppm, 350 to 2000ppm, 100 to 1000ppm, 150 to 1000ppm, 200 to 1000ppm, 250 to 1000pm, 300 to 1000ppm, 350 to 1000ppm, or the like.

contains steviol glycoside in an amount of 500ppm or less based on the total weight of the enzyme treatment composition. In this embodiment, the steviol glycoside may be 400ppm or less, 300ppm or less, 200ppm or less, 100ppm or less, 90ppm or less, 80ppm or less, 70ppm or less, 60ppm or less, 50ppm or less, or the like.

Containing 100 to 10000ppm of amino acids relative to the total weight of the enzyme treatment composition,

the amino acid is more than 1 selected from arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan and cystine. In this embodiment, the amino acid may be 100 to 2000ppm, 150 to 2000ppm, 200 to 2000ppm, 250 to 2000pm, 300 to 2000ppm, 350 to 2000ppm, 100 to 1000ppm, 150 to 1000ppm, 200 to 1000ppm, 250 to 1000pm, 300 to 1000ppm, 350 to 1000ppm, or the like.

contains 100 to 10000ppm of amino acid and 500ppm or less of steviol glycoside relative to the total weight of the enzyme treatment composition. In this embodiment, the amino acid may be 100 to 2000ppm, 150 to 2000ppm, 200 to 2000ppm, 250 to 2000pm, 300 to 2000ppm, 350 to 2000ppm, 100 to 1000ppm, 150 to 1000ppm, 200 to 1000ppm, 250 to 1000pm, 300 to 1000ppm, 350 to 1000ppm, or the like. In the present embodiment, steviol glycoside may be 400ppm or less, 300ppm or less, 200ppm or less, 100ppm or less, 90ppm or less, 80ppm or less, 70ppm or less, 60ppm or less, 50ppm or less, or the like.

the amino acid content of the enzyme treatment composition is 0.1 to 50% by weight relative to the solid content of the enzyme treatment composition. In this embodiment, the amino acid content may be 1.0 to 50 wt%, 2.0 to 40 wt%, 3.0 to 30 wt%, 3.0 to 20 wt%, 3.5 to 20 wt%, 4.0 to 20 wt%, 4.5 to 20 wt%, 5.0 to 20 wt%, 5.5 to 20 wt%, or 6.0 to 20 wt%.

the amino acid content of the enzyme treatment composition is 0.1 to 60% by weight relative to the total weight of the enzyme treatment composition, based on the dry weight. In this embodiment, the amino acid content may be 1.0 to 50 wt%, 2.0 to 40 wt%, 3.0 to 30 wt%, 3.0 to 20 wt%, 3.5 to 20 wt%, 4.0 to 20 wt%, 4.5 to 20 wt%, 5.0 to 20 wt%, 5.5 to 20 wt%, or 6.0 to 20 wt%.

In one embodiment of the present invention, there is provided a method for producing an enzyme-treated composition of stevia plant, characterized by comprising,

comprises a step of extracting stevia plant with a solvent,

a step of preparing a slurry from the residue obtained after extraction of stevia plant,

and a step of 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% or 7 to 30%.

comprises a step of extracting stevia plant with a solvent,

and a step of subjecting the extracted residue to an enzyme treatment using hemicellulase and/or protease, wherein the Brix of the reaction solution in the enzyme treatment is 0.25 to 30. In this embodiment, the Brix of the reaction solution in the enzyme treatment may be 0.25 to 10, 0.25 to 8.0, 0.25 to 6.0, 0.25 to 5.0, 0.5 to 10, 0.5 to 8.0, 0.5 to 6.0, 0.5 to 5.0, or the like.

Examples

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Production example

1. Extraction of stevia plants

The dried stevia leaves (water content: 10% by weight or less) were immersed in ion-exchanged water heated to 60.+ -. 5 ℃ and allowed to stand for 20 minutes. Then, heated ion-exchanged water was introduced from the lower part through a column extractor (column capacity: 1L,GE Health Care system), and 30 times the amount of the extract (1 st) of the dried leaves was collected. Then, ion exchange water at 90.+ -. 5 ℃ was introduced from the lower part, and 45 times the amount of the extract (2 nd) of the dried stevia leaves was collected. After extraction, ion-exchanged water and stevia leaves in the column were transferred to a container for enzyme treatment and used in the subsequent steps. The results of analyzing the amount of amino acids contained in each 100g of stevia dry leaf used as a raw material in advance by the acid decomposition method are shown in Table 1. Regarding the amount of amino acids, 15 kinds of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, valine, alanine, glycine, proline, glutamic acid, serine, threonine and aspartic acid were added to the sample, and the sample was subjected to hydrolysis after degassing and sealing, diluted or concentrated appropriately after constant volume, and the solution with pH adjusted to 2.2 was used as a test solution and analyzed by an amino acid autoanalyzer. In the case of cystine and methionine, a performic acid solution was added to a sample, the oxidized solution was concentrated and dried under reduced pressure, hydrochloric acid was added to hydrolyze the solution, the solution was diluted or concentrated appropriately after constant volume, and the solution with pH adjusted to 2.2 was used as a test solution and analyzed by an amino acid automatic analyzer. Tryptophan was prepared by adding barium hydroxide octahydrate, thiodiglycol and water to a sample, heating and dissolving, adding hydrochloric acid, degassing and sealing the tube, hydrolyzing the tube, adjusting the tube to slightly alkaline, fixing the volume, and appropriately diluting the tube, and analyzing the tube by high performance liquid chromatography.

TABLE 1

Amino acids	Content (mg/100 g)
		Arginine (Arg)	615
Lysine	764
		Histidine	286
Phenylalanine (Phe)	681
		Tyrosine	424
Leucine (leucine)	1070
		Isoleucine (Ile)	579
Methionine	232
		Valine (valine)	704
Alanine (Ala)	803
		Glycine (Gly)	654
Proline (proline)	660
		Glutamic acid	1460
Serine amino acid	1230
		Threonine (Thr)	608
Aspartic acid	1160
		Tryptophan	220
Cystine (cystine)	133
		Total	12283

2. Enzymatic treatment of extraction residues

Hemicellulase (manufactured by Tian Ye, hemicellulase "Amano" 90) and protease (manufactured by Tian Ye, protease A Amano) were added to the residue (slurry) obtained in the above "extraction of stevia rebaudiana plant", and the mixture was subjected to enzyme treatment at pH5.84 and 42℃for 16 hours with stirring. At this time, the slurry concentration was about 11.2%. The amount of enzyme added was such that it was 4% by weight relative to the dry weight of stevia plant. In addition, brix in the enzyme treatment is shifted as shown in FIG. 1. Then, the reaction solution was heated with a warm bath to inactivate the enzyme. Further, solid-liquid separation was performed by centrifugation, and an enzyme-treated composition of stevia plant was obtained as an extract.

EXAMPLE A confirmation of the amount of amino acids and steviol glycoside in the enzyme-treated composition of stevia plant

The enzyme-treated composition of stevia plant obtained by the method described in the above production example was analyzed for the amount of amino acids and steviol glycoside. The results are shown in fig. 2 and 3, respectively. In addition, 17 kinds of amino acids, i.e., arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, cystine, valine, alanine, glycine, proline, glutamic acid, serine, threonine and aspartic acid, were added and sulfosalicylic acid was extracted, then the pH was adjusted to 2.2, and the test solution was diluted and adjusted appropriately to be analyzed by an amino acid autoanalyzer. The test solutions were adjusted to be slightly alkaline, and the tryptophan was subjected to constant volume and analysis by high performance liquid chromatography in the same manner as the above 17 samples. The steviol glycoside amount was analyzed by a liquid chromatography mass spectrometry (column: shim-pack XR-ODS II (2.0 mm (i.d.)) x 150mm (L) (Shimadzu corporation), mobile phase: 30% acetonitrile/70% Milli-Q water (containing 0.1% formic acid), flow rate: 0.34 ml/min.) in addition, in FIGS. 2 and 3, samples of "1st" and "2nd" were the results of analyzing the 1st extract and the 2nd extract obtained in the above production example (the same as described below), and samples of "3rd (before)" and "3rd (after)" were the samples before and after the enzyme treatment, respectively (the same as described below).

As shown in fig. 2, it was confirmed that the amino acid concentration of the resulting composition was significantly increased by enzyme treatment with hemicellulase and protease, as compared with that before enzyme addition. The composition of each amino acid in the amino acid amounts shown in FIG. 2 is shown in Table 2 below.

TABLE 2

In addition, as shown in FIG. 3, the resulting composition contained a small amount of steviol glycoside (1 st:2231.42ppm,2nd:719.78ppm,3rd (front): 28.51ppm,3rd (rear): 55.53 ppm). Here, the steviol glycoside amount shown in FIG. 3 is the total steviol glycoside content composed of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside I, rebaudioside M, rebaudioside N, stevioside, steviolbioside, dulcoside A, and rubusoside. The proportions of each steviol glycoside in the extract solutions of the 1st and 2nd th times and the total steviol glycosides in the samples after the enzyme treatment are shown in Table 3 below.

TABLE 3

EXAMPLE B confirmation of amino acid amount when various proteases were used

Enzyme-treated compositions of stevia plants which were subjected to enzyme treatment with various enzymes were obtained in the same manner as in the above production examples, except that the conditions for the enzyme reaction were as shown in Table 4 below. For each composition, the amino acid amount was analyzed under the conditions of reference example a. The results are shown in FIG. 4. The composition of each amino acid shown in FIG. 4 is shown in Table 5 below.

TABLE 4

Example(s)	Hemicellulase(s)	ProteinsEnzymes	pH	Temperature (. Degree. C.)
					1	Hemicellulase Amano 90	Peptidase R	7	37
2	Hemicellulase Amano 90	Protease A Amano SD	7	50
					3	Hemicellulase Amano 90	ProteAX	8	70
4	Hemicellulase Amano 90	Protease M Amano SD	6	50
					5	Hemicellulase Amano 90	Protease P Amano 3SD	7	40
6	Hemicellulase Amano 90	Protease HFAmano 150SD	3	50

TABLE 5

As shown in fig. 4, it was confirmed that each of the compositions obtained in example B had substantially the same amino acid composition as the composition obtained in the above production example even when the enzyme used was changed.

[ embodiment C ] evaluation of sweetness enhancing Effect of rebaudioside A

An aqueous solution was prepared by adding rebaudioside a (manufactured by the chemical industry of gaku, J-100 (purity 95% or more)) and the enzyme-treated composition of stevia plant obtained in the above manufacturing example to pure water so as to reach the concentration of table 6 below. Regarding the enzyme treatment composition, the amino acid concentration of Table 6 was obtained by adding the enzyme treatment composition. In addition, sample 1 was a control sample containing no enzyme treatment composition. Each sample was evaluated as a professional panel with respect to the sensory subjects (3) by the following criteria. The results are shown in Table 7.

TABLE 6

Sample of	1	2	3	4
					Reb.A(ppm)	280	280	280	280
Amino acid (ppm)	0	80	160	332

Evaluation criteria

O: compared with the control sample, intense sweet taste is perceived

Delta: slightly perceived intense sweetness compared to the control sample

X: the sweetness was weaker than the control sample

TABLE 7

Sample of	2	3	4
				Panel a	△	○	○
Panel B	×	○	○
				Panel C	○	○	×

EXAMPLE D evaluation of sweet taste enhancing Effect of sucrose

An aqueous solution was prepared by adding sucrose (Sanjing sweetened) and the enzyme-treated composition of stevia plant obtained in the above-mentioned production example to pure water in such a manner as to reach the concentrations shown in Table 8 below. Regarding the enzyme treatment composition, the amino acid concentration of Table 8 was obtained by adding the enzyme treatment composition. In addition, sample 1 was a control sample containing no enzyme treatment composition. Each sample was evaluated as a professional panel with respect to the sensory subjects (3) by the following criteria. The results are shown in Table 9.

TABLE 8

Sample of	1	2	3	4
					Sucrose (weight%)	7	7	7	7
Amino acid (ppm)	0	80	160	332

Evaluation criteria

O: compared with the control sample, intense sweet taste is perceived

Delta: slightly perceived intense sweetness compared to the control sample

X: the sweetness was weaker than the control sample

TABLE 9

Sample of	2	3	4
				Panel a	Δ	Δ	○
Panel B	○	○	Δ
				Panel C	○	○	Δ

Claims

1. A composition which is an enzyme-treated composition of stevia plant, characterized in that,

2. The composition according to claim 1, wherein the amino acid content is 100 to 10000ppm relative to the total weight of the enzyme treatment composition.

3. The composition according to claim 1 or 2, wherein the amino acid is 1 or more selected from arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, aspartic acid, tryptophan and cystine.

4. A composition according to any one of claims 1 to 3, wherein the amino acid is an amino acid from a stevia plant.

5. The composition according to any one of claims 1-4, wherein the steviol glycoside is 1 or more selected from the group consisting of rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside M, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudioside R, du Kegan a, dulcoside C, rubusoside, steviolmonoside, steviolbioside, and stevioside.

6. The composition according to any one of claims 1 to 5, wherein the enzyme treatment composition is a composition which is enzymatically treated with hemicellulases and/or proteases.

7. The composition of any one of claims 1-6, wherein the stevia plant comprises leaves, stems and/or tissue of a stevia plant.

8. A food or beverage comprising the composition according to any one of claims 1 to 7.

9. A method for producing an enzyme-treated composition of stevia plant, characterized by comprising the steps of,

comprises the step of extracting stevia plant with solvent,

10. The method of claim 9, wherein the residue is a residue after at least 1 or more extractions.

11. The method according to claim 9 or 10, further comprising a step of performing solid-liquid separation after the enzyme treatment.

12. The method according to any one of claims 9 to 11, wherein the hemicellulase and/or proteinase is added in an amount of 0.5 to 15 wt% relative to the dry weight of the stevia plant.

13. The method according to any one of claims 9 to 12, wherein the enzyme treatment time is 1 to 48 hours.

14. The method according to any one of claims 9 to 13, wherein the pH at the time of the enzyme treatment is 2 to 10.

15. The method according to any one of claims 9 to 14, wherein the temperature at which the enzyme is treated is between 10 and 80 ℃.

16. A composition obtainable by the process of any one of claims 9 to 15.

17. A food or beverage comprising the composition of claim 16.

18. A method for enhancing sweetness of a food or beverage, comprising the step of blending the composition according to any one of claims 1 to 7 and 16 into a food or beverage.