Novel steviol glycoside and method for producing same, and sweetener composition containing same

Abstract

A novel steviol glycoside has been demanded. According to one aspect of the present invention, there is provided a compound represented by formula (1) (wherein (i) R1 represents Xyl(1-2)Glc1- and R2 represents Glc(1-2)Glc1-; or (ii) R1 represents Glc(1-2)Glc1- and R2 represents Glc(1-2)[Xyl(1-3)]Glc1- wherein Glc represents glucose and Xyl represents xylose), or a salt or hydrate thereof.

Description

Novel steviol glycosides, methods for producing the same, and sweetener compositions containing the same

[0001] This invention relates to a novel steviol glycoside, a method for producing the same, and a sweetener composition containing the same. The invention further relates to food and beverages and flavor modifiers containing the novel steviol glycoside.

[0002] The leaves of the stevia plant (Stevia rebaudiana), a member of the Asteraceae family, contain a secondary metabolite called steviol, a type of diterpenoid. Steviol glycosides are approximately 100 to 300 times sweeter than sugar and are used in the food industry as a calorie-free sweetener. Obesity is a serious social problem that is developing internationally, and the demand for calorie-free sweeteners is growing daily from the perspective of promoting health and reducing medical costs. Currently, artificially synthesized amino acid derivatives such as aspartame and acesulfame potassium are used as artificial sweeteners, but naturally occurring calorie-free sweeteners like steviol glycosides are expected to be safer and more easily accepted by consumers.

[0003] The main steviol glycosides of stevia are ultimately modified with sugars to form a glycoside called rebaudioside A (Reb. A), which has four sugars attached (Figure 1). Its precursor, the steviol trisaccharide glycoside stevioside, is the most abundant, and these two are the central substances responsible for the sweetness of stevia. Stevioside is found in the highest concentration in stevia leaves and is known to be more than 200 times sweeter than sugar. Reb. A is also a steviol tetrasaccharide glycoside that is highly sweet (more than 200 times sweeter than sugar) and has a good taste quality, and these are attracting attention as calorie-free sweeteners. In addition to these, the existence of glycosides thought to be reaction intermediates and analogs with different sugar types is known. For example, Reb. All four glycoside sugars in A are glucose, but among them, rebaudioside C (Reb. C) is known in which rhamnose is added instead of glucose at position 2 of the glucose at position 13, and rebaudioside F (Reb. F) is known in which xylose is added at the same position.

[0004] To date, attempts have been made to obtain stevia plants with a higher content of Reb. A than wild-type stevia plants through selective breeding, etc., given that Reb. A, in which all four glycoside sugars are glucose, has a good taste (for example, Patent Document 1). Attempts have also been made to obtain new steviol glycosides by decomposing known steviol glycosides such as rebaudioside M (Reb. M (also called Reb. X)), which has a good taste, with acid (for example, Patent Document 2). In addition, several steviol glycosides with added xylose molecules have also been reported (Patent Document 3).

[0005] Japanese Patent No. 3436317, International Publication No. 2014 / 146135, International Publication No. 2021 / 020516

[0006] Because the sweetness and flavor of steviol glycosides can vary greatly depending on the number and type of sugar molecules attached to the diterpene structure that forms their backbone, there is a need for novel steviol glycosides.

[0007] The present invention provides the following steviol glycosides, sweetener compositions containing the same, and food and beverages. [1] Formula (1): (wherein, (i) R １ However, it represents Xyl(1-2)Glc1- and R ２ This represents Glc(1-2)Glc1-; or (ii)R １ However, Glc(1-2)Glc1- represents and R ２ A compound represented by Glc(1-2)[Xyl(1-3)]Glc1-, where Glc represents glucose and Xyl represents xylose, or a salt or hydrate thereof. [2] The following formula (2) or (3): The compound shown in [1], or a salt or hydrate thereof. [3] Formula (4) or (5) below: [1] or [2] as shown in [1] or [2], or a salt or hydrate thereof. [4] Food and beverages comprising any of the compounds or salts or hydrates thereof described in [1] to [3]. [5] Food and beverages according to [4], wherein the content of any of the compounds or salts or hydrates thereof described in [1] to [3] is 1 to 600 ppm by mass. [6] Sweetener composition comprising any of the compounds or salts or hydrates thereof described in [1] to [3]. [7] Sweetener composition according to [6], wherein the content of any of the compounds or salts or hydrates thereof described in [1] to [3] is 1 to 99% by weight. [8] A sweetener composition according to [6] or [7], further comprising one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside. [9] A food or beverage containing the sweetener composition according to any one of [6] to [8].

[10] A food or beverage according to any one of [4], [5], and [9], which is a beverage.

[11] Use of any of the compounds described in [1] to [3] or their salts or hydrates as sweeteners.

[12] A method for producing any of the compounds described in [1] to [3], comprising: (1) preparing leaves of Stevia rebaudiana; (2) crushing the leaves in a solvent to obtain a leaf crushing liquid; (3) precipitating and filtering the leaf crushing liquid to obtain a filtrate; (4) gel filtering the filtrate to obtain a fraction containing 10 to 100 kDa of protein; (5) adding a steviol glycoside and UDP-glucose and / or UDP-xylose as substrates to the fraction; and (6) reacting at 20 to 37°C for 30 minutes or more.

[13] The production method according to

[12] , wherein the steviol glycoside serving as the substrate contains one or more steviol glycosides selected from the group consisting of stevioside, rebaudioside A, rebaudioside D, rebaudioside E, and rebaudioside M.

[0008] According to one aspect of the present invention, a novel steviol glycoside, a method for producing the same, and a sweetener composition, food and drink product, flavor modifier, etc. containing the novel steviol glycoside can be provided. The steviol glycoside of a preferred aspect of the present invention has little sweet aftertaste.

[0009] It is a diagram showing the structure and name of steviol glycoside. (A) is a photograph showing the SDS-PAGE electrophoresis image of each fraction separated using Sephadex G75, and (B) is a graph showing the protein concentration of each fraction separated using Sephadex G75. It is a mass chromatogram of the steviol glycoside produced when stevioside is used as substrate 1. The MS / MS and MS ３ spectrum of the ion at m / z 935.4 (Rt 28.7 minutes). It is a mass chromatogram of the steviol glycoside produced when Reb. E is used as substrate 1. The MS / MS and MS3 spectra of the ion at m / z 1097.4 (Rt 28.40 minutes). The １ 1H-NMR spectrum (400 MHz, Pyr-d5) of Compound 3. The １３ 13C-NMR spectrum (100 MHz, Pyr-d5) of Compound 3. The １ 1H-NMR spectrum (400 MHz, Pyr-d5) of Compound 6. The １３ 13C-NMR spectrum (100 MHz, Pyr-d5) of Compound 6. The １ 1H-NMR spectrum (400 MHz, Pyr-d5) of Compound 1. The １３ 13C-NMR spectrum (100 MHz, Pyr-d5) of Compound 1. It is an MS chromatogram at m / z 935.4 of in vitro synthesis and chemical synthesis using a stevia-derived enzyme. The MS / MS and MS of in vitro synthesis (Rt 28.76 minutes) ３This is the spectrum. MS / MS and MS of a chemically synthesized sample (Rt 28.8 min). ３ This is a spectrum. This is a graph showing the sensory evaluation results (average score). This is a graph showing the sensory evaluation results (standard score).

[0010] The present invention will now be described in detail. The following embodiments are illustrative for illustrating the present invention and are not intended to limit the invention to these embodiments only. Any paragraph headings used herein are for structural purposes only and should not be construed as limiting the subject matter described. The present invention can be carried out in various forms without departing from its essence. All documents cited herein, as well as published gazettes, patent gazettes and other patent documents, are incorporated herein by reference. Furthermore, this specification includes the contents of the specification and drawings of the Japanese Patent Application (Japanese Patent Application No. 2024-230838), filed on December 26, 2024, on which this application claims priority.

[0011] In this specification, "rebaudioside," "Reb," and "Reb." all have the same meaning and refer to "rebaudioside." Similarly, in this specification, "dulcoside" refers to "dulcoside."

[0012] 1. Novel Steviol Glycoside The inventors have for the first time identified the structure of a novel steviol glycoside containing xylose. According to one aspect of the present invention, a novel steviol glycoside containing xylose (hereinafter also referred to as "the glycoside of the present invention") is provided. The novel steviol glycoside according to one aspect of the present invention is given by formula (1): (wherein, (i) R １ However, it represents Xyl(1-2)Glc1- and R ２ This represents Glc(1-2)Glc1-; or (ii)R １ However, Glc(1-2)Glc1- represents and R ２It is a compound represented by Glc(1-2)[Xyl(1-3)]Glc1- (where Glc represents glucose and Xyl represents xylose), or a salt or hydrate thereof. The glucose molecule and xylose molecule in the sugar chain are also called glucopyranosyl and xylopyranosyl, respectively.

[0013] As described above, a steviol glycoside according to one aspect of the present invention includes a steviol glycoside having a sugar chain containing two molecules of glucose at the 13th position of steviol and one molecule of glucose and one molecule of xylose at the 19th position (hereinafter also referred to as "glycoside A of the present invention") and a steviol glycoside having a sugar chain containing two molecules of glucose and one molecule of xylose at the 13th position of steviol and a sugar chain containing two molecules of glucose at the 19th position (hereinafter also referred to as "glycoside B of the present invention").

[0014] Furthermore, as stated above, Glc represents glucose and Xyl represents xylose. In this specification, Glc may be α-glucose or β-glucose, and Xyl may be α-xylose or β-xylose. Alternatively, in this specification, Glc may be α-glucose and β-glucose, and Xyl may be α-xylose and β-xylose. "Glc1-" indicates that the carbon atom at position 1 of glucose is glycosidically bonded to steviol, and "Glc(1-2)Glc1-" indicates that the carbon atom at position 1 of another glucose is glycosidically bonded to the carbon atom at position 2 of the glucose indicated by "Glc1-". Also, "Xyl(1-2)Glc1-" indicates that the carbon atom at position 1 of xylose is glycosidically bonded to the carbon atom at position 2 of the glucose indicated by "Glc1-". Furthermore, "Glc(1-2)[Xyl(1-3)]Glc1-" indicates that the carbon atom at position 1 of glucose is glycosidically bonded to the carbon atom at position 2 of glucose, indicated by "Glc1-", and that the carbon atom at position 1 of xylose is glycosidically bonded to the carbon atom at position 3 of glucose, indicated by "Glc1-".

[0015] Glycoside A includes, for example, glycosides having the structures shown by formula (2), formula (2)', formula (4), and formula (4)'. In glycoside A represented by formula (2), glucose is β-glucosidically bonded to the carboxyl group at position 19 of steviol, and xylose is β1-2 bonded to that glucose. In glycoside A represented by formula (2)', glucose is β-glucosidically bonded to the carboxyl group at position 19 of steviol, and xylose is α1-2 bonded to that glucose. Formulas (4) and (4)' show structures that further specify the conformations of glycoside A of formulas (2) and (2)', respectively.

[0016] Glycoside B includes, for example, glycosides having the structures shown by formula (3), formula (3)', formula (5), and formula (5)'. Glycoside B, represented by formula (3), has glucose β-glucosidically bonded to the hydroxyl group at position 13 of steviol, with other glucose molecules β1-2-linked to that glucose molecule, and xylose molecules β1-3-linked to it. Glycoside B, represented by formula (3)', has glucose β-glucosidically bonded to the hydroxyl group at position 13 of steviol, with other glucose molecules β1-2-linked to that glucose molecule, and xylose molecules α1-3-linked to it. Formulas (5) and (5)' show structures that further specify the conformations of glycoside B of formulas (3) and (3)', respectively.

[0017] In a preferred embodiment of the present invention, formula (2) or (3): Compounds represented by, or salts or hydrates thereof, are provided. In a more preferred embodiment of the present invention, the following formula (4) or (5): A compound represented by, or a salt or hydrate thereof, is provided.

[0018] The glycosides according to one aspect of the present invention include isomers such as the α-form and the β-form as described above. Therefore, the glycoside of the present invention may be only the α-form, only the β-form, or a mixture of the α-form and the β-form. Preferably, the proportion of the β-form in the glycoside of the present invention is 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 99% or more. The α-form and the β-form can be isolated and purified by using known methods such as high performance liquid chromatography (HPLC) and ultra (high) performance liquid chromatography (UPLC).

[0019] The glycoside according to one aspect of the present invention may be not only the compound represented by the formula (1) but also a salt or a hydrate thereof. In the present specification, the "salt of the compound of the formula (1)" means a physiologically acceptable salt of the compound of the formula (1), such as a sodium salt. Further, the "hydrate of the compound of the formula (1)" in the present specification means a compound in which water molecules are added to the compound of the formula (1).

[0020] The glycoside according to one aspect of the present invention is not particularly limited and may be a chemically synthesized product or a biosynthesized product. For example, the glycoside according to one aspect of the present invention may be obtained by chemical synthesis or biosynthesis. Details of the method for producing the glycoside of the present invention will be described later in this specification.

[0021] The glycoside according to one aspect of the present invention has a sweetness similar to known steviol glycosides such as Reb. A, Reb. D, and Reb. M, and can affect the sweetness by being added to foods and drinks, etc. Therefore, the glycoside of the present invention can be used as a novel sweetener. According to one aspect of the present invention, there is provided the use of the compound represented by the formula (1), or a salt or a hydrate thereof, as a sweetener.

[0022] The glycoside of a preferred embodiment of the present invention is selected from glycoside A or glycoside B. Glycoside A has a lower sweetness than known steviol glycosides such as Reb. A, Reb. D, and Reb. M, and has less sweetness aftertaste. Similarly, glycoside B also has a lower sweetness than known steviol glycosides, and one or more taste qualities among the onset of sweetness, sweetness intensity, sweetness aftertaste, bitterness, and bitterness aftertaste may be good. Therefore, the steviol glycoside according to one embodiment of the present invention can be suitably used for various applications as a sweetener as described below.

[0023] 2. Sweetener composition containing a novel steviol glycoside According to one aspect of the present invention, there is provided a sweetener composition (also referred to herein as "the sweetener composition of the present invention") containing a compound represented by formula (1), or a salt or hydrate thereof. The sweetener composition according to one embodiment of the present invention is not particularly limited as long as it contains a compound represented by formula (1), or a salt or hydrate thereof, and may be a composition containing an extract containing a compound represented by formula (1), or a salt or hydrate thereof. The sweetener composition according to another embodiment of the present invention is a composition containing an extract containing a compound represented by formula (2) or (3), or a compound represented by formula (4) or (5), or a salt or hydrate thereof.

[0024] The amount of the glycoside according to one embodiment of the present invention contained in the sweetener composition according to one embodiment of the present invention is not particularly limited. For example, it may be 1 to 99% by weight, 5 to 95% by weight, 10 to 90% by weight, 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30 to 70% by weight, based on the total amount of the sweetener composition. 35 to 65% by weight, 40 to 60% by weight, 45 to 55% by weight, 1 to 5% by weight, 1 to 10% by weight, 1 to 15% by weight, 1 to 20% by weight, 1 to 25% by weight, 1 to 30% by weight, 1 to 35% by weight, 1 to 40% by weight, 1 to 45% by weight, or 1 to 50% by weight.

[0025] A sweetener composition according to one aspect of the present invention may further contain other steviol glycosides. For example, a sweetener composition according to one aspect of the present invention may further contain, in addition to the glycoside of the present invention, one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside.

[0026] If other steviol glycosides are included, the composition ratio of the glycoside according to one embodiment of the present invention to the other steviol glycosides may be 1:99 to 99:1, 1:99 to 5:95, 1:99 to 10:90, 1:99 to 20:80, 1:99 to 30:70, 1:99 to 40:60, 1:99 to 50:50, 5:95 to 95:5, 10:90 to 90:10, 15:85 to 85:15, 20:80 to 80:20, 25:75 to 75:25, 30:70 to 70:30, 35:65 to 65:35, 40:60 to 60:40, 45:55 to 55:45, or 50:50. Furthermore, only one type of glycoside may be used, or multiple types may be used.

[0027] A sweetener composition according to one aspect of the present invention may further contain sweeteners other than steviol glycosides. Examples of such sweeteners include natural sweeteners such as fructose, sugar, fructose-glucose liquid sugar, glucose, maltose, high-fructose liquid sugar, sugar alcohols, oligosaccharides, honey, sugarcane juice (brown sugar syrup), starch syrup, monk fruit powder, monk fruit extract, licorice powder, licorice extract, thaumatococcus danieli seed powder, and thaumatococcus danieli seed extract, as well as artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, and saccharin. Among these, natural sweeteners are preferred from the viewpoint of providing a refreshing taste, ease of drinking, a natural flavor, and a moderate richness, and fructose, glucose, maltose, sucrose, and sugar are particularly suitable. These sweetener components may be used individually or in combination.

[0028] A method for producing a sweetener composition according to one aspect of the present invention is not particularly limited as long as a sweetener composition having the above composition can be obtained. According to one aspect of the present invention, a method for producing a sweetener composition of the present invention is provided, comprising the steps of obtaining the glycoside of the present invention and mixing the glycoside with another steviol glycoside or a sweetener other than a steviol glycoside. The step of obtaining the glycoside of the present invention may be carried out by chemical synthesis or biosynthesis, and the glycoside of the present invention obtained by this step may be obtained as a mixture with other steviol glycosides (for example, Reb. A or Reb. D).

[0029] 3. Food and beverages containing novel steviol glycosides According to one aspect of the present invention, a food or beverage (hereinafter also referred to as "food or beverage of the present invention") is provided that contains the compound represented by formula (1), or a salt or hydrate thereof, or the sweetener composition of the present invention. A food or beverage according to one aspect of the present invention is not particularly limited as long as it contains the compound represented by formula (1), or a salt or hydrate thereof, and may also contain an extract or sweetener composition containing the compound represented by formula (1), or a salt or hydrate thereof. Here, food and beverage means beverages and food. Accordingly, in one embodiment, the present invention provides a novel beverage or food, and also provides a method for producing said beverage or food. A food or beverage according to another aspect of the present invention is a food or beverage that contains an extract containing the compound represented by formula (2) or (3), or formula (4) or (5), or a salt or hydrate thereof.

[0030] The amount of the glycoside according to an aspect of the present invention contained in the food or drink according to an aspect of the present invention varies depending on the specific food or drink. In the case of beverages, it is preferably about 1 to 600 ppm by mass, for example, 20 to 550 ppm by mass, 25 to 550 ppm by mass, 30 to 550 ppm by mass, 35 to 550 ppm by mass, 40 to 550 ppm by mass, 45 to 550 ppm by mass, 50 to 550 ppm by mass, 55 to 550 ppm by mass, 20 to 540 ppm by mass, 25 to 540 ppm by mass, 30 to 540 ppm by mass, 35 to 540 ppm by mass, 40 to 540 ppm by mass, 45 to 540 ppm by mass, 50 to 540 ppm by mass, 55 to 540 ppm by mass, 20 to 530 ppm by mass, 25 to 530 ppm by mass, 30 to 530 ppm by mass, 35 to 530 ppm by mass, 40 to 530 ppm by mass, 45 to 530 ppm by mass, 50 to 530 ppm by mass, 55 to 530 ppm by mass, 20 to 520 ppm by mass, 25 to 520 ppm by mass, 30 to 520 ppm by mass, 35 to 520 ppm by mass, 40 to 520 ppm by mass, 45 to 520 ppm by mass, 50 to 520 ppm by mass, 55 to 520 ppm by mass, 20 to 510 ppm by mass, 25 to 510 ppm by mass, 30 to 510 ppm by mass, 35 to 510 ppm by mass, 40 to 510 ppm by mass, 45 to 510 ppm by mass, 50 to 510 ppm by mass, 55 to 510 ppm by mass, 20 to 505 ppm by mass, 25 to 505 ppm by mass, 30 to 505 ppm by mass, 35 to 505 ppm by mass, 40 to 505 ppm by mass, 45 to 505 ppm by mass, 50 to 505 ppm by mass, 55 to 505 ppm by mass, 20 to 500 ppm by mass, 25 to 500 ppm by mass, 30 to 500 ppm by mass, 35 to 500 ppm by mass, 40 to 500 ppm by mass, 45 to 500 ppm by mass, 50 to 500 ppm by mass, 55 to 500 ppm by mass, 20 to 495 ppm by mass, 25 to 495 ppm by mass, 30 to 495 ppm by mass, 35 to 495 ppm by mass, 40 to 495 ppm by mass, 45 to 495 ppm by mass, 50 to 495 ppm by mass, 55 to 495 ppm by mass, 20 to 490 ppm by mass, 25 to 490 ppm by mass, 30 to 490 ppm by mass, 35 to 490 ppm by mass, 40 to 490 ppm by mass, 45 to 490 ppm by mass, 50 to 490 ppm by mass, 55 to 490 ppm by mass, 100 to 400 ppm by mass, 150 to 400 ppm by mass, 200 to 400 ppm by mass,The amount may be 250-400 ppm by mass, 300-400 ppm by mass, 100-150 ppm by mass, 100-200 ppm by mass, 100-250 ppm by mass, or 100-300 ppm by mass. Setting the content within this range has the advantage of providing a moderate sweetness. It also has the advantage of suppressing lingering aftertaste. Alternatively, the amount of the glycoside according to one aspect of the present invention contained in a food or beverage according to another aspect of the present invention may be 1-50 ppm by mass, 1-45 ppm by mass, 1-40 ppm by mass, 1-35 ppm by mass, 1-30 ppm by mass, 1-25 ppm by mass, 1-20 ppm by mass, 1-15 ppm by mass, 1-10 ppm by mass, or 1-5 ppm by mass. Including the glycoside according to one aspect of the present invention at such low concentrations can improve the flavor of other sweeteners contained in the beverage. The glycoside content can be measured by HPLC. Alternatively, if the amounts of glycosides are known, values ​​calculated from those amounts may be used. In this specification, "ppm" means "mass ppm" unless otherwise specified.

[0031] Food and beverages according to one aspect of the present invention may further contain other steviol glycosides. For example, in addition to the glycoside of the present invention, food and beverages according to one aspect of the present invention may further contain one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside.

[0032] If other steviol glycosides are present, the composition ratio of the glycoside according to one embodiment of the present invention to the other steviol glycosides may be 1:99 to 99:1, 1:99 to 5:95, 1:99 to 10:90, 1:99 to 20:80, 1:99 to 30:70, 1:99 to 40:60, 1:99 to 50:50, 5:95 to 95:5, 10:90 to 90:10, 15:85 to 85:15, 20:80 to 80:20, 25:75 to 75:25, 30:70 to 70:30, 35:65 to 65:35, 40:60 to 60:40, 45:55 to 55:45, or 50:50 by mass.

[0033] Food and beverages according to one aspect of the present invention may further contain sweeteners other than steviol glycosides. Examples of such sweeteners include natural sweeteners such as fructose, sugar, fructose-glucose liquid sugar, glucose, maltose, sucrose, high-fructose liquid sugar, sugar alcohols, oligosaccharides, honey, sugarcane juice (brown sugar syrup), starch syrup, monk fruit powder, monk fruit extract, licorice powder, licorice extract, thaumatococcus danieli seed powder, and thaumatococcus danieli seed extract, as well as artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, and saccharin. Among these, natural sweeteners are preferred from the viewpoint of providing a refreshing taste, ease of drinking, a natural flavor, and a moderate richness, and fructose, glucose, maltose, sucrose, and sugar are particularly suitable. Only one type of these sweetener may be used, or multiple types may be used.

[0034] Examples of foods according to one aspect of the present invention are not limited to confectionery, bread, flour, noodles, rice dishes, processed agricultural and forestry products, processed livestock products, processed marine products, milk and dairy products, oils and fats and processed oils and fats, seasonings or other food ingredients.

[0035] Examples of beverages according to one aspect of the present invention include, but are not limited to, carbonated beverages, non-carbonated beverages, alcoholic beverages, non-alcoholic beverages, beer-flavored beverages such as beer and non-alcoholic beer, coffee beverages, tea beverages, cocoa beverages, nutritional beverages, and functional beverages.

[0036] A beverage according to one aspect of the present invention may be prepared as a packaged beverage that has been heat-sterilized and filled into a container. The container is not particularly limited and can be, for example, a PET bottle, aluminum can, steel can, paper carton, chilled cup, or glass bottle. When heat sterilization is performed, the type is not particularly limited and can be carried out using conventional methods such as UHT sterilization and retort sterilization. The temperature of the heat sterilization process is not particularly limited, but can be, 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, sterilization at an appropriate temperature for a few seconds, for example, 5 to 30 seconds, is also acceptable.

[0037] A method for producing food or beverages according to one aspect of the present invention is not particularly limited as long as a food or beverage having the above-mentioned components can be obtained. According to one aspect of the present invention, a method for producing food or beverages according to the present invention is provided, comprising the steps of: obtaining the glycoside or sweetener composition of the present invention; and adding the glycoside or sweetener composition to food or beverages or their raw materials. The step of obtaining the glycoside or sweetener composition of the present invention is described in "2. Sweetener composition containing a novel steviol glycoside" and "4. Method for producing a novel steviol glycoside," respectively. The step of adding the glycoside or sweetener composition according to one aspect of the present invention to food or beverages or their raw materials can be performed at any step in the food or beverage manufacturing process, for example, when mixing the raw materials of food or beverages, or when making final adjustments to the taste of food or beverages.

[0038] 4. Method for Producing Novel Steviol Glycosides The novel steviol glycosides of the present invention can be produced by (A) chemical synthesis or (B) biosynthesis. Each method is described below.

[0039] (A) Chemical Synthesis Steviol glycosides according to one aspect of the present invention can be produced, for example, by the method described in the examples below. Specifically, glycoside A can be obtained by using stevioside, a natural product, as a starting material, removing the glucose at position 19 by hydrolysis, and instead attaching a disaccharide hemiacetal composed of glucose and xylose. Furthermore, glycoside B can be synthesized by using rebaudioside F2, a known steviol glycoside, as a starting material, removing the glucose at position 19 by hydrolysis, and instead attaching a disaccharide hemiacetal composed of two glucose molecules.

[0040] (B) Biosynthesis The steviol glycoside according to one aspect of the present invention can also be produced by biosynthesis. According to one aspect of the present invention, the steviol glycoside according to one aspect of the present invention can be biosynthesized by a production method comprising: (1) preparing the leaves of Stevia rebaudiana; (2) crushing the leaves in a solvent to obtain a leaf crushed material-containing liquid; (3) precipitating and filtering the leaf crushed material-containing liquid to obtain a filtrate; (4) gel filtering the filtrate to obtain a fraction containing 10 to 100 kDa of protein; (5) adding the steviol glycoside and UDP-glucose and / or UDP-xylose, which are substrates, to the fraction; and (6) reacting at 20 to 37°C for 30 minutes or more.

[0041] The leaves of Stevia rebaudiana are not particularly limited, and commercially available plant leaves may be used, or leaves of plants grown from commercially available plants through crossbreeding may be used. For example, plants obtained by the method described in the examples below may be used. The leaves of Stevia rebaudiana used in the production method according to one aspect of the present invention are not particularly limited as long as they contain steviol glycosides, but preferably have a higher content of rebaudioside D or rebaudioside M than the leaves of natural Stevia plants. Such leaves of Stevia plants can be obtained, for example, by the method described in International Publication No. 2019 / 074089.

[0042] The leaves you prepare can be dried leaves or fresh leaves before drying.

[0043] The prepared leaves can be crushed in a solvent to obtain a liquid containing crushed leaves. The solvent is not particularly limited and can be water, extraction buffer, alcohol, or a mixture thereof. Preferred solvents include extraction buffer, deionized water, pure water (e.g., Milli-Q water), and aqueous ethanol solution. As the extraction buffer, an aqueous solution of trishydroxymethylaminomethane (Tris), phosphate buffers such as potassium phosphate and sodium phosphate, or the extraction buffer described in the examples can be used. The crushing method is also not particularly limited and can be any known means such as a juicer, blender, or ball mill.

[0044] A filtrate can be obtained by precipitating and filtering the leaf-crushed material-containing liquid. The precipitation and filtration methods are not particularly limited, and known means can be used. In one embodiment of the present invention, precipitation may be performed using ammonium sulfate (ammonium sulfate precipitation). Before the precipitation treatment, the filtrate may be pre-filtered using a nylon mesh or the like, then centrifuged, and the supernatant may be subjected to the precipitation treatment. After precipitation, the filtrate can be obtained by filtering using a syringe filter or the like.

[0045] The obtained filtrate can be separated into multiple fractions according to the molecular weight of the proteins contained in the filtrate by gel filtration. For example, by performing gel filtration using the method described in the examples, a fraction containing proteins of 10 to 100 kDa can be obtained. The proteins of 10 to 100 kDa contained in the fraction include glycosyltransferases derived from stevia.

[0046] The obtained fraction can be supplemented with a substrate steviol glycoside and UDP-glucose and / or UDP-xylose. This allows glucose and / or xylose to be added to the substrate steviol glycoside by glycoside-forming enzymes derived from stevia contained in the fraction. Various steviol glycosides can be used as the substrate steviol glycoside. In one aspect of the present invention, the substrate steviol glycoside comprises one or more steviol glycosides selected from the group consisting of stevioside, rebaudioside A, rebaudioside D, rebaudioside E, and rebaudioside M. In a preferred aspect of the present invention, the substrate steviol glycoside comprises stevioside or rebaudioside E.

[0047] By adding a substrate to the above fraction and reacting it at 20-37°C for 30 minutes or more, the addition of sugar by glycoside enzymes can be further advanced. The reaction temperature is preferably 25°C to 35°C, more preferably 28-32°C. The reaction time is not particularly limited as long as it is 30 minutes or more, but from the viewpoint of efficiency, it is preferably 2-48 hours, more preferably 4-36 hours, and even more preferably 6-24 hours.

[0048] After the reaction is complete, the reaction may be stopped by adding a reaction stopper such as acetonitrile, if desired. After stopping the reaction, the mixture may be filtered.

[0049] 5. Flavoring agent containing a novel steviol glycoside According to one aspect of the present invention, a flavoring agent containing the compound represented by formula (1) above, or a salt or hydrate thereof, is provided. In one aspect of the present invention, a composition having the composition described in "2. Sweetener composition containing a novel steviol glycoside" can also be used as a flavoring agent.

[0050] In this specification, "flavoring agent" refers to a substance that adjusts the flavor of food or beverages when added to them. A flavoring agent according to one aspect of the present invention can adjust the flavor of food or beverages without consumers noticing the taste of the flavoring agent itself when added to them. For example, a steviol glycoside according to one aspect of the present invention has the characteristic of having less lingering sweetness compared to conventional steviol glycosides, and can therefore be used as a flavoring agent to adjust the lingering sweetness of food or beverages.

[0051] A flavor modifier according to one aspect of the present invention preferably contains one or more other sweeteners in addition to the compound represented by formula (1) or its salt or hydrate. Such sweeteners are selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside. Examples include one or more steviol glycosides, natural sweeteners such as fructose, sugar, fructose-glucose syrup, glucose, maltose, sucrose, high-fructose corn syrup, sugar alcohols, oligosaccharides, honey, sugarcane juice (brown sugar syrup), starch syrup, monk fruit powder, monk fruit extract, licorice powder, licorice extract, thaumatococcus danieli seed powder, and thaumatococcus danieli seed extract, as well as artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, and saccharin.

[0052] [Exemplary Embodiments of the Invention] The following are exemplary embodiments of the invention, but the invention is not limited to these embodiments. Exemplary embodiment 1 of the invention is the following formula (4): The present invention provides a compound represented by formula (4) or a salt thereof. Exemplary embodiment 2 of the present invention provides a beverage containing a compound represented by formula (4) or a salt thereof according to Exemplary embodiment 1 of the present invention. Exemplary embodiment 3 of the present invention provides a beverage containing 1 to 50 ppm by mass, 1 to 25 ppm by mass, or 1 to 10 ppm by mass of the compound represented by formula (4) according to Exemplary embodiment 1 of the present invention. Exemplary embodiment 4 of the present invention provides a beverage containing 50 to 500 ppm by mass, 100 to 400 ppm by mass, or 100 to 300 ppm by mass of the compound represented by formula (4) according to Exemplary embodiment 1 of the present invention. Exemplary embodiment 5 of the present invention provides a beverage containing 1 to 50 ppm by mass of the compound represented by formula (4) according to Exemplary embodiment 1 of the present invention, and further containing one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside.

[0053] Exemplary embodiment 6 of the present invention provides a beverage containing 50 to 500 ppm by mass of the compound represented by formula (4) according to Exemplary embodiment 1 of the present invention, and further containing one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside. Exemplary embodiment 7 of the present invention contains 1 to 50 ppm by mass of the compound represented by formula (4) according to Exemplary embodiment 1 of the present invention, and includes rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, The present invention provides a beverage further comprising one or more other steviol glycosides selected from the group consisting of lucoside A, lucoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside, wherein the composition ratio of the compound represented by formula (4) to the other steviol glycosides is 1:99 to 99:1, 1:99 to 5:95, 1:99 to 10:90, or 1:99 to 50:50 by mass.Exemplary embodiment 8 of the present invention contains 50 to 500 ppm by mass of the compound represented by formula (4) according to Exemplary embodiment 1 of the present invention, and includes rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, and Zul The present invention provides a beverage further comprising one or more other steviol glycosides selected from the group consisting of coside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside, wherein the composition ratio of the compound represented by formula (4) to the other steviol glycosides is 1:99 to 99:1, 20:80 to 80:20, 30:70 to 70:30, or 45:55 to 55:45 by mass.

[0054] An exemplary embodiment 9 of the present invention is given by the following formula (5): The present invention provides a compound represented by formula (5) or a salt thereof. Exemplary embodiment 10 of the present invention provides a beverage containing a compound represented by formula (5) or a salt thereof according to Exemplary embodiment 9 of the present invention. Exemplary embodiment 11 of the present invention provides a beverage containing 1 to 50 ppm by mass, 1 to 25 ppm by mass, or 1 to 10 ppm by mass of the compound represented by formula (5) according to Exemplary embodiment 9 of the present invention. Exemplary embodiment 12 of the present invention provides a beverage containing 50 to 500 ppm by mass, 100 to 400 ppm by mass, or 100 to 300 ppm by mass of the compound represented by formula (5) according to Exemplary embodiment 9 of the present invention. Exemplary embodiment 13 of the present invention provides a beverage containing 1 to 50 ppm by mass of the compound represented by formula (5) according to Exemplary embodiment 9 of the present invention, and further containing one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside.

[0055] Exemplary embodiment 14 of the present invention provides a beverage containing 50 to 500 ppm by mass of the compound represented by formula (5) according to Exemplary embodiment 9 of the present invention, and further containing one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside. Exemplary embodiment 15 of the present invention contains 1 to 50 ppm by mass of the compound represented by formula (5) according to Exemplary embodiment 9 of the present invention, and includes rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, The present invention provides a beverage further comprising one or more other steviol glycosides selected from the group consisting of lucoside A, lucoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside, wherein the composition ratio of the compound represented by formula (5) to the other steviol glycosides is 1:99 to 99:1, 1:99 to 5:95, 1:99 to 10:90, or 1:99 to 50:50 by mass.Exemplary embodiment 16 of the present invention contains 50 to 500 ppm by mass of the compound represented by formula (5) according to Exemplary embodiment 9 of the present invention, and includes rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, and Zul The present invention provides a beverage further comprising one or more other steviol glycosides selected from the group consisting of coside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside, wherein the composition ratio of the compound represented by formula (5) to the other steviol glycosides is 1:99 to 99:1, 20:80 to 80:20, 30:70 to 70:30, or 45:55 to 55:45 by mass.

[0056] In this specification, the term "approximately" means that the subject lies within ±25%, ±10%, ±5%, ±3%, ±2%, or ±1% of the number following "approximately." For example, "approximately 10" means in the range of 7.5 to 12.5. Also, in this specification, weight percent may be considered equivalent to mass percent.

[0057] The present invention will be described in detail below with reference to examples, but the content of the present invention is not limited thereto.

[0058] [Preparation of Stevia Plants Used in the Examples] In the examples described below, Stevia plants (Variety A) developed by crossbreeding at Suntory Global Innovation Center Limited (SIC) were used. These Stevia plants are the self-pollinated progeny of a variety (Variety B) developed at SIC. Variety B is the second self-pollinated generation of a commercially available Stevia variety, and was developed by selecting lines with high RebD and RebM content. In other words, the Stevia plants (Variety A) used in the examples correspond to the third self-pollinated generation of a commercially available Stevia variety.

[0059] The content of steviol glycosides in the dried leaves of Stevia plants (variety A) is shown in the table below. The content of steviol glycosides in the dried leaves was measured by liquid chromatography-mass spectrometry (LC-MS / MS).

[0060] [Purification and Reaction of Stevia-Derived Glycoside-Transferase] Glycoside-transferase was obtained from the leaves of the stevia plant (variety A) described above. The raw materials used and the procedure for purifying the glycoside-transferase are shown below. [Raw Materials Used] ・Stevia leaves used 14.7 g FW leaf (fresh weight leaves) (stevia plant (variety A)) ・Extraction buffer 100 mM Tris-Cl pH 8 0.25% PVP (polyvinylpyrrolidone) 1 x complete (registered trademark) EDTA-free (protease inhibitor cocktail) 0.1% b-ME (β-mercaptoethanol) 50 mM NaCl [Purification of Glycoside-Transferase] The stevia leaves were crushed in 200 ml of the above extraction buffer (4°C) using a juicer and filtered through a nylon mesh. The obtained filtrate was separated by centrifugation (12,000x g, 10 min, 4°C), and the supernatant was precipitated with ammonium sulfate (30-70% saturation). The obtained precipitate was suspended in 5 ml of extraction buffer and filtered through a 0.22 μm syringe filter. The obtained filtrate was then gel filtered using a Sephadex G75 under the following conditions (4 ml / fraction): Gel filtration conditions: Filtration rate: 1 ml / min Fraction: 4 ml Bed volume: 70 ml Solvent: Extraction buffer

[0061] Enzyme activity was measured by adding the following substrates 1 and 2 to each obtained fraction. Enzyme activity measurement study 1 Substrate 1: 1 mM substrate: RebD Substrate 2: 0.2 mM UDP-Glc ( １３ (Labeled with C) Reaction conditions: 30°C, 2 hours Scale: 100 μl After reacting under the above conditions, the reaction was stopped by mixing with 100 μl of acetonitrile, filtered, and then analyzed by LC-MS.

[0062] When fractions #4-7 of the enzyme gel filtration fraction were added to a substrate containing Rev. D, a reaction was observed in which Rev. D was converted to Rev. M. The Rev. M obtained in this way was １３ Because it contains glucose labeled with C, Table 2 contains "RebD +13 It was labeled "C-Glc". Fractions #8 and later contained endogenous rebaudioside M. Based on the above results, we investigated changes in the reaction conditions using fraction #4, and further re-examined the results by adding UDP-Xyl to substrate 2.

[0063] Enzyme activity measurement study 2 Substrate 1: 200 μM Substrate: Stevioside 50 μM Substrate: Reb. E Substrate 2: 0.5 mM UDP-Glc ( １３ (Labeled with 1C) and UDP-Xyl Reaction conditions: 30°C, 20 hours Scale: 50 μl After reacting under the above conditions, the reaction was stopped by mixing with 50 μl of acetonitrile, filtered, and analyzed by LC-MS. The analytical results will be described later.

[0064] [Structural Analysis of Novel Steviol Glycosides] The structural analysis of novel steviol glycoside 1E, detected after enzymatic treatment in Enzyme Activity Measurement Study 2, was performed using the following procedure: (i) High-performance liquid chromatography (HPLC) - high-resolution mass spectrometry (MS) and MS / MS, ion fragmentation up to three steps (MS) ３ (ii) Structural estimation by fragmentation analysis of fragmented (i) chemical synthesis of presumed steviol glycoside standards by chemical reaction, (iii) HPLC-high-resolution MS and MS of chemically synthesized standards ３ Structural confirmation by matching fragmentation retention time and fragmentation pattern.

[0065] Each of the above steps (i) to (iii) will be explained in detail below.

[0066] (i) High-performance liquid chromatography (HPLC) - high-resolution mass spectrometry (MS) and MS / MS, up to three-step ion fragmentation (MS) ３Structural estimation by fragmentation analysis of fragmented material: In the high-performance liquid chromatography-electrospray ionization precise mass spectrometry (HPLC-ESI-HRMS) instrumentation, the HPLC instrumentation consisted of a Prominence LC-20AD (Shimadzu Corporation) for the liquid delivery unit LC pump and an SM-C18 (4.6 × 250 mm) (Intact Corporation) for the separation column. For LC mobile phase delivery, 0.2% acetic acid-containing Milli-Q water was used as mobile phase A and methanol as mobile phase B. The binary gradient was kept constant at a mobile phase B concentration of 10% for 0-5 minutes, then the mobile phase B concentration was changed from 10% to 70% over 15 minutes, and then from 70% to 100% over another 5 minutes. Finally, the mobile phase B concentration was maintained at 100% for 5 minutes to terminate. The mobile phase flow rate was 0.4 mL / min, and 20 μL of diluted and filtered stevia leaf extract was injected. The mass spectrometry section used an Orbitrap Elite MS (Thermo Fisher Scientific) equipped with an ESI ion source. Mass spectrometry measurements were performed in negative ion measurement mode, with a m / z range of 150–2000 and a set resolution of 60,000. MS / MS measurements were performed in CID mode, where the target m / z was selected and fragmentation was achieved by collision with an inert gas. ３ The target ion was selected as the ion with the strongest intensity in the MS / MS spectrum. The irradiation required for fragmentation was performed at a collision energy of 35, which is the instrument's inherent reference energy.

[0067] To understand the fragmentation pattern of the novel steviol glycoside 1E, MS / MS and MS of structurally known standard rebaudiosides A, D, and M were performed. ３ Fragmentation pattern analysis was performed. As a result, in MS / MS of the novel steviol glycoside, the strongest ionic intensity was observed in the peak where all ester-bonded sugar chains at position 19 were removed. This result indicates the total molecular weight of the sugar chains ester-bonded to the carbon at position 19.

[0068] Figure 3 shows the mass chromatogram of the steviol glycoside produced when stevioside is used as substrate 1. An unidentified peak (Rt: 28.74 and Rt: 29.97) was detected at m / z 935.4 (corresponding to stevioside + 1 molecule Xyl). MS / MS and MS ３ Multi-step fragmentation (Figure 4) suggests that the structure of the compound with the Rt:28.74 peak is one in which xylose is β1-2 bonded to glucose at position 19 of stevioside. The structure of this compound was determined by comparing it with the chemically synthesized product described later.

[0069] Figure 5 shows the mass chromatogram of the steviol glycoside produced when Reb. E is used as substrate 1. An unidentified peak (Rt: 28.40) was detected at m / z 1097.4 (corresponding to Xyl of Reb. E + 1 molecule). MS / MS and MS ３ Multi-step fragmentation (Figure 6) suggests that the structure of the compound at this peak is one in which xylose is β1-3 linked to the glucose at position 13 of rebaudioside E.

[0070] (ii) Chemical synthesis of a presumed steviol glycoside standard (novel steviol glycoside 1S) by chemical reaction [Synthesis of novel steviol glycoside 1S] (1) Outline of the synthesis route As shown in Synthesis Scheme 1, in the synthesis of the novel steviol glycoside 1S(1), the framework of the target compound, the novel steviol glycoside 1S(1), was obtained by condensing intermediate (3) and the disaccharide hemiacetal (2) via the Mitsunobu reaction. In the synthesis of intermediate (3), the ester bond at the 19th position of steviol of the known natural product stevioside (4) was alkaline hydrolyzed, and then the hydroxyl group of the sugar chain was protected with an acetyl (Ac) group to obtain intermediate (3). The disaccharide hemiacetal (2) was synthesized by the method described in Patent Document 3. When the obtained intermediate (3) and the disaccharide hemiacetal (2) were condensed via the Mitsunobu reaction, the reaction proceeded with a good yield of 46% (β-isomer only) and complete β-selectivity. By deprotecting the protecting group of the obtained compound, the novel steviol glycoside 1S(1) was obtained. Next, each synthesis step will be described.

[0071] (2) Synthesis of intermediate (3) As shown in Synthesis Scheme 2, in the synthesis of intermediate (3), stevioside (4) (5 g, 6.22 mmol) was dissolved in a mixed solvent of ethanol (62 mL) and water (5 mL), 4 mol / L sodium hydroxide (15 mL) was added at room temperature, and the mixture was stirred at 75 °C for 24 hours. After confirming the completion of the reaction by TLC (chloroform / methanol / water = 5 / 4 / 0.1, Rf value = 0.9), the reaction mixture was neutralized with ion exchange resin (Dowex MAC-3 (H)) (pH 7), the resin was filtered off, and the mixture was concentrated under reduced pressure to obtain compound 5 (4 g).

[0072] Next, the obtained compound 5 (4 g) was dissolved in pyridine (62 mL), acetic anhydride (9 mL) was added at room temperature, and the mixture was stirred at room temperature for 18 hours. After confirming the completion of the reaction by TLC (ethyl acetate / hexane = 2 / 1, Rf value = 0.7), saturated aqueous sodium bicarbonate solution (40 mL) was added at 0°C, and the mixture was extracted three times with ethyl acetate. The organic layer was concentrated under reduced pressure, and the resulting syrup was subjected to silica gel column chromatography, and intermediate 3 (3.3 g, 57% (2 steps)) was obtained from the eluate (ethyl acetate / hexane = 1 / 1).

[0073] The NMR spectrum of the obtained intermediate compound 3 was obtained using an "AVANCE III HD 400 spectrometer" manufactured by Bruker. １ H-NMR and １３ 13C-NMR was measured. The results are shown in Figures 7 and 8. The obtained data is summarized below. The solvent and frequency used for the measurement are as follows. The same apparatus was used to measure the NMR spectrum for the other compounds described later. [Intermediate 3] 1H-NMR (CDCl3, 400 MHz) δ 0.81 (m, 2H), 0.92-1.07 (complex, 6H), 1.12 (m, 1H), 1.23 (m, 8H), 1.41-2.23 (complex, 49H), 3.61-3.80 (complex, 4H), 4.05-4.19 (complex, 7H), 4.59 (d, J = 7.6 Hz, 1H), 4.67 (d, J = 7.9 Hz, 1H), 4.85-4.99 (complex, 5H), 5.11-5.19 (complex, 4H); 13 C-NMR (CDCl3, 100 MHz) δ 14.3, 16.4, 19.1, 20.4, 20.6, 20.7×2, 20.8, 20.9, 21.0, 21.2, 22.1, 28.5, 38.3, 38.5, 39.7, 41.0, 41.3, 41.5, 43.8, 44.7, 47.4, 53.2, 56.6, 60.5, 62.7, 62.9, 69.0, 69.8, 71.4, 71.6, 71.8, 71.9, 73.0, 75.4, 88.0, 96.0, 100.3, 104.7, 151.4, 169.6, 170.0, 170.1, 170.5, 170.8, 170.9, 171.3, 179.6.

[0074] (3) Synthesis of disaccharide hemiacetal compound The disaccharide hemiacetal compound (2) was synthesized by the method described in Patent Document 3 (International Publication No. 2021 / 020516).

[0075] (4) Synthesis of novel steviol glycoside 1S As shown in Synthesis Scheme 3, in the synthesis of compound 6, the disaccharide hemiacetal (2) (2 g, 3.55 mmol) and intermediate (3) (2.2 g, 2.36 mmol) were dissolved in 1,4-dioxane (118 mL), tributylphosphine (1.8 mL, 7.08 mmol) and 1,1'-azobis(N,N'-dimethylformamide) (TMAD) (1.2 g, 7.08 mmol) were added at room temperature, and the mixture was stirred at 60 °C for 16 hours. After confirming the completion of the reaction by TLC (toluene / ethyl acetate = 1 / 1, Rf value = 0.4), the mixture was diluted with ethyl acetate, and the organic layer was washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, and dried over magnesium sulfate. After filtering off the magnesium sulfate, the syrup obtained by concentration under reduced pressure was subjected to silica gel column chromatography, and compound 6 (1.6 g, 46%) was obtained from the eluate (toluene / ethyl acetate = 3 / 2).

[0076] The NMR spectrum of compound 6 obtained was obtained using an "AVANCE III HD 400 spectrometer" manufactured by Bruker. １ H-NMR and １３ 13C-NMR was measured. The results are shown in Figures 9 and 10. The obtained data is summarized below. The solvent and frequency used for the measurement are as follows. The same apparatus was used to measure the NMR spectrum for the other compounds described later. [Compound 6] 1 H-NMR (CDCl3, 400 MHz) δ 0.70-1.17 (complex, 10H), 1.32-1.66 (complex, 6H), 1.69-2.28 (complex, 60H), 3.29 (t, 1H), 3.56-3.87 (complex, 5H), 4.00-4.28 (complex, 7H), 4.57 (d, J = 7.1 Hz, 1H), 4.61-4.88 (complex, 7H), 4.98-5.18 (complex, 7H), 5.56 (d, J = 7.8 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) δ 16.1, 19.3, 20.2×2, 20.3, 20.4×2, 20.5×3, 20.6×2, 20.7, 21.2, 21.3, 29.0, 36.6×2, 39.2, 40.2, 41.1, 42.3, 43.1, 43.8, 47.2, 53.4, 57.3, 60.9, 61.9, 62.0, 62.4, 68.1×2, 68.6, 68.8, 70.5, 71.0, 71.4, 71.5, 71.6, 71.7, 73.0, 74.7, 74.8, 85.9, 91.3, 96.0, 100.5, 100.9, 104.4, 125.2, 128.1, 128.9, 137.6, 152.6, 169.2, 169.3, 169.4, 169.6×2, 169.8×2, 169.9, 170.1, 174.5.

[0077] Compound (6) (1.5 g, 1.01 mmol) was dissolved in a mixed solvent of methanol (5 mL) and THF (5 mL), and sodium methoxide (0.5 M in methanol) (2 mL) was added at room temperature. The mixture was stirred at room temperature for 18 hours. After confirming the completion of the reaction by TLC (chloroform / methanol / water = 5 / 4 / 1, Rf value = 0.6), an ion exchange resin (Dowex MAC-3 (H)) was added to neutralize the mixture, and the syrup obtained by concentrated under reduced pressure was filtered through a gel filtration column (GE Healthcare, Sephadex LH-20, H ２ The mixture was subjected to (O) to obtain a novel steviol glycoside 1S (compound (1)) (500 mg, 53%).

[0078] The NMR spectrum of the obtained novel steviol glycoside 1S (compound (1)) was obtained using an "AVANCE III HD 400 spectrometer" manufactured by Bruker. １ H-NMR and １３ 13C-NMR was measured. The results are shown in Figures 11 and 12. The obtained data is summarized below. The solvent and frequency used for the measurement are as follows. The same apparatus was used to measure the NMR spectrum for other compounds described later. [Novel steviol glycoside 1S (compound (1))] 1H-NMR (D2O, 400 MHz) δ 0.81-1.20 (complex, 8H), 1.27 (m, 3H), 1.37-2.38 (complex, 17H), 3.24-3.96 (complex, 25H), 4.65 (d, J = 7.6 Hz, 1H), 4.69-4.79 (complex, 2H), 4.95 (s, 1H), 5.21 (s, 1H), 5.59 (d, J = 7.7 Hz, 1H); 13 C-NMR (D2O, 100 MHz) δ 15.8, 16.7, 19.1, 20.1, 21.3, 24.9, 28.5, 36.3, 36.6, 39.1, 40.2, 41.0, 41.6, 43.9, 44.3, 47.0, 53.4, 57.3, 60.6, 61.2, 65.3, 67.7, 69.2, 69.3, 69.9, 73.8, 74.2, 75.6, 76.2, 76.5, 79.9, 80.2, 87.0, 92.5, 102.9, 104.2, 104.9, 152.7, 177.5.

[0079] (iii) HPLC-High-resolution MS / MS and MS of chemically synthesized standards 3 Structural determination by matching fragmentation retention time and fragmentation pattern: Under the conditions described in (i), an Orbitrap Elite MS (Thermo Fisher Scientific) equipped with an HPLC-ESI ion source was used to perform HPLC-high-resolution MS / MS and MS. ３ A comparison was made between the reaction product using a stevia-derived enzyme via fragmentation (novel steviol glycoside 1E) and the chemically synthesized novel steviol glycoside 1S (β-form of compound 1). As a result, peaks at retention times of 28.76 minutes and 28.80 minutes were detected for both the reaction product using the stevia-derived enzyme and the chemically synthesized product, respectively (Figure 13). Furthermore, MS / MS and MS analysis of each were performed. ３ The fragmentation mass spectra (Figures 14 and 15) also matched. These results confirmed that the reaction product using the stevia-derived enzyme and the chemically synthesized novel steviol glycoside 1S (β-form of compound 1) have the same structure.

[0080] Sensory Evaluation of Novel Steviol Glycoside A (Compound 1) To evaluate the taste quality of various steviol glycosides, beverage samples were prepared by adding Rev. A, Rev. D, Rev. M, sugar, and novel steviol glycoside A (commonly referred to as Compound 1 or Novel Glycoside A) to pure water. Samples of Rev. A, Rev. D, Rev. M, and novel steviol glycoside A (Compound 1) were all prepared at a concentration of 300 ppm.

[0081] The obtained beverage samples were subjected to sensory evaluation using the following indices: sweetness onset, sweetness intensity, sweetness lingering, bitterness, bitterness lingering, and overall evaluation. Sensory evaluation was performed by spitting. Seven individuals trained in the sensory evaluation of sweeteners served as panelists and conducted the evaluation, with the following evaluation criteria. For each evaluation item, a 5 wt / wt% sugar aqueous solution was set to 0 points, and each steviol glycoside was scored in increments of 0.5 points. Higher values ​​indicate a faster sweetness onset, stronger sweetness intensity, shorter sweetness lingering, less bitterness, shorter bitterness lingering, and a higher overall evaluation. The results are shown in Figure 16 and Table 3. The scores shown in the figures and tables are the average values ​​of the scores from the seven panelists. Data obtained by standardizing the scores are shown in Figure 17 and Table 4. Based on the sensory evaluation results, it is inferred that the novel steviol glycoside A had a lower sweetness level compared to the other glycosides, and this mainly influenced the lower overall evaluation. Furthermore, the lingering sweetness of the novel steviol glycoside A was slightly less than that of the other glycosides. Therefore, it is expected that the novel steviol glycoside A (compound 1) can be used to improve the lingering sweetness.

Claims

1. Formula (1): (wherein, (i) R １ However, it represents Xyl(1-2)Glc1- and R ２ This represents Glc(1-2)Glc1-; or (ii)R １ However, Glc(1-2)Glc1- represents and R ２ A compound represented by Glc(1-2)[Xyl(1-3)]Glc1- (where Glc represents glucose and Xyl represents xylose), or a salt or hydrate thereof.

2. The following formula (2) or (3): The compound according to claim 1, or a salt or hydrate thereof.

3. The following formula (4) or (5): The compound according to claim 1 or 2, or a salt or hydrate thereof, represented by the formula.

4. A food or beverage comprising a compound according to any one of claims 1 to 3, or a salt or hydrate thereof.

5. The food or beverage according to claim 4, wherein the content of the compound or salt or hydrate described in any one of claims 1 to 3 is 1 to 600 ppm by mass.

6. A sweetener composition comprising a compound according to any one of claims 1 to 3, or a salt or hydrate thereof.

7. The sweetener composition according to claim 6, wherein the content of any one of claims 1 to 3, or a salt or hydrate thereof, is 1 to 99% by weight.

8. The sweetener composition according to claim 6 or 7, further comprising one or more steviol glycosides selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, dulcoside C, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside.

9. Food and beverages comprising the sweetener composition according to any one of claims 6 to 8.

10. A food or beverage according to any one of claims 4, 5, and 9, which is a beverage.

11. Use of the compound or a salt or hydrate described in any one of claims 1 to 3 as a sweetener.

12. A method for producing the compound according to any one of claims 1 to 3, comprising: (1) preparing leaves of Stevia rebaudiana; (2) crushing the leaves in a solvent to obtain a leaf crushed material-containing liquid; (3) precipitating and filtering the leaf crushed material-containing liquid to obtain a filtrate; (4) gel filtering the filtrate to obtain a fraction containing 10 to 100 kDa of protein; (5) adding a steviol glycoside and UDP-glucose and / or UDP-xylose as substrates to the fraction; and (6) reacting at 20 to 37°C for 30 minutes or more.

13. The method for producing the product according to claim 12, wherein the steviol glycoside that serves as the substrate comprises one or more steviol glycosides selected from the group consisting of stevioside, rebaudioside A, rebaudioside D, rebaudioside E, and rebaudioside M.

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