Method for producing rebaudioside D-containing crystallized material and rebaudioside D-containing crystallized material

Abstract

The present invention provides a method for producing a rebaudioside D-containing crystallized product, which allows for a high yield of rebaudioside D and for obtaining a rebaudioside D-containing crystallized product containing rebaudioside D at high purity, and also provides a rebaudioside D-containing crystallized product. [Solution] A method for producing a RebD-containing crystallized product, in which the total steviol glycoside content in a crude product derived from a stevia plant is 50 to 95% by mass and the crude product contains at least RebA and RebD, includes the steps of: preparing a crystallization solution by mixing the crude product with a solvent containing methanol or ethanol so that the degree of supersaturation of RebD at 10°C is 10 or more and the degree of supersaturation of RebA at 10°C is 18 or less; and cooling the crystallization solution while stirring to precipitate RebD.

Description

[Technical field]

[0001] The present invention relates to a method for producing a rebaudioside D-containing crystallized product, which has a high yield of rebaudioside D and is capable of obtaining a rebaudioside D-containing crystallized product containing rebaudioside D at high purity, and to a rebaudioside D-containing crystallized product. [Background technology]

[0002] The leaves of Stevia rebaudiana (Asteraceae) contain a secondary metabolite called steviol, a type of diterpenoid. Steviol glycoside is used in the food industry as a calorie-free sweetener because it is about 300 times sweeter than sugar. Obesity has become a serious social problem internationally, and the demand for calorie-free sweeteners is increasing day by day from the perspective of health promotion and medical cost reduction. Currently, artificially synthesized amino acid derivatives such as aspartame and acesulfame potassium are used as artificial sweeteners. However, naturally occurring calorie-free sweeteners such as steviol glycoside are expected to be safer and easier to gain public acceptance.

[0003] There have been several reports on the purification of steviol glycosides. For example, Patent Document 1 discloses a method for purifying rebaudioside D by dissolving an extract of Stevia rebaudiana Bertoni plant in an aqueous solution of an organic solvent and crystallizing it three or more times. Patent Document 2 discloses a method for obtaining a steviol glycoside composition by preparing a starting material containing at least one steviol glycoside of Stevia rebaudiana plant and an aqueous alcohol solution, dissolving the starting material in the aqueous alcohol solution, maintaining the solution at about 24 to 80°C for about 1 minute to about 240 hours, separating the solid and liquid, and drying the solution, in which the steviol glycoside composition has a solubility of at least 0.01% in water at 20°C. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2013-507914 A [Patent Document 2] JP 2019-518065 A Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above circumstances, a new method for producing RebD crystallized products is currently required. [Means for solving the problem]

[0006] One aspect of the present invention provides the following: [1] A method for producing a rebaudioside D-containing crystallized product using a crude product obtained by crude purification of an extract from a stevia plant, comprising the steps of: the total steviol glycoside content in the crude product is 50 to 95% by mass, and the crude product contains at least rebaudioside A and rebaudioside D; preparing a crystallization solution by mixing the crude product with a solvent containing methanol or ethanol so that the degree of supersaturation of rebaudioside D at 10° C. is 10 or more and the degree of supersaturation of rebaudioside A at 10° C. is 18 or less; A method for producing a rebaudioside D-containing crystallized product, comprising the step of cooling the crystallization solution with stirring to precipitate rebaudioside D. [2] The method of producing the above-mentioned [1], wherein the total steviol glycosides are rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, stevioside, rebaudioside F and rebaudioside M. [3] The method according to the above [1] or [2], wherein the rebaudioside A content in the crude product is 5 to 70% by mass and the rebaudioside D content is 2 to 70% by mass. [4] The method according to any one of the above [1] to [3], wherein the concentration of the methanol or ethanol in the solvent is 99.9 mass % or less. [5] The method according to any one of the above [1] to [4], wherein the solvent is maintained at a temperature of 40 to 80° C. when the roughly purified product is mixed. [6] The method according to any one of the above [1] to [5], wherein rebaudioside D is used as the seed crystal. [7] The method according to any one of the above [1] to [6], wherein the crystallization solution is cooled to a temperature of 35° C. or lower while stirring to precipitate rebaudioside D. [8] The method according to any one of the above [1] to [7], wherein the crystallization solution is cooled at a rate of 0.002 to 1.37° C. / min while stirring to precipitate rebaudioside D. [9] The method according to any one of the above [1] to [8], wherein the crystallization solution is cooled with stirring for 1 to 48 hours to precipitate rebaudioside D.

[10] The crude purified product is an extraction step of extracting dried leaves of the stevia plant with a solvent to obtain an extract; A solid-liquid separation step of subjecting the extract to solid-liquid separation to obtain a clear liquid; a flocculation step of adding a flocculant to the clarified liquid to flocculate the liquid and obtain a treated liquid; a resin purification step of treating the treatment liquid with a hydrophobic porous resin; and A concentration step in which the solution after resin purification is concentrated. The method for producing the composition according to any one of the above [1] to [9], wherein the composition is obtained by a method comprising the steps of:

[11] The method according to any one of the above [1] to

[10] , further comprising the steps of separating the precipitated rebaudioside D from a liquid phase and drying the separated rebaudioside D.

[12] Furthermore, separating and drying the precipitated rebaudioside D to obtain a primary crystallization product; a step of mixing the primary crystallized product with a solvent containing methanol or ethanol so that the degree of supersaturation of rebaudioside D at 10° C. is 10 or more and the degree of supersaturation of rebaudioside A at 10° C. is 18 or less, thereby preparing a solution of the primary crystallized product; cooling the primary crystallization solution with stirring to precipitate rebaudioside D; A process of separating and drying the precipitated Rebaudioside D. The method for producing a compound according to any one of the above [1] to

[10] ,

[13] The method according to any one of [1] to

[12] above, wherein the ratio of rebaudioside D to total steviol glycosides in the rebaudioside D-containing crystallization product is 35 to 95% by mass.

[14] The method of producing the rebaudioside D-containing crystallized product according to the above

[13] , wherein the ratio of rebaudioside A to total steviol glycosides in the rebaudioside D-containing crystallized product is 10 to 50% by mass.

[15] The method according to any one of [1] to

[14] above, wherein the ratio of crystallized rebaudioside D to the rebaudioside D contained in the crude product is 70 to 99 mass% when the number of crystallizations is one.

[16] A rebaudioside D-containing crystallized product produced by the production method according to any one of [1] to

[15] above.

[17] A food or beverage comprising the rebaudioside D-containing crystallized product described in

[16] above.

[18] The food or beverage described in

[17] above, which is a beverage. Effect of the Invention

[0007] In the present invention, a RebD-containing crystallized product can be obtained with high purity and high yield. [Brief description of the drawings]

[0008] [Figure 1] 1 shows solubility curves in ethanol solvents of various concentrations in Experimental Example 1. [Diagram 2] 1 shows the relationship between the RebD addition rate and the yield in Experimental Example 2. [Diagram 3] 1 shows the effect of the concentration of an ethanol solvent on the composition of the RebD crystallized product in Experimental Example 3. [Figure 4] 1 shows the effect of the concentration of an ethanol solvent on the yield (amount of crystals) in Experimental Example 3. [Diagram 5] 1 shows a temperature profile in Experimental Example 4. [Figure 6] 1 shows the effect of the cooling rate on the composition of the RebD crystallized product in Experimental Example 4. [Figure 7] 4 shows the effect of the cooling rate on the yield (amount of crystals) in Experimental Example 4. [Figure 8] 1 shows a temperature profile in Experimental Example 5. [Figure 9] 1 shows the effect of crystallization time on the composition of RebD crystallized products in Experimental Example 5. [Figure 10] 1 shows the effect of crystallization time on yield (amount of crystals) in Experimental Example 5. [Figure 11] 1 shows the effect of the concentration of an ethanol solvent on the composition of a RebD crystallized product when secondary crystallization is performed in Experimental Example 6. [Figure 12] 1 shows the effect of the concentration of an ethanol solvent on the yield (amount of crystals) when secondary crystallization is performed in Experimental Example 6. [Figure 13] 1 shows a temperature profile during secondary crystallization in Experimental Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will be described in detail below. The following embodiments are illustrative for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be implemented in various forms without departing from the gist of the present invention. All documents cited in this specification, as well as published publications, patent publications, and other patent documents, are incorporated herein by reference.

[0010] As used herein, "rebaudioside," "rebaudioside," "Reb," and "Reb." have the same meaning and all mean "rebaudioside." Similarly, as used herein, "dulcoside" means "dulcoside."

[0011] The present invention relates to a method for producing a rebaudioside D (RebD)-containing crystallized product by dissolving a crude product (hereinafter, sometimes simply referred to as a crude product) obtained by crudely purifying an extract from a stevia plant in a solvent of methanol or ethanol to precipitate RebD as crystals.

[0012] In the conventional method, RebA is first purified from the extract of the stevia plant, and RebD is purified from the remaining mother liquor. This is because most available stevia plant leaves contain more RebA than RebD. However, in the present invention, RebD is crystallized from the primary crystallization stage. Considering that RebA is contained at a higher concentration than RebD in the crude product of the extract from the stevia plant, it is surprising that a highly pure RebD-containing crystallized product can be obtained even when such a crude product is used as a raw material.

[0013] Since the content of RebA is overwhelmingly higher than that of RebD in the leaves of stevia plants, it is believed that RebD is entrapped during RebA crystallization in the conventional method. Therefore, when attempting to obtain a high-purity RebD preparation by purifying RebD from the mother liquor after RebA crystallization in the conventional method, the yield is inevitably low because RebD is entrapped during RebA crystallization. In the present invention, by carrying out purification targeting RebD, both of the above problems are solved, and both high purity and high yield of RebD are achieved.

[0014] <Rough purification> In the production method of the present invention, a crude product of a stevia plant is used. The crude product of a stevia plant may be a commercially available product, or may be obtained by purifying an extract from a stevia plant, particularly from the leaves of a stevia plant. In addition, the crude product may be a commercially available product or a product obtained by crude purification of an extract to which RebD has been further added. When the extract from the stevia plant is roughly purified to obtain a crude product, the extract from the stevia plant may be a commercially available product, or may be obtained by subjecting the stevia plant, particularly the leaves of the stevia plant, to an extraction treatment. (A) an extraction step in which dried leaves of a stevia plant are extracted with a solvent to obtain an extract; (B) a solid-liquid separation step of subjecting the extract to solid-liquid separation to obtain a clear liquid; (C) a flocculation step in which a flocculant is added to the clarified liquid to flocculate it and obtain a treated liquid; (D) a resin purification step of treating the treatment liquid with a hydrophobic porous resin; and (E) a concentration step of concentrating the solution after the resin purification treatment; Hereinafter, this method may be referred to as the crude purification method of the present invention. Each step will be described below.

[0015] In one embodiment of the crude purification method of the present invention, the moisture content of the dried leaves of the stevia plant is 1 to 11% by weight.

[0016] (A) Extract from the dried leaves of the stevia plant One embodiment of the crude purification method of the present invention includes extracting dried leaves of a stevia plant with an aqueous solvent to obtain an extract (extract solution). In this specification, the dried leaves of a stevia plant refer to fresh leaves of a stevia plant that have been dried to reduce their moisture content. The moisture content of the dried leaves of a stevia plant is preferably 1 to 10% by weight, more preferably 2 to 8% by weight, and particularly preferably 3 to 4% by weight. The dried leaves of a stevia plant are not particularly limited as long as they contain steviol glycosides, but preferably have a higher content of RebD or rebaudioside M (RebM) than the dried leaves of a natural stevia plant. Such dried leaves of a stevia plant can be obtained, for example, by a method described in International Publication No. WO 2019 / 074089.

[0017] The dried leaves of the stevia plant preferably used in the production method of the present invention contain 5.0 to 25 g of steviol glycoside per 100 g of dried leaves when the moisture content is 3 to 4% by weight. In other embodiments, the dried leaves may contain 6.0 to 24 g, 7.0 to 23 g, 8.0 to 22 g, 9.0 to 21 g, 10 to 20 g, or 11 to 19 g of steviol glycoside per 100 g of dried leaves when the moisture content is 3 to 4% by weight.

[0018] In another preferred embodiment, the dried leaves of the stevia plant used in the production method of the present invention contain 8 to 17 g of steviol glycosides per 100 g of the dried leaves when the moisture content is 3 to 4% by weight.

[0019] In this embodiment, the preferred dried leaves contain 0.5 g or more of RebD per 100 g of dried leaves when the moisture content is 3 to 4% by weight. In another embodiment, the preferred dried leaves contain 0.19 g or more of RebM per 100 g of dried leaves when the moisture content is 3 to 4% by weight. In another embodiment, the preferred dried leaves contain 3.0 g or more of rebaudioside A (RebA) per 100 g of dried leaves when the moisture content is 3 to 4% by weight. In another preferred embodiment, when the moisture content of the dried leaves is 3 to 4% by weight, the amount of RebD per 100 g of the dried leaves is 0.6 g or more, 0.7 g or more, 0.8 g or more, 1.0 g or more, 1.1 g or more, 1.2 g or more, 1.3 g or more, 1.4 g or more, 1.5 g or more, 1.6 g or more, 1.7 g or more, 1.8 g or more, 1.9 g or more, 2.0 g or more, 2.1 g or more, 2.2 g or more, 2.3 g or more, 2.4 g or more, It may be present in an amount of 2.5g or more, 2.6g or more, 2.7g or more, 2.8g or more, 2.9g or more, 3.0g or more, 3.1g or more, 3.2g or more, 3.3g or more, 3.4g or more, 3.5g or more, 3.6g or more, 3.7g or more, 3.8g or more, 3.9g or more, 4.0g or more, 4.1g or more or 4.2g or more, for example in an amount of 6.0g or less, 5.5g or less or 5.0g or less. Or 2.7~8.0g, 3.2~8.0g, 3.7~8.0g, 4.2~8.0g, 4.7~8.0g, 5.2~8.0g, 5.7~8.0g, 6.2~8.0g, 6.7~8.0g, 2.7~7.5g, 3.2~7.5g, 3.7~7.5g, 4.2~7.5g, 4.7~7.5g, It may be present in an amount of 5.2-7.5g, 5.7-7.5g, 6.2-7.5g, 6.7-7.5g, 2.7-7.0g, 3.2-7.0g, 3.7-7.0g, 4.2-7.0g, 4.7-7.0g, 5.2-7.0g, 5.7-7.0g, 6.2-7.0g or 6.7-7.0g.In other preferred embodiments, the dried leaves contain RebM in an amount of 0.20 g or more, 0.25 g or more, 0.30 g or more, 0.35 g or more, 0.40 g or more, 0.45 g or more, 0.50 g or more, 0.55 g or more, 0.60 g or more, 0.65 g or more, 0.70 g or more, 0.75 g or more, 0.80 g or more, 0.85 g or more, 0.90 g or more, 0.95 g or more, 1.00 g or more, 1.05 g or more, 1.10 g or more, 1.15 g or more, 1.20 g or more, 1.25 g or more, 1.30 g or more, 1.35 g or more, 1.40 g or more, or 1.45 g or more per 100 g of dried leaves, and may be present in an amount of 1.50 g or less, 1.30 g or less, or 1.20 g or less. In other preferred embodiments, when the moisture content of the dried leaves is 3 to 4% by weight, RebA is present in an amount of 3.0 g or more, 3.5 g or more, 4.0 g or more, 4.5 g or more, 5.0 g or more, 5.5 g or more, 6.0 g or more, 6.5 g or more, 7.0 g or more, 7.5 g or more, 8.0 g or more, 8.5 g or more, 9.0 g or more, 9.5 g or more, 10 g or more, 11 g or more, 12 g or more, 13 g or more, or 14 g or more per 100 g of dried leaves, and may be present in an amount of, for example, 17 g or less, 16 g or less, or 15 g or less.

[0020] In another preferred embodiment, the dried leaves may contain 0.5 to 0.9 g or 0.6 to 0.85 g of RebD per 100 g of dried leaves when the moisture content is 3 to 4% by weight. RebM may be contained in any of the amounts of 0.2 to 1.5 g, 0.2 to 1.3 g, 0.2 to 1.2 g, 0.2 to 1 g, 0.2 to 0.8 g, 0.2 to 0.7 g, 0.3 to 1.5 g, 0.3 to 1.3 g, 0.3 to 1.1 g, 0.3 to 0.9 g, and 0.3 to 0.7 g per 100 g of dried leaves having a moisture content of 3 to 4% by weight. RebA may be contained in any of the amounts of 3 to 17 g, 3 to 16 g, 3 to 15 g, 3 to 14 g, 3 to 13 g, 3 to 12 g, 5 to 17 g, 5 to 16 g, 5 to 15 g, 5 to 14 g, 5 to 13 g, 5 to 12 g, 6 to 17 g, 6 to 16 g, 6 to 15 g, 6 to 14 g, 6 to 13 g, 6 to 12 g, or 6 to 11 g per 100 g of dried leaves having a moisture content of 3 to 4% by weight.

[0021] Extraction of steviol glycosides from dried leaves can be carried out using a solvent such as water, alcohol, or a mixture thereof. Preferred extraction solvents include ion-exchanged water, pure water (e.g., Milli-Q water), and an aqueous ethanol solution. During extraction, the dried leaves may or may not be crushed. If crushing is required, crushing may be performed using a ball mill or the like. Alternatively, extraction may be performed using a kneader extractor (e.g., SKN-R100, manufactured by Sanyu Kiki Co., Ltd.).

[0022] Steviol glycosides can be extracted more efficiently by heating the aqueous solvent during extraction. The temperature during extraction may be, for example, 0 to 100°C, 20 to 90°C, 40 to 80°C, 25 to 80°C, 30 to 75°C, 35 to 70°C, 40 to 65°C, or 45 to 70°C, and is preferably 45 to 70°C.

[0023] Extraction may be performed not only once, but multiple times. By performing extraction multiple times, more steviol glycosides contained in the leaves can be extracted. From the viewpoint of efficiency, extraction is preferably performed about twice.

[0024] (B) Solid-liquid separation treatment In one embodiment of the crude purification method of the present invention, the obtained extract can be subjected to solid-liquid separation to obtain a clear liquid. The solid-liquid separation treatment is not particularly limited as long as the solid and liquid are sufficiently separated, but examples thereof include treatment with a centrifuge, treatment with a mesh, and treatment with a filter press. The solid-liquid separation treatment may use a plurality of means. For example, a first solid-liquid separation treatment may be followed by a second solid-liquid separation treatment to obtain a clear liquid.

[0025] In addition to the above-mentioned treatments, the solid-liquid separation treatment may also include a treatment using a microfiltration membrane.

[0026] (C) Coagulation treatment In one embodiment of the crude purification method of the present invention, a treated liquid can be obtained by adding a flocculant to the clear liquid obtained by the solid-liquid separation treatment. The flocculant is not particularly limited, and known inorganic flocculants or organic polymer flocculants can be used. In another embodiment of the present invention, the flocculant is one or more selected from aluminum sulfate, polyaluminum chloride, iron (III) chloride or its hydrate, synthetic polymer flocculants (such as polyacrylamide high polymers and partial hydrolysates of polyacrylamide), alginic acid, chitin, chitosan, and calcium hydroxide. It is sufficient that one or more of these flocculants are contained as the flocculant, and other flocculants may also be contained. Two or more types of flocculants may be used in combination, for example, a combination of calcium hydroxide and iron chloride, or a combination of calcium hydroxide, iron chloride, and chitosan.

[0027] The amount of the flocculant to be added is not particularly limited as long as it is an amount that causes flocculation for each flocculant, but for example, it can be added in an amount of 3.0 to 50% by weight based on the soluble solid content contained in the clear liquid. For example, calcium hydroxide can be added in an amount equivalent to 10 to 30% by weight of the solid content in the clear liquid, preferably 12 to 28% by weight, more preferably 14 to 25% by weight. In the case of iron (III) chloride hexahydrate, it can be added in an amount equivalent to 15 to 40% by weight of the solid content in the clear liquid, preferably 18 to 38% by weight, more preferably 20 to 35% by weight. In the case of a 0.5% (w / v) chitosan solution, it can be added in an amount equivalent to 3.0 to 10% by weight of the solid content in the clear liquid, preferably 4.0 to 8.0% by weight, more preferably 4.5 to 7.0% by weight.

[0028] The pH during the flocculation treatment is not particularly limited and can be appropriately selected so that flocculation is optimized depending on the type of flocculant. In one embodiment of the present invention, the pH of the clear liquid during the flocculation treatment may be 2.0 to 13, 3.0 to 13, 4.0 to 13, 5.0 to 13, or 6.0 to 13.

[0029] The temperature for the aggregation treatment is not particularly limited, and the treatment may be carried out at room temperature (about 25° C.) without heating or cooling.

[0030] After the flocculation treatment, the aggregates contained in the treatment liquid may be removed before the resin purification treatment described below. The removal of the aggregates can be performed by any method such as filtration.

[0031] In addition to the above methods, the method for removing aggregates also includes centrifugation.

[0032] (D) Resin purification treatment In one embodiment of the crude purification method of the present invention, the treatment liquid obtained by the coagulation treatment is treated with a hydrophobic porous resin. Steviol glycoside is amphiphilic having a hydrophilic group and a hydrophobic group in its molecular structure, and has a molecular weight of about 1,000. It is also known that it is stable at pH 2.5 to 9.0 and does not ionize even in acidic or alkaline conditions. On the other hand, the treatment liquid after the coagulation treatment contains a large amount of components other than steviol glycoside. Without being bound by theory, such components include components with molecular weights different from steviol glycoside, such as iron ions, and components that ionize, such as amino acids, and it is believed that these components can be removed by treatment with a hydrophobic porous resin.

[0033] Steviol glycosides having a hydrophobic steviol skeleton are hydrophobically bound to the synthetic resin and captured. On the other hand, highly hydrophilic impurities do not bind to the resin and are transferred to the through fraction and removed, so it is believed that the purity of steviol glycosides can be improved by loading the treated solution that has undergone the coagulation treatment into a column packed with the above-mentioned resin and then washing with water. In addition, the bond between the functional group of steviol glycoside and the synthetic resin is dissociated by a low-polarity solvent, which has the advantage that steviol glycosides can be finally recovered in high yield.

[0034] The porous resin used in one embodiment of the crude purification method of the present invention is not particularly limited as long as it has low affinity to water, but for example, a porous resin of one or more hydrophobic resins selected from a copolymer of styrene and divinylbenzene, polyethylene, polypropylene, polystyrene, poly(meth)acrylonitrile, polyamide and polycarbonate is preferred. In a preferred embodiment of the present invention, it is preferred that the copolymer of styrene and divinylbenzene is not subjected to an introduction treatment of an ion exchange group (i.e., does not have an ion exchange group). In general, when producing an ion exchange resin, styrene and divinylbenzene are copolymerized to form a three-dimensional network structure, and then an ion exchange group is introduced into the resin, but "no introduction treatment of an ion exchange group" means that such treatment has not been performed.

[0035] In one embodiment of the crude purification method of the present invention, the hydrophobic porous resin has a hydrophobic group, and the hydrophobic group includes one or more selected from an aryl group, an alkyl group, an alkylsilyl group, an ester group, and an epoxy group. If one or more hydrophobic groups selected from these are included, other hydrophobic groups may be further included. Examples of the aryl group include a phenyl group, a benzyl group, a tolyl group, and a xylyl group, and examples of the alkyl group include a C1-20 alkyl group, such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and an octadecyl group.

[0036] In a preferred embodiment of the crude purification method of the present invention, the most frequent pore radius of the hydrophobic porous resin is 10 to 200 Å. In a preferred embodiment, the most frequent pore radius is 10 to 150 Å, 15 to 100 Å, or 20 to 80 Å. It is believed that such pore characteristics allow steviol glycosides to be efficiently adsorbed into the pores and efficiently separated from other components.

[0037] In addition, the treatment liquid may be treated with an anion exchange resin before treatment with the hydrophobic porous resin. By performing a treatment in advance with an anion exchange resin, components that bind to the hydrophobic resin, such as dyes and catechins, can be effectively removed. Such an anion exchange resin is not particularly limited, but may be, for example, a basic anion exchange resin. As such a basic anion exchange resin, a weakly basic anion exchange resin having a primary or secondary amino group introduced as a functional group or a strongly basic anion exchange resin having a quaternary ammonium group (for example, a trimethylammonium group or a dimethylethanolammonium group) may be used.

[0038] (E) Concentration treatment The solution obtained from the resin purification treatment may be further concentrated to remove the aqueous solvent. Such a treatment is not particularly limited, but examples thereof include a method of evaporating the aqueous solvent by heating, or a method of removing the aqueous solvent by drying under reduced pressure.

[0039] After the concentration treatment, spray drying may be performed. The temperature conditions for spray drying are usually an inlet temperature of about 120 to 200°C and an outlet temperature of about 80 to 120°C.

[0040] (crude purified product) The crude product used in the production method of the present invention contains total steviol glycosides in an amount of 50 to 95% by mass relative to the total weight of the crude product, and preferably contains total steviol glycosides in any of the following amounts (mass%): 55 to 95, 60 to 95, 70 to 95, 80 to 95, 90 to 95, 50 to 90, 50 to 80, 50 to 70, 50 to 65, 50 to 60, 55 to 90, 55 to 80, 55 to 70, 55 to 60, 60 to 70, 60 to 80, 50 to 94, 55 to 94, or 60 to 94.

[0041] In the present specification, the total steviol glycosides preferably consist of seven types of steviol glycosides, namely, RebA, RebB, RebC, RebD, stevioside, RebF and RebM. In the present specification, the total steviol glycosides may be abbreviated as TSG.

[0042] The crude product contains at least RebA and RebD. Preferably, the content (mass%) of RebA in the crude product is any one of 5 to 70, 15 to 70, 20 to 70, 25 to 70, 35 to 70, 45 to 70, 55 to 70, 65 to 70, 5 to 65, 5 to 45, 5 to 25, 15 to 65, 25 to 45, 40 to 75, and 45 to 65, and the content (mass%) of RebD in the crude product is any one of 1 to 70, 2 to 70, 5 to 75, and 45 to 65. The values ​​are: 0, 15-70, 25-70, 35-70, 45-70, 55-70, 65-70, 1-65, 1-45, 1-25, 2-65, 2-45, 2-25, 5-65, 5-45, 5-25, 15-65, 25-45, 40-75, 45-65, 1-20, 1-15, 1-10, 1-8, 2-20, 2-15, 2-10, 2-8.

[0043] In another preferred embodiment, the RebA content in the crude purified product is 1 to 30 mass %, and the RebD content is any one of the above.

[0044] <Preparation of crystallization solution> Next, the solvent is placed in an arbitrary container, for example, a crystallizer equipped with an agitator, and the crude product is mixed into the solvent to prepare a crystallization solution. At this time, the degree of supersaturation of RebA (10°C) is set within a specific range while the degree of supersaturation of RebD (10°C) is set within a specific range so that RebA does not crystallize. Then, the crude product is mixed into the solvent in an amount that achieves such a degree of supersaturation. In addition, the degree of supersaturation (10°C) means the degree of supersaturation when 10°C is used as the reference temperature. There is no intention to require cooling to 10°C.

[0045] The degree of supersaturation is expressed by the following formula: σ = (CC * ) / C * In the formula, σ is the degree of supersaturation, C is the ratio (ppm) of the amount of the substance added to the solvent, and C * indicates the saturated soluble concentration (ppm). In this specification, C is expressed as "the ratio of the amount of the substance added to the solvent" rather than simply "the concentration of the substance" because it is assumed that the added substance does not completely dissolve and remains in a suspended state. In this specification, the ratio of the amount of the added substance to the solvent is used as the standard for the degree of supersaturation, regardless of whether the added substance is dissolved or not and the degree of dissolution. In the present invention, seed crystals may be used, but the amount of the seed crystals added is not included in the calculation of the degree of supersaturation.

[0046] In this specification, the saturated solubility is measured by the following method. That is, 100 ml of a solvent with a predetermined methanol or ethanol concentration (w / w) is placed in a container (volume 100 ml or 200 ml, preferably 200 ml), the liquid temperature is brought to 10°C in advance in a water bath, and then the mixture is stirred at 200 rpm, and an excess amount (enough to suspend) of the target substance is added. After 24 hours, the filtrate is collected using a 0.45 um membrane filter, and the amount of the target substance (RebA or RebD) contained in the filtrate is measured by LCMS. As the LCMS, preferably, LCMS8050 manufactured by Shimadzu Corporation is used.

[0047] The degree of supersaturation of RebD (10°C) is 10 or more, and is preferably 10 to 200, 10 to 180, 10 to 160, 10 to 140, 10 to 120, 10 to 100, 10 to 80, 10 to 60, 10 to 40, 10 to 20, 15 to 200, 35 to 200, 55 to 200, 75 to 200, 95 to 200, 115 to 200, 135 to 200, 155 to 200, 175 to 200, 15 to 180, 15 to 16 The range is 0, 15-140, 15-120, 15-100, 15-80, 15-60, 15-40, 15-20, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, 20-60, 20-40, 80-180, 80-160, 80-140, 80-120, 80-100, 100-180, 100-160, 100-140, or 100-120.

[0048] In another preferred embodiment, the degree of supersaturation of RebD (10° C.) is 10 to 350, 20 to 350, 50 to 350, 10 to 300, 20 to 300, 50 to 300, 10 to 205, or 20 to 205.

[0049] The degree of supersaturation of RebA (10°C) is 18 or less, including 0 or less. In one embodiment of the present invention, the degree of supersaturation of RebA (10°C) is more than 0 and 18 or less. More preferably, it is any one of 3 to 18, 3 to 13, 3 to 8, 5 to 18, 7 to 18, 9 to 18, 11 to 18, 13 to 18, 15 to 18, 17 to 18, 5 to 13, 7 to 11, 5 to 15, 7 to 15, 9 to 15, 11 to 15, and 13 to 15.

[0050] In another preferred embodiment, the degree of supersaturation of RebA (10° C.) is 16 or less, minus 1 to 16, or 0 to 16.

[0051] Under the condition that the degree of supersaturation of RebA is small and the degree of supersaturation of RebD is large, RebA is difficult to precipitate and RebD is easy to precipitate. When the degrees of supersaturation (10°C) of RebA and RebD are both within the above range and the degree of supersaturation of RebA is greater than 0, the ratio of the degree of supersaturation of RebD, σD, to the degree of supersaturation of RebA, σD / σA, is 4 or more, 5 or more, 4 to 40, 5 to 40, 7 to 40, 12 to 40, 17 to 40, 22 to 40, 27 to 40, 32 to 40, 37 to 40, 4 to 30, 5 to 30, 7 to 30, It is preferable that the range is any one of 12 to 30, 17 to 30, 22 to 30, 27 to 30, 4 to 35, 5 to 35, 8 to 35, 13 to 35, 18 to 35, 23 to 35, 28 to 35, 33 to 35, 8 to 25, 13 to 25, 18 to 25, 23 to 25, 10 to 40, 15 to 40, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 10 to 30, 15 to 30, 20 to 30, and 25 to 30.

[0052] In another preferred embodiment, σD / σA is any one of 40,000 to 1,000,000, 50,000 to 1,000,000, 40,000 to 750,000, and 50,000 to 750,000.

[0053] The solvent used to prepare the crystallization solution is a methanol or ethanol solvent. Preferably, it is an aqueous methanol solution or an aqueous ethanol solution. More preferably, it is an aqueous ethanol solution. Particularly preferably, an ethanol solvent is used without using a methanol solvent. "Using an ethanol solvent without using a methanol solvent" preferably means that the methanol concentration in the solvent is 1 mg / L or less, more preferably 0.5 mg / L or less, and particularly preferably 0.1 mg / L or less. The lower limit of the methanol concentration is 0 mg / L. Ethanol has a lower environmental impact than methanol, and also has a lower impact on the human body. When using methanol, it is mandatory to install ventilation equipment and ventilation capacity based on laws and regulations at the manufacturing site, and to conduct special health examinations for employees, but ethanol has a great advantage in industrial use in that there are no such obligations.

[0054] The amount of the crude product added is appropriately set so that the degree of supersaturation falls within the above-mentioned range, and is preferably 1 to 30 mass % per solvent, and more preferably any of the following amounts (mass %): 1 to 25, 1 to 20, 1 to 15, 1 to 10, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 30, 15 to 30, 20 to 30, 25 to 29, 10 to 25, or 15 to 20.

[0055] In another preferred embodiment, the amount of the crude product added is 1 to 28 mass %, 1 to 6 mass %, 6 to 25 mass %, or 6 to 28 mass % based on the solvent.

[0056] The amount of the crude product added is appropriately set so that the degree of supersaturation falls within the above-mentioned range. Preferably, the crude product is added in an amount of 0.15 to 3.50, 0.30 to 3.50, 0.45 to 3.50, 0.60 to 3.50, 0.75 to 3.50, 0.90 to 3.50, 1.05 to 3.50, 1.20 to 3.50, 1.50 to 3.50, 1.80 to 3.50, 2.10 to 3.50, 2.40 to 3.50, 2.70 to 3.50, 3.00 to 3.50, 0.15 to 3.00, 0.30 to 3.00, 0.45 to 3.0 The amount added is 0, 0.60 to 3.00, 0.75 to 3.00, 0.90 to 3.00, 1.05 to 3.00, 1.20 to 3.00, 1.50 to 3.00, 1.80 to 3.00, 2.10 to 3.00, 2.40 to 3.00, 2.70 to 3.00, 0.15 to 2.50, 0.15 to 2.00, 0.15 to 1.50, 0.15 to 1.00, 0.15 to 0.70, 0.15 to 0.40, 0.30 to 2.50, 0.45 to 2.00, 0.60 to 1.50, or 0.75 to 1.00 (mass%).

[0057] In another preferred embodiment, the crude product is added in an amount such that the amount of RebD per solvent is any of 0.20 to 3.50 and 0.25 to 3.50 (mass %).

[0058] The concentration (mass %) of the solvent containing methanol or ethanol is preferably 99.9 mass % or less, more preferably 92 mass % or less. In one embodiment of the present invention, the concentration is any one of 50 to 99, 60 to 99, 70 to 99, 80 to 99, 90 to 99, 50 to 89, 60 to 79, 50 to 92, 60 to 92, 70 to 92, 80 to 92, 90 to 92, 50 to 82, 60 to 72, 70 to 90, 80 to 90, and 70 to 80 mass %.

[0059] In another preferred embodiment, the concentration (mass %) of the solvent containing methanol or ethanol is either 73 mass % or more and less than 95 mass %, or 73 to 92 mass %.

[0060] The liquid temperature (°C) of the solvent when the crude product is mixed with the solvent is preferably maintained at any one of 40 to 80, 42 to 80, 44 to 80, 46 to 80, 48 to 80, 50 to 80, 52 to 80, 57 to 80, 62 to 80, 67 to 80, 72 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 42 to 75, 44 to 70, 46 to 65, 48 to 60, and 50 to 55.

[0061] It is preferable to mix seed crystals together with the crude product when mixing it with the solvent. As the seed crystals, it is preferable to use RebD. There is no particular limitation on the particle size of the seed crystals.

[0062] When seed crystals are added, the solution is preferably at a temperature such that the solution is supersaturated in order to avoid dissolution of the seed crystals.

[0063] The amount of seed crystals added may be appropriately determined depending on other conditions, but the yield tends to improve with a larger amount of addition. .900~10.000, 1.000~10.000, 2.000~10.000, 3.000~10.000, 4.000~10.000, 5.000~10.000, 6.000~10.000, 7.000~10.000, 8.000~10.000, 0.050~9.000, 0.050~8.000, 0.050~7.000, 0.050~6.000, 0.050~5.000, 0.050~4.000, 0.050~3.000, 0.050~2.000, 0.050~1.000, 0.050~0.700, 0.050~0.400, 0.050~0.100, 0.075~9.000, 0.100~8.000, 0.200~7.000, 0.300~6. It is preferable that the value be any one of the values ​​of 0.000, 0.400 to 5.000, 0.500 to 4.000, 0.600 to 3.000, 0.700 to 2.000, 0.800 to 1.000, 5.500 to 9.500, 6.500 to 9.500, 7.500 to 9.500, 8.500 to 9.500, 5.500 to 8.500, and 6.500 to 7.500. Cs=Ws / W th In the formula, Ws is the amount of seed crystals (unit: g), W th indicates expected yield (unit: g). W th =(CC * ) / 1,000,000 x volume of solvent In the formula, C is the ratio (ppm) of the amount of RebD added to the solvent, C * indicates the saturated solubility (unit: ppm). The amount of solvent is in ml. C does not include the amount of seed crystals.

[0064] <Precipitation> The crystallization solution is cooled with stirring in any crystallization apparatus, for example, a cylindrical rotary crystallizer, to precipitate RebD.

[0065] Since rapid cooling may result in a crystallized product with low RebD purity or may not improve the yield, it is preferable to cool gradually. A suitable cooling rate (°C / min) is 0.002°C / min or more, more preferably 0.002 to 1.37°C / min, and particularly preferably 0.002 to 1.37, 0.002 to 0.87, 0.002 to 0.37, 0.002 to 0.27, 0.002 to 0.17, 0.002 to 0.07, 0.002 to 0.04, 0.002 to 0.01, 0.00 2~0.007, 0.007~1.37, 0.017~1.37, 0.027~1.37, 0.057~1.37, 0.087~1.37, 0.387~1.37, 0.687~1.37, 0.987~1.37, 0.007~0.87, 0.017~0.37, 0.027~0.27, 0.057~0.17.

[0066] In another preferred embodiment, the cooling rate (° C. / min) is 0.02 to 0.2° C. / min.

[0067] The stirring and cooling time (unit: hour) may be appropriately set depending on other conditions such as whether or not seed crystals are used, but from the viewpoint of improving the yield, any of 1 to 48, 6 to 48, 11 to 48, 16 to 48, 21 to 48, 26 to 48, 31 to 48, 36 to 48, 41 to 48, 46 to 48, 1 to 43, 1 to 38, 1 to 33, 1 to 28, 1 to 23, 1 to 18, 1 to 13, 1 to 8, 1 to 3, 6 to 43, 11 to 38, 16 to 33, and 21 to 28 is preferable.

[0068] In another preferred embodiment, the stirring and cooling time (unit: hour) is 6 to 24 hours, 12 hours or more, or 12 to 24 hours.

[0069] The cooling temperature (°C) in the precipitation step is appropriately determined depending on other conditions such as whether or not seed crystals are used, but it is preferable to cool to any of 3 to 40, 6 to 40, 9 to 40, 12 to 40, 15 to 40, 18 to 40, 21 to 40, 24 to 40, 27 to 40, 30 to 40, 33 to 40, 20 to 40, 35°C or less, 3 to 35, 6 to 35, 9 to 35, 12 to 35, 15 to 35, 18 to 35, 21 to 35, 24 to 35, 27 to 35, 30 to 35, and 25 to 35.

[0070] In another preferred embodiment, the cooling temperature (°C) is 9 to 20°C, 9 to 17°C, 4 to 20°C, or 4 to 17°C.

[0071] Furthermore, the temperature (°C) at the start of cooling is preferably any one of 30 to 85, 40 to 85, 50 to 85, 60 to 85, 70 to 85, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 30 to 40, 40 to 80, 40 to 70, and 40 to 60.

[0072] The stirring speed (rpm) is preferably any one of 5 to 600, 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 5 to 20, 55 to 600, 105 to 600, 155 to 600, 205 to 600, 255 to 600, 305 to 600, 355 to 600, 405 to 600, 455 to 600, 505 to 600, 555 to 600, 50 to 600, 200 to 600, 350 to 600, 500 to 600, 100 to 450, 100 to 300, 100 to 150, 200 to 500, and 300 to 400.

[0073] <Post-process> The solid composition containing the precipitated RebD is separated from the liquid phase by centrifugation, filtration, or the like, and the separated RebD is dried. The separation means is not particularly limited as long as the solid and liquid are sufficiently separated, and examples include treatment with a centrifuge, treatment with a membrane filter, and treatment with a mesh. If necessary, after separation and before drying, the surface of the RebD crystals may be washed by spraying a small amount of the same solvent as used for crystallization onto the crystals.

[0074] <Other> The mother liquor after the solid-liquid separation treatment may be used for the crystallization of other components such as RebA and RebM.

[0075] In one aspect of the production method of the present invention, from the perspective of attaching more importance to the yield than the purity, the above-mentioned rough purification step, crystallization solution preparation step, precipitation step, and post-treatment step may be carried out once each, and the production may be considered complete (single crystallization). In another aspect, in order to further increase the purity of the RebD-containing crystallized product, the crystallization solution preparation step to the post-treatment step may be repeated multiple times (multiple crystallizations). From the perspective of the balance between the yield and the purity, double crystallization is preferred.

[0076] <Double crystallization> When performing double crystallization, the above-mentioned rough purification step to the precipitation step are carried out, the precipitated rebaudioside D is separated and dried to obtain a primary crystallized product. Then, instead of the above-mentioned rough purified product, the primary crystallized product is mixed in a solvent containing methanol or ethanol so that the supersaturation of rebaudioside D at 10 °C is 10 or more and the supersaturation of rebaudioside A at 10 °C is 18 or less to prepare a primary crystallized product solution. Instead of the above-mentioned crystallization solution, the primary crystallized product solution is cooled with stirring to precipitate RebD, and the precipitated RebD is separated and dried to obtain a RebD-containing crystallized product. The procedures and conditions in the double crystallization may be the same as the above-mentioned crystallization procedures and conditions. In that case, the "rough purified product" in the above description shall be read as the "primary crystallized product".

[0077] In double crystallization, the purity can be further increased by lowering the concentration of the solvent containing methanol or ethanol used for preparing the crystallization solution compared to the case of single crystallization. Also, in the second crystallization, the supersaturation of RebA may be set lower than that in the first crystallization.

[0078] In the present invention, crystallization may be carried out any number of times, but high-purity purification is possible even with a small number of crystallization times of 2 or less. Therefore, there are various advantages such as reduction of defects, shortening of cycle time, and energy saving.

[0079] <RebD-containing crystallized product> The RebD-containing crystallized product obtained by the method of the present invention (hereinafter, sometimes referred to as the RebD-containing crystallized product of the present invention) contains a large amount of RebD. Preferably, the ratio (mass%) of RebD to TSG in the RebD-containing crystallized product of the present invention is 35 to 99, 45 to 99, 55 to 99, 65 to 99, 75 to 99, 85 to 99, 35 to 89, 35 to 79, 35 to 69, 35 to 59, 35 to 95, 45 to 95, 55 to 95, 65 to 95, 75 to 95, 85 to 95, 40 to 99, 50 to 99, 60-99, 70-99, 80-99, 90-99, 40-99, 40-89, 40-79, 40-69, 40-59, 50-89, 60-79, 40-95, 50-95, 60-95, 70-95, 80-95, 90-95, 40-85, 40-75, 40-65, 40-55, 50-85, 60-75.

[0080] The RebD-containing crystallization product of the present invention has a low content of RebA. Preferably, under the condition that the ratio of RebD is within any of the above-mentioned numerical ranges, the ratio (mass%) of RebA to TSG is any of 5 to 80, 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 10 to 40, 20 to 40, 20 to 30, 30 to 40, 10 to 30, 10 to 20, 12 to 18, 5 to 30, 5 to 20, and 5 to 10.

[0081] In another preferred embodiment, the ratio (mass %) of RebA to TSG is 3.5-80, 3.5-35, 3.5-30, or 3.5-12.

[0082] In the method of the present invention, RebD can be crystallized in high yield. Preferably, the ratio (mass%) of crystallized RebD to RebD contained in the crude product when the number of crystallizations is one is any one of 70 to 99, 75 to 99, 80 to 99, 85 to 99, 90 to 99, 70 to 94, 70 to 89, 70 to 84, 70 to 79, 70 to 74, 75 to 94, 80 to 89, 50 to 99, 55 to 99, 60 to 99, 65 to 99, 50 to 94, 50 to 89, 50 to 84, 50 to 79, 50 to 74, 50 to 69, 55 to 94, 60 to 89, and 65 to 84.

[0083] In another preferred embodiment, the ratio (mass %) of crystallized RebD to the RebD contained in the crude product, when the number of crystallizations is considered to be one, is 39 to 85, more preferably 39 to 82.

[0084] In addition, the recovery rate of RebD from leaf extraction to after crystallization (sometimes referred to as total yield) is high, and is preferably 35 to 90, 45 to 90, 55 to 90, 65 to 90, or 75 to 90 mass%.

[0085] The ratio (mass%) of TSG in the RebD-containing crystallized product of the present invention is preferably 50 to 99, 60 to 99, 70 to 99, 80 to 99, 90 to 99, 50 to 90, 50 to 70, 50 to 60, 60 to 80, 60 to 90, 60 to 70, or 70 to 80. In another embodiment, the proportion (mass %) of TSG in the RebD-containing crystallized product of the present invention is preferably 50 or more, 60 or more, 70 or more, or 75 or more.

[0086] In one embodiment of the present invention, when an ethanol solvent is used during production and a methanol solvent is not used, the amount of methanol contained in the RebD-containing crystallized product is small, preferably less than 10 ppm, particularly preferably less than 5 ppm, and most preferably 2 ppm or less. The methanol content can be measured by headspace GCMS. The lower limit is preferably 1 ppm.

[0087] In another preferred embodiment, when an ethanol solvent is used but no methanol solvent is used during production, the lower limit of the amount of methanol contained in the RebD-containing crystallized product is 0 ppm.

[0088] In one embodiment of the present invention, when an ethanol solvent is used during production and a methanol solvent is not used, the ratio of methanol to ethanol (MeOH / EtOH) in the RebD-containing crystallized product is also small, preferably 0.00010 to 0.00080, more preferably 0.00010 to 0.00070, particularly preferably 0.00010 to 0.00050, and most preferably 0.00010 to 0.00030.

[0089] According to one aspect of the present invention, the RebD-containing crystallized product of the present invention may be used as a sweetener composition. Such a sweetener composition may contain, in addition to the RebD-containing crystallized product of the present invention, other sweeteners other than steviol glycosides. Examples of other sweeteners include natural sweeteners such as fructose, sugar, high fructose glucose liquid sugar, glucose, maltose, high fructose liquid sugar, sugar alcohol, oligosaccharides, honey, sugarcane juice (brown sugar syrup), starch syrup, Monk fruit powder, Monk fruit extract, licorice powder, licorice extract, Thaumatococcus daniellii seed powder, Thaumatococcus daniellii seed extract, and sucrose, and artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, and saccharin. Among them, from the viewpoints of providing a refreshing taste, easy drinking, a natural taste, and a moderate richness, it is preferable to use natural sweeteners, and in particular, fructose, glucose, maltose, sucrose, and sugar are preferably used. These sweetening components may be used alone or in combination.

[0090] Moreover, according to one aspect of the present invention, there are provided foods, beverages, fragrances and pharmaceuticals (also referred to herein as "foods, beverages and the like of the present invention", "flavors, and pharmaceuticals of the present invention", respectively) that contain the RebD-containing crystallized product of the present invention. The foods, beverages, fragrances and pharmaceuticals of the present invention are not particularly limited as long as they contain the RebD-containing crystallized product of the present invention. Here, "foods, beverages" means beverages and foods, and in a preferred embodiment, the foods, beverages and the like are beverages. Thus, in one embodiment, the present invention provides novel beverages or foods.

[0091] The amount (ppm by mass) of total steviol glycosides contained in the food, beverage, flavoring, and medicine of the present invention varies depending on the specific food, beverage, and medicine. In the case of beverages, the amount is preferably about 1 to 800 ppm by mass, for example, 20 to 750, 20 to 700, 20 to 650, 20 to 600, 20 to 550, 25 to 550, 30 to 550, 35 to 550, 40 to 550, 45 to 550, 50 to 550, 55 to 550, 2 0~540, 25~540, 30~540, 35~540, 40~540, 45~540, 50~540, 55~540, 20~530, 25~530, 30~530, 35~530, 40~530, 45~530, 50~530, 55~530, 20~520, 25~520, 30~520, 35~520, 40~520, 45~520, 50~520, 55~520, 20~510, 25 ~510, 30~510, 35~510, 40~510, 45~510, 50~510, 55~510, 20~505, 25~505, 30~505, 35~505, 40~505, 45~505, 50~505, 55~505, 20~500, 25~500, 30~500, 35~500, 40~500, 45~500, 50~500, 55~500, 20~495, 25~495, 30~ The content may be 495, 35-495, 40-495, 45-495, 50-495, 55-495, 20-490, 25-490, 30-490, 35-490, 40-490, 45-490, 50-490, 55-490, 100-400, 150-400, 200-400, 250-400, 300-400, 100-150, 100-200, 100-250, or 100-300. By setting the content within this range, there is an advantage that it is possible to impart a moderate sweetness. In this specification, "ppm" means "ppm by mass" unless otherwise specified.

[0092] The food, beverage, flavoring, and pharmaceutical of the present invention may further contain a sweetener other than steviol glycoside. Such sweeteners include natural sweeteners such as fructose, sugar, fructose glucose liquid sugar, glucose, maltose, sucrose, high fructose liquid sugar, sugar alcohol, oligosaccharide, honey, sugarcane juice (brown sugar syrup), starch syrup, Monk fruit powder, Monk fruit extract, licorice powder, licorice extract, Thaumatococcus daniellii seed powder, and Thaumatococcus daniellii seed extract, and artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, and saccharin. Among them, it is preferable to use natural sweeteners from the viewpoint of refreshingness, ease of drinking, natural taste, and imparting a moderate richness, and in particular, fructose, glucose, maltose, sucrose, and sugar are preferably used. Only one type of these sweet components may be used, or multiple types may be used.

[0093] Examples of foods of the present invention include, but are not limited to, confectionery, bread, flour, noodles, rice, processed agricultural and forestry foods, processed livestock products, processed marine products, milk and dairy products, oils and fats, processed oils and fats, seasonings, or other food ingredients.

[0094] Examples of beverages 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, functional beverages, etc. EXAMPLES

[0095] The present invention will be explained in more detail below with reference to experimental examples.

[0096] In the following experimental examples, unless otherwise specified, TSG refers to seven types: RebA, RebB, RebC, RebD, Stevioside, RebF, and RebM.

[0097] In order to find the optimal RebD crystallization conditions, we first carried out small-scale experiments in the laboratory to examine the relationship between various experimental conditions and the results. The small-scale experiments were conducted to obtain a rough relationship between the conditions and the results.

[0098] <Experimental Example 1> The degree of supersaturation has a significant effect on the phenomenon of nucleation and crystal growth. Therefore, we measured the solubility of RebA, the main component of Stevia leaves, and RebD, the target component, in each solvent. Specifically, 100 ml of a solvent with a given ethanol concentration (w / w) was placed in a 50 ml or 100 ml glass container (separable round-bottom flask, Tokyo Rikakikai Co., Ltd.), and the temperature was adjusted to a given level in advance in a water bath (PCC-7000, Tokyo Rikakikai Co., Ltd.), followed by stirring at 200 rpm (SPZ-100, Tokyo Rikakikai Co., Ltd.), and adding an excess amount (enough to suspend) of RebA (J-100, Morita Chemical Co., Ltd.) or RebD (Jining Renewal & Joint International, China). After 24 hours, the filtrate was collected using a 0.45 um membrane filter, and the amount of RebA or RebD contained in the filtrate was measured using an LCMS (Shimadzu, LCMS8050), and a solubility curve was created. The results are shown in Figure 1. According to Figure 1, the saturated solubility concentrations of RebA and RebD in a 90% ethanol solvent at 10°C were 8,250 ppm and 99.3 ppm, respectively.

[0099] <Experimental Example 2> In this experiment, we examined the effect of the ratio of RebD to solvent on the recovery rate of RebD.

[0100] An experiment was carried out using a single component system of RebD. Specifically, 99.9% by mass grade undenatured alcohol was used, and water was appropriately added to adjust the ethanol concentration to prepare a 50% by mass ethanol solvent. Using a separable round-bottom flask (manufactured by Tokyo Rikakikai Co., Ltd.), RebD was dissolved in a 50% by mass ethanol solvent under the conditions in the table below. At this time, an amount of RebD was added as seed crystals such that the seed crystal addition rate relative to the expected yield was 0.05. Then, the mixture was cooled under the following conditions. (Crystallization conditions) Initial Temp 45℃ End Temp 10℃ RebD Jining Renewal & Joint International Made of aluminum Agitation speed 100rpm [Table 1] The seed crystal addition rate is calculated by the following formula. C s =W s / W th In the formula, W s is the seed crystal weight (g), W th is the expected yield (g), C s indicates the seed crystal addition rate (unitless). W th =(CC * ) / 1,000,000 x volume of solvent In the formula, C is the ratio (ppm) of the amount of RebD added (excluding the amount of seed crystals) to the solvent, and C * indicates saturated solubility (ppm). The unit of solvent volume is ml. Then, crystals were obtained by performing solid-liquid separation. The filter used for solid-liquid separation was a 0.45 um membrane filter manufactured by Advantec. The RebD crystals were then washed with 99% ethanol and dried at 50°C.

[0101] The results are shown in Figure 2. It was found that in all ratios, the recovery rate (amount of RebD recovered relative to the amount of RebD used (including seed crystals)) was about 75%.

[0102] <Experimental Example 3> In this experiment, we investigated the effect of the concentration of ethanol used as a solvent on the TSG (total steviol glycosides) composition and yield of RebD crystallized products.

[0103] A crude product having the following composition (unit: mass%) was prepared. In this crude product, the RebD purity per TSG was 1.8 mass%. The TSG content in the crude product was 59.7 mass%. [Table 2]

[0104] The following amount of crude product was dissolved in the following concentration and amount of ethanol solvent (78°C). Next, the mixture was cooled under the following temperature profile and stirring conditions to crystallize RebD crystals. A separable round-bottom flask (manufactured by Tokyo Rikakikai Co., Ltd.) was used for the crystallization. In addition, 99.9% by mass grade undenatured alcohol was used as ethanol, and water was added appropriately to adjust the ethanol concentration. The ratio of RebD to the solvent was 0.25% by mass, the ratio of TSG to the solvent was 13.8% by mass, the degree of supersaturation (10°C) of RebA was 5.10 (when using 90.50% by mass ethanol solvent), the degree of supersaturation (10°C) of RebD was 24.37 (when using 90.50% by mass ethanol solvent), and σD / σA was 4.78 (when using 90.50% by mass ethanol solvent). The degree of supersaturation is calculated using the following formula: The amount of seed crystals is not included in the initial concentration (the ratio of the amount of substance added to the solvent). σ = (initial concentration - saturation solubility at 10°C) / (saturation solubility at 10°C) [Table 3]

[0105] Then, solid-liquid separation was performed to obtain RebD crystals. The filter used for solid-liquid separation was a 0.45 um membrane filter manufactured by Advantec. The RebD crystals were then washed with 99% by mass ethanol and dried at 50° C. to obtain a RebD crystallization product. The glycoside ratios of the obtained RebD crystals and the filtrate were analyzed by LCMS (Shimadzu Corporation, LCMS8050). The results are shown in Figures 3 and 4.

[0106] Under the conditions of this experiment, when ethanol with a concentration of 95% by mass was used, the viscosity of the slurry increased and solid-liquid separation was not possible, whereas when the ethanol concentration was about 70% by mass, no crystals were precipitated. Figure 4 shows that crystal precipitation was poor when the ethanol concentration was low, but Figure 3 suggests that the purity of RebD is not significantly affected by the ethanol concentration. It was found that there is an optimal ethanol solvent concentration for the ratio of TSG to the solvent used in this experiment (13.8%).

[0107] <Experimental Example 4> The cooling rate has a significant effect on the appearance of crystal nuclei and crystal growth. For example, when the cooling rate is fast, when crystals grow from the initial state of few nuclei, the supersaturation cannot be consumed completely, and nucleation often prevails. On the other hand, when the temperature drop is slow, crystal growth prevails. However, in that case, the crystallization time tends to be longer, depending on the consumption rate of supersaturation. In this experiment, the effect of cooling rate on the TSG composition and yield was investigated.

[0108] Specifically, primary crystallization was carried out in the same manner as in Experimental Example 3, except that the conditions in the table below were used instead of the conditions in Table 3, to obtain a RebD crystallization product. [Table 4]

[0109] The results are shown in Figures 6 and 7. Figures 6 and 7 show that when the temperature drop rate was faster, a precipitate with a lower RebD purity relative to TSG was obtained. The yield also decreased. Regarding the properties of the precipitate, when the temperature drop rate was faster, a viscous precipitate was obtained. On the other hand, when the temperature drop rate was slower, no viscosity was observed.

[0110] <Experimental Example 5> The crystallization time has a significant effect on crystal growth and also affects the time cycle of the entire purification process. As a result, the crystallization time also affects the cost and product characteristics (yield, purity, etc.). Therefore, in this experiment, we investigated the effect of crystallization time on TSG composition and yield.

[0111] Specifically, primary crystallization was carried out in the same manner as in Experimental Example 3, except that the conditions in the table below were used instead of the conditions in Table 3, to obtain a RebD crystallization product. [Table 5]

[0112] The results are shown in Figures 9 and 10. It was found that the crystallization time does not affect the glycoside composition, but does affect the yield. In the 6-hour crystallization, further crystals precipitated from the filtrate after crystallization. This suggested that a crystallization time of at least 12 hours was necessary. However, it is possible that the time could be further shortened by using or not using seed crystals.

[0113] <Experimental Example 6> In this experiment, in order to further increase the purity of RebD, the pros and cons of carrying out secondary crystallization using the primary crystallized product were examined. Specifically, the primary crystallization conditions were determined as shown in the table below, with reference to the results of Experimental Examples 2 to 5. Then, the primary crystallization was carried out in the same manner as in Experimental Example 3, except that the conditions in the table below were adopted instead of the conditions in Table 3, to obtain a primary crystallized product of RebD. [Table 6]

[0114] Thereafter, secondary crystallization was carried out in the same manner as in Experimental Example 3, except that the conditions in the following table were used instead of the conditions in Table 3, to obtain a secondary crystallized product of RebD. The ratio of RebD to the solvent was 0.56% by mass. The degree of supersaturation of RebA (10°C) was -0.66, and the degree of supersaturation of RebD (10°C) was 53.6. [Table 7]

[0115] The results are shown in Figures 11 and 12. As in the case of investigating the solvent for primary crystallization in Experimental Example 3, it was found that the yield also decreased with a decrease in the ethanol concentration in secondary crystallization.

[0116] In this experimental example, the final crystal composition and yield information was that the yield was approximately 43% by mass when the secondary crystallization was carried out under conditions that achieved a purity of approximately 90% (i.e., an ethanol concentration of 70% by mass in the secondary crystallization).

[0117] <Experimental Example 7> In this experimental example, the case where RebA, rather than RebD, was crystallized in the primary crystallization was examined.

[0118] An extraction operation was performed using dried leaves of the stevia plant, and the obtained extract was subjected to a solid-liquid separation operation. The content of each steviol glycoside in the dried leaves was as follows (unit: mass%). The TSG content in the dried leaves used in this experiment was 16.6 g per 100 g. [Table 8]

[0119] The clear liquid after solid-liquid separation was subjected to coagulation, resin purification, and evaporation concentration processes. As a result, a crude product having the composition (unit: mass%) shown in the table below was obtained. The proportion of RebD per TSG in the crude product was 3.8 mass%. [Table 9]

[0120] The following amount of the crude product was dissolved in the following concentration and amount of ethanol solvent (40° C.). The addition rate of RebD to the solvent was 0.22% by mass. Next, the mixture was cooled under the following temperature profile and stirring conditions to crystallize RebA. A separable round-bottom flask (manufactured by Tokyo Rikakikai Co., Ltd.) was used for the crystallization. Furthermore, 99.9% by mass grade undenatured alcohol was used as the ethanol. Then, solid-liquid separation was performed to obtain RebA crystals. The filter used for solid-liquid separation was a 0.45 um membrane filter manufactured by Advantec. The RebA crystals were then washed with 99% ethanol and dried at 50°C. [Table 10]

[0121] The primary crystallized product was analyzed using a Shimadzu LCMS8050. Table 11 below shows the ratio (unit: mass%) of each steviol glycoside to TSG in the primary crystallized product. Table 12 shows the yield information (unit: mass%). The RebA purity relative to TSG was 98.6%, and the RebA yield was 41.8%. The RebA crystals were contaminated with about 11.5% by mass of the RebD yield. [Table 11] [Table 12]

[0122] Next, RebD crystallization was attempted using RebD seed crystals from the mother liquor of the primary crystallization. As shown in the table below, the purity of RebD relative to TSG was 94% by mass or more. [Table 13]

[0123] <Experimental Example 8> In this experiment, RebD crystallization was performed using the optimal conditions obtained from small-scale experiments conducted in the laboratory.

[0124] An extraction operation was performed using dried leaves of the stevia plant, and the obtained extract was subjected to a solid-liquid separation operation. The content of each steviol glycoside in the dried leaves was as follows. The TSG content in the dried leaves used in this experiment was 8.7 g per 100 g. [Table 14]

[0125] The clear liquid after solid-liquid separation was subjected to coagulation, resin purification, and evaporation concentration processes. As a result, a crude product having the composition (unit: mass%) shown in the table below was obtained. In the crude product, the ratio of RebD per TSG was 4.6 mass%. The ratio of TSG in the crude product was 54.0 mass%. [Table 15]

[0126] The following amount of crude product (shown as sample in the table) was dissolved in the following concentration and amount of ethanol solvent (40° C.). The ratio of TSG to the solvent was 12.5% ​​by mass, the ratio of RebD to the solvent was 0.58% by mass, the degree of supersaturation of RebA (10° C.) was 9.98, and the degree of supersaturation of RebD (10° C.) was 57.64. The mixture was then cooled and crystallized under the following temperature profile and stirring conditions. A separable round-bottom flask (Tokyo Rikakikai Co., Ltd.) was used for the crystallization. As the ethanol, undenatured alcohol of 99.9% by mass grade was used, and water was appropriately added thereto to adjust the ethanol concentration. Then, crystals were obtained by performing solid-liquid separation. The filter used for solid-liquid separation was a 0.45 um membrane filter manufactured by Advantec. The crystals were then washed with 99% by mass ethanol and dried at 50°C. [Table 16]

[0127] As a result of the primary crystallization, 343 mg of crystals were obtained with a TSG purity (ratio of TSG per crystallized product) of 76.2% and a RebD purity (ratio of RebD per TSG) of 70.5%. The yield (unit step yield) was 39.5%.

[0128] <Experimental Example 9> An extraction operation was performed using dried leaves of the stevia plant, and the obtained extract was subjected to a solid-liquid separation operation. In the dried leaves, the ratio (mass%) of each steviol glycoside per TSG was as follows. The TSG content in the dried leaves used in this experiment was 11.8 g per 100 g. [Table 17]

[0129] The clear liquid after solid-liquid separation was subjected to flocculation, resin purification, and evaporation concentration steps. The obtained steviol glycoside composition was spray-dried to obtain a crude product. The composition of the crude product after spray drying is as follows. [Table 18]

[0130] Undenatured alcohol 99.9% by weight was used, and water was added appropriately to adjust the following concentration and amount of ethanol. Ethanol solvent was placed in a crystallization vessel (volume: 1 liter) equipped with a stirrer and heated. When the temperature of the ethanol solvent reached about 55°C, the crude product was added to the vessel. The temperature was lowered while stirring at about 350 rpm. At this time, the mixture was cooled evenly over a cooling time (i.e., 11 hours) - 1 hour (i.e., 10 hours). After the crystallization, solid-liquid separation was performed using a 0.45 um membrane filter manufactured by Advantec. The obtained crystals were dried at 60-70°C. [Table 19]

[0131] The unit step yield (the ratio of crystallized RebD to the RebD contained in the crude product before crystallization) was 80.0% by mass. The TSG purity (the ratio of TSG to the crystallized product) was 63.2% by mass, and the RebD purity (the ratio of RebD to TSG) was 35.8% by mass.

[0132] <Experimental Example 10> This experiment investigated the crystallization of RebD when a large amount of RebA was present in the ethanol solvent, i.e., when the degree of supersaturation of RebA was set high.

[0133] 1.Extraction / solid-liquid separation Ion-exchanged water of 15 times the amount (mass) of dried stevia leaves (water content: 3-4% by weight) was heated to 60°C ± 5°C, and the dried stevia leaves were immersed in the water. Then, extraction was performed for 60 minutes in a kneader extractor (SKN-R100, manufactured by Sanyu Kiki Co., Ltd.) while stirring with a stirring rod at 8 rpm. Next, the mixture was filtered through 18 mesh and 140 mesh meshes, cooled in a heat exchanger using cold water, and the filtrate was subjected to solid-liquid separation in a disk-type centrifuge (9150 rpm (11601G), 24 L / min) to obtain a primary extract. Meanwhile, the filtered leaves were extracted again under the same conditions, and a transparent secondary extract was obtained by solid-liquid separation, which was added to the primary extract to obtain a clear liquid.

[0134] The ratio of each steviol glycoside per TSG in the dried leaves was measured by liquid chromatography mass spectrometry (LC / MS / MS) (Shimadzu Corporation, LCMS8050). The results are shown in the table below (unit: mass%). [Table 20]

[0135] 2.Agglutination Ca(OH) equivalent to 16.16% by mass of the soluble solids in the clarified liquid 2 (calculated from Brix (soluble solids concentration)) was added to the clear liquid, and the resulting mixture was stirred for 15 minutes. Then, FeCl was added in an amount equivalent to 28.28 mass% of the soluble solids in the clear liquid. 3 6H 2 O was added, the mixture was stirred for 30 minutes, the pH was adjusted to 7 with citric acid, and then a volume (mL) of 0.5% (w / v) chitosan solution equivalent to 5.63 times the soluble solids (g) in the clear liquid was added. The mixture was stirred vigorously for 3 minutes, weakly for 2 minutes, and then allowed to stand for 10 minutes. The electrically neutral coagulated precipitate was then removed by centrifugation. As a result, a clear treated liquid was obtained.

[0136] 3. Resin Purification Resin purification was carried out by using (i) an anion exchange resin and (ii) a hydrophobic porous resin (one in which no ion exchange groups had been introduced). (i) Purification using an anion exchange resin A column was filled with a highly porous basic anion exchange resin (manufactured by Mitsubishi Chemical Corporation), and the treated solution after flocculation and separation was poured into the column for purification. After the treated solution after flocculation and separation was poured into the column, it was pushed out with ion exchange water in an amount twice the volume of the column, and a solution containing the purified steviol glycoside composition was recovered. This purification removed black impurities and colored components from the treated solution.

[0137] (ii) Purification using hydrophobic porous resin A column was filled with a hydrophobic porous resin (manufactured by Mitsubishi Chemical Corporation), and the sample purified using the anion exchange resin (i) was added to the column for purification. The hydrophobic porous resin used was a copolymer of styrene and divinylbenzene, which had no ion exchange groups and had a most frequent pore radius of 45 Å. After the solution purified in (i) above was added to the column, the column was washed with a 0.01M aqueous citric acid solution in an amount three times the volume of the column and a 0.01M aqueous sodium hydroxide solution in an amount three times the volume of the column. The steviol glycoside composition was then eluted with a 60% aqueous ethanol solution in an amount four times the volume of the column and collected.

[0138] 4. Evaporation and concentration The solution was evaporated and concentrated to remove ethanol using a centrifugal thin-film vacuum evaporator Evapor (manufactured by Okawara Manufacturing Co., Ltd.) Water remained even after the evaporation and concentration process, and the composition was in a liquid state.

[0139] The resulting steviol glycoside composition was spray-dried to obtain a crude product. The composition of the crude product after spray-drying (unit: mass %) is as follows: [Table 21]

[0140] Except for using the conditions in the table below, crystallization was carried out in the same manner as in Experimental Example 9. The results are shown in Table 31 below. [Table 22]

[0141] <Experimental Example 11> Extraction and solid-liquid separation were performed in the same manner as in Experimental Example 10. The ratio (mass%) of each steviol glycoside per TSG in the dried leaves was measured in the same manner as in Experimental Example 10, and the results are shown in the table below. [Table 23]

[0142] The flocculation, resin purification, and evaporation concentration steps were carried out in the same manner as in Experimental Example 10. The obtained steviol glycoside composition was spray-dried to obtain a crude product. The composition (mass%) of the crude product after spray drying is as follows: [Table 24]

[0143] Primary crystallization was carried out in the same manner as in Experimental Example 9, except that the conditions in the table below were used. [Table 25]

[0144] The secondary crystallization was carried out in the same manner as the primary crystallization, except that the conditions in the table below were used. The results are shown in Tables 31 to 33 below. [Table 26]

[0145] <Experimental Example 12> Extraction and solid-liquid separation were performed in the same manner as in Experimental Example 10. The ratio (mass%) of each steviol glycoside per TSG in the dried leaves was measured in the same manner as in Experimental Example 10, and the results are shown in the table below. [Table 27]

[0146] The flocculation, resin purification, and evaporation concentration steps were carried out in the same manner as in Experimental Example 10. The obtained steviol glycoside composition was spray-dried to obtain a crude product. The composition (mass%) of the crude product after spray drying is as follows: [Table 28]

[0147] Except for using the conditions in the table below, crystallization was carried out in the same manner as in Experimental Example 9. The degree of supersaturation (10° C.) of RebA was 8.2, and the degree of supersaturation (10° C.) of RebD was 160.1. [Table 29]

[0148] The secondary crystallization was carried out in the same manner as the primary crystallization except that the conditions in the table below were adopted. The results are shown in Tables 32 and 33. [Table 30]

[0149] The purity, degree of supersaturation, and unit step yield of RebA and unit step yield of RebD of the various components in the crude products used in Experimental Examples 10 to 12 are shown in the table below. The purity of RebA and the purity of RebD were measured using an LCMS8050 (Shimadzu Corporation) and expressed as a ratio (w / w) to TSG. The formula for calculating the degree of supersaturation is as described above. In calculating the degree of supersaturation, the saturated solubilities of RebA and RebD (90 mass% EtOH, 10°C) were as follows: RebA=8270.5ppm RebD=99.33ppm The RebA unit step yield indicates the ratio of the amount of RebA contained in the crystallized product to the amount of RebA in the raw material used in the crystallization. The same applies to the RebD unit step yield. [Table 31] [Table 32] [Table 33] Although it depends on the degree of supersaturation of RebD, it is estimated that the precipitation of RebA becomes intense at a RebA supersaturation of 15 to 18. In addition, in the above table, some purities exceeding 100% are thought to be due to weight increase caused by residual solvent or analytical errors.

[0150] <Experimental Example 13> This experiment was conducted to examine whether the composition of the RebD crystallized product would change when ethanol was used as the crystallization solvent compared to when methanol was used.

[0151] Two types of RebD crystallization products were obtained. Both are believed to have been crystallized using methanol solvent. Sample No. 1 was a RebD crystallization product manufactured by GLG, and Sample No. 2 was a RebD crystallization product manufactured by JNRJ.

[0152] The secondary crystallized product of RebD obtained in Experimental Example 11 was designated as Sample No. 3.

[0153] Extraction and solid-liquid separation were performed in the same manner as in Experimental Example 10. The ratio (mass%) of each steviol glycoside per TSG in the dried leaves was measured in the same manner as in Experimental Example 10, and the results are shown in the table below. [Table 34] The flocculation, resin purification, and evaporation concentration steps were carried out in the same manner as in Experimental Example 10. The obtained steviol glycoside composition was spray-dried to obtain a crude product. The composition (mass%) of the crude product after spray drying is as follows: [Table 35] Except for using the conditions in the table below, primary crystallization was carried out in the same manner as in Experimental Example 9. The degree of supersaturation of RebA (10°C) was 16.1, and the degree of supersaturation of RebD (10°C) was 160.2. [Table 36] The secondary crystallization was carried out in the same manner as the primary crystallization, except that the conditions in the table below were used. [Table 37] The obtained secondary crystallized product of RebD was designated as Sample No. 4. [Table 38]

[0154] The secondary crystallized product of RebD obtained in Experimental Example 12 was designated as Sample No. 5.

[0155] Sample No. 4 was further dried at 60 to 70° C. for 3 days to obtain Sample No. 6.

[0156] Sample No. 5 was further dried at 60 to 70° C. for 3 days to obtain Sample No. 7.

[0157] Each sample was dissolved in dimethylformamide and the methanol and ethanol contents were measured using HS (headspace)-GC / MS. The results are shown in the table below. [Table 39]

[0158] <Experimental Example 14> 1.Extraction / solid-liquid separation Water 15 times the amount (mass) of dried stevia leaves (moisture content: 9-11% by weight) was heated to 60-65°C, and dried stevia leaves were immersed in the water. Then, extraction was performed for 60 minutes in a stirring tank (volume: 400 L) while stirring at 75 rpm using stirring blades (radius 27 cm x 2 pieces x 2 stages). Next, the mixture of dried stevia leaves and water was filtered through a 100-mesh nylon mesh set in a Buchner funnel with a diameter of 95 cm, diatomaceous earth (Celite 503) was added, filtered with a filter press, and filtered with a microfiltration membrane (pore size: 10 μm) to separate the solid and liquid to obtain a primary extract. Meanwhile, the filtered leaves were extracted again under the same conditions, and a transparent secondary extract was obtained by solid-liquid separation, which was added to the primary extract to obtain a clear liquid.

[0159] 2.Agglutination Ca(OH) equivalent to 16.16% by mass of the soluble solids in the clarified liquid 2(calculated from Brix (soluble solids concentration)) was added to the clear liquid, and the resulting mixture was stirred for 15 minutes. Then, FeCl was added in an amount equivalent to 28.28 mass% of the soluble solids in the clear liquid. 3 6H 2 O was added, the mixture was stirred for 30 minutes, the pH was adjusted to 7 with citric acid, and then a volume (mL) of 0.5% (w / v) chitosan solution equivalent to 5.63 times the soluble solids (g) in the clear liquid was added. The mixture was stirred vigorously for 3 minutes, weakly for 2 minutes, and then allowed to stand for 10 minutes. The electrically neutral coagulated precipitate was then removed by centrifugation. As a result, a clear treated liquid was obtained.

[0160] 3. Resin Purification Resin purification was carried out by using (i) an anion exchange resin and (ii) a hydrophobic porous resin (one in which no ion exchange groups had been introduced). (i) Purification using an anion exchange resin A column was filled with a highly porous basic anion exchange resin (manufactured by Mitsubishi Chemical Corporation), and the treated solution after flocculation and separation was poured into the column for purification. After the treated solution after flocculation and separation was poured into the column, it was pushed out with ion exchange water in an amount twice the volume of the column, and a solution containing the purified steviol glycoside composition was recovered. This purification removed black impurities and colored components from the treated solution.

[0161] (ii) Purification using hydrophobic porous resin A column was filled with a hydrophobic porous resin (manufactured by Mitsubishi Chemical Corporation), and the sample purified using the anion exchange resin (i) was added to the column for purification. The hydrophobic porous resin used was a copolymer of styrene and divinylbenzene, which had no ion exchange groups and had a most frequent pore radius of 45 Å. After the solution purified in (i) above was added to the column, the column was washed with a 0.01M aqueous citric acid solution in an amount three times the volume of the column and a 0.01M aqueous sodium hydroxide solution in an amount three times the volume of the column. The steviol glycoside composition was then eluted with a 60% aqueous ethanol solution in an amount six times the volume of the column and collected.

[0162] 4. Evaporation and concentration The solution was evaporated and concentrated using a centrifugal thin-film vacuum evaporator (Okawahara Manufacturing Co., Ltd.) to remove ethanol. Next, the solution was concentrated in two stages using an evaporator to remove ethanol. Water remained even after the evaporation and concentration process, and the composition was in a liquid state.

[0163] The resulting steviol glycoside composition was spray-dried to obtain a crude product. The composition of the crude product after spray-drying (unit: mass %) is as follows: [Table 40]

[0164] Undenatured alcohol 99.9% by weight was used, and water was added appropriately to adjust the following concentration and amount of ethanol. Ethanol solvent was placed in a separable flask and heated. When the temperature of the ethanol solvent reached about 71°C, the crude product and seed crystals were added to the vessel. The degree of supersaturation of RebD (10°C) was 162.854, and the degree of supersaturation of RebA (10°C) was 0.793. The temperature was reduced while stirring. After the crystallization, solid-liquid separation was performed using a 0.45 um membrane filter manufactured by Advantec. The obtained crystals were dried at 60-70°C. The seed crystal addition rate (Cs) was 0.08. [Table 41]

[0165] The ratio (mass%) of each steviol glycoside per TSG (here, RebA, RebB, RebC, RebD, stevioside, RebF, RebM, RebN, dulcoside A, RebI, RebG, rubusoside, steviobioside, RebE) in the primary crystallization product was as shown in the table below. The unit process yield of RebD up to the primary crystallization was 86.39 mass%. The ratio of TSG in the primary crystallization product was 97.4 mass%. [Table 42]

[0166] The secondary crystallization was carried out in the same manner as the primary crystallization, except that the conditions in the table below were adopted. The details of the temperature profile are shown in Figure 13. The degree of supersaturation of RebD (10°C) was 342.254, and the degree of supersaturation of RebA (10°C) was -0.47. [Table 43]

[0167] The ratio of each steviol glycoside per TSG in the secondary crystallization product was as shown in the table below. The unit process yield of RebD up to the secondary crystallization was 98.9% by mass. The ratio of TSG in the secondary crystallization product was 95.9% by mass. [Table 44]

[0168] <Experimental Example 15> Extraction and solid-liquid separation were performed in the same manner as in Experimental Example 10. The ratio (mass%) of each steviol glycoside per TSG in the dried leaves was measured in the same manner as in Experimental Example 10, and the results are shown in the table below. [Table 45]

[0169] The flocculation, resin purification, and evaporation concentration steps were carried out in the same manner as in Experimental Example 10. The obtained steviol glycoside composition was spray-dried to obtain a crude product. The ratio (mass%) of each steviol glycoside per TSG (here, eight types: RebA, RebB, RebC, RebD, stevioside, RebF, RebM, and RebN) in the crude product after spray drying is as follows. The ratio of TSG per crude product was 95.75%. [Table 46]

[0170] Except for using the conditions in the table below, crystallization was performed in the same manner as in Experimental Example 9. The degree of supersaturation of RebA (10°C) was 5.7, and the degree of supersaturation of RebD (10°C) was 160.1. [Table 47]

[0171] The secondary crystallization was carried out in the same manner as the primary crystallization, except that the conditions in the table below were used. [Table 48]

[0172] The purity, degree of supersaturation, and unit step yield of RebA and unit step yield of RebD of each component were calculated in the same manner as in Experimental Examples 10 to 12. The results are shown in the table below. Note that in the table below, some purity values ​​exceed 100%, but this is thought to be due to weight increase caused by residual solvent or analytical error. [Table 49] [Table 50]

[0173] <Experimental Example 16> Extraction and solid-liquid separation were performed in the same manner as in Experimental Example 10. The ratio (mass%) of each steviol glycoside per TSG in the dried leaves was measured in the same manner as in Experimental Example 10, and the results are shown in the table below. [Table 51]

[0174] The flocculation, resin purification, and evaporation concentration steps were carried out in the same manner as in Experimental Example 10. The obtained steviol glycoside composition was spray-dried to obtain a crude product. The composition (mass%) of the crude product after spray drying is as follows: [Table 52]

[0175] Primary crystallization was carried out in the same manner as in Experimental Example 9, except that the conditions in the table below were used. [Table 53]

[0176] The secondary crystallization was carried out in the same manner as the primary crystallization, except that the conditions in the table below were used. [Table 54]

[0177] The purity, degree of supersaturation, and unit step yield of RebA and unit step yield of RebD of each component were calculated in the same manner as in Experimental Examples 10 to 12. The results are shown in the table below. In the table below, some purities exceed 100%. This is thought to be due to weight increase caused by residual solvent or analytical error. [Table 55] [Table 56]

Claims

1. A method for producing a rebaudioside D-containing crystallized product using a crude product obtained by crude purification of an extract from a stevia plant, comprising: preparing a crystallization solution by mixing the crude product with a solvent containing methanol or ethanol so that the degree of supersaturation of rebaudioside D at 10° C. is 10 or more and the degree of supersaturation of rebaudioside A at 10° C. is 18 or less; and cooling the crystallization solution with stirring to precipitate rebaudioside D; The content of rebaudioside D in the crude product is 1 to 20% by mass, A method for producing a rebaudioside D-containing crystallized product, wherein the ratio of rebaudioside D to total steviol glycosides in the rebaudioside D-containing crystallized product after the first crystallization is 35 to 95% by mass.

2. The method according to claim 1, wherein the content of rebaudioside A in the crude product is 5 to 70% by mass.

3. The method according to claim 2, wherein the content of rebaudioside A in the crude product is 20 to 70% by mass.

4. The method according to any one of claims 1 to 3, wherein rebaudioside D is used as a seed crystal.

5. The method according to any one of claims 1 to 4, wherein the degree of supersaturation of rebaudioside D at 10°C is 20 or more and the degree of supersaturation of rebaudioside A at 10°C is 18 or less.

6. A method for producing a food or drink, comprising adding a rebaudioside D-containing crystallized product produced by the method according to any one of claims 1 to 5.

7. The method according to claim 6 , wherein the food or drink is a beverage.

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