WO2021085643A1 - レバウディオサイドd含有晶析物の製造方法およびレバウディオサイドd含有晶析物 - Google Patents
レバウディオサイドd含有晶析物の製造方法およびレバウディオサイドd含有晶析物 Download PDFInfo
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- WO2021085643A1 WO2021085643A1 PCT/JP2020/040974 JP2020040974W WO2021085643A1 WO 2021085643 A1 WO2021085643 A1 WO 2021085643A1 JP 2020040974 W JP2020040974 W JP 2020040974W WO 2021085643 A1 WO2021085643 A1 WO 2021085643A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/60—Sweeteners
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/30—Artificial sweetening agents
- A23L27/33—Artificial sweetening agents containing sugars or derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/30—Artificial sweetening agents
- A23L27/33—Artificial sweetening agents containing sugars or derivatives
- A23L27/36—Terpene glycosides
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/0004—Crystallisation cooling by heat exchange
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/02—Crystallisation from solutions
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
Definitions
- a rebaudioside D-containing crystallization product having a high yield of rebaudioside D and containing rebaudioside D in high purity can be obtained.
- the present invention relates to a method for producing a product, and a crystallized product containing rebaudioside D.
- Steviol glycosides are used in the food industry as calorie-free sweeteners because they have about 300 times the sweetness of sugar. Obesity has become a serious social problem internationally, and the demand for calorie-free sweeteners is increasing day by day from the viewpoint of improving health and reducing medical costs.
- artificially synthesized amino acid derivatives Aspartame and Acesulfame Potassium are used as artificial sweeteners.
- naturally occurring calorie-less sweeteners such as steviol glycosides are expected to be safer and easier to obtain consumer understanding (Public Acceptance).
- Patent Document 1 discloses a method for purifying rebaugioside D by dissolving an extract of a Stevia, rebaudiana, and bertoni plant in an aqueous solution of an organic solvent and crystallizing it three or more times.
- Patent Document 2 prepares a starting material containing at least one steviol glycoside of a Stevia-Levaudiana plant and an aqueous alcohol solution, and dissolves the starting material in the aqueous alcohol solution for about 1 minute to about 240 hours.
- a method for producing a rebaudioside D-containing crystallized product which uses a crude product obtained by crudely purifying an extract from a Stevia plant.
- 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.
- the crude product is mixed with a solvent containing methanol or ethanol so that the supersaturation degree of rebaudioside D at 10 ° C. is 10 or more and the supersaturation degree of rebaudioside A at 10 ° C. is 18 or less.
- the process of preparing a crystallization solution by mixing in, and A method for producing a rebaudioside D-containing crystallized product which comprises a step of cooling the crystallization solution with stirring to precipitate rebaudioside D.
- the total steviol glycosides are rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, stevioside, rebaudioside F and rebaudioside M.
- the manufacturing method described in Crab. [11] The production according to any one of the above [1] to [10], further comprising a step of separating the precipitated rebaudioside D and the liquid phase and drying the separated rebaudioside D. Method. [12] In addition A step of separating the precipitated rebaudioside D and drying it to obtain a primary crystallized product.
- the primary crystallized product contains methanol or ethanol so that the supersaturation degree of rebaudioside D at 10 ° C.
- a step of preparing a solution of a primary crystallized product by mixing in a solvent The step of cooling the primary crystallized product solution with stirring to precipitate rebaudioside D, and The production method according to any one of the above [1] to [10], which comprises a step of separating the precipitated rebaudioside D and drying it. [13] The production according to any one of the above [1] to [12], wherein the ratio of rebaudioside D to the total steviol glycoside in the rebaudioside D-containing crystallized product is 35 to 95% by mass. Method.
- a RevD-containing crystallized product can be obtained with high purity and high yield.
- the solubility curves in the ethanol solvent of various concentrations in Experimental Example 1 are shown.
- the relationship between the RebD addition rate and the yield in Experimental Example 2 is shown.
- the effect of the concentration of the ethanol solvent on the composition of the RebD crystallized product in Experimental Example 3 is shown.
- the effect of the concentration of the ethanol solvent on the yield (crystal amount) in Experimental Example 3 is shown.
- the temperature profile in Experimental Example 4 is shown.
- the effect of the cooling rate on the composition of the RebD crystallized product in Experimental Example 4 is shown.
- the influence of the cooling rate on the yield (crystal amount) in Experimental Example 4 is shown.
- the temperature profile in Experimental Example 5 is shown.
- the effect of the crystallization time on the composition of the RebD crystallized product in Experimental Example 5 is shown.
- rebaudioside In the present specification, "rebaudioside”, “rebaudioside”, “Reb” and “Reb.” Have the same meaning, and all of them mean “rebaudioside”. Similarly, as used herein, “zulcoside” means “dulcoside”.
- a crude product obtained by crudely purifying an extract from a Stevia plant (hereinafter, may be simply referred to as a crude product) is dissolved in a solvent of methanol or ethanol to form RebD as crystals.
- the present invention relates to a method for producing a rebaudioside D (RebD) -containing crystallized product by precipitating.
- RebA was first purified from the extract of Stevia plant, and RevD was purified from the remaining mother liquor. This is because the leaves of most available Stevia plants contain more RebA than RebD.
- RebD is crystallized from the stage of primary crystallization. Considering that RebA is contained in a higher concentration than RebD in the crude product of the extract from Stevia plant, despite using such a crude product as a raw material, it contains highly pure RebD. It is surprising that the crystallized product is obtained.
- a crude product of Stevia plant is used in the production method of the present invention.
- a commercially available product may be used, or an extract from the leaves of Stevia plant, particularly Stevia plant, may be purified and obtained.
- a commercially available product or a product obtained by crudely purifying an extract may be further added with RebD.
- the extract from the Stevia plant may be a commercially available product, or may be obtained by extracting the leaves of the Stevia plant, particularly the Stevia plant. Good.
- a suitable method for obtaining a crude product is (A) Extraction step of extracting dried leaves of Stevia plant with a solvent to obtain an extract. (B) Solid-liquid separation step of obtaining a clarified liquid by solid-liquid separation treatment of the extract. (C) A coagulation step in which a coagulant is added to the clarification liquid to coagulate and a treatment liquid is obtained. (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. Examples include methods including. Hereinafter, this method may be referred to as a crude purification method of the present invention. Each step will be described below.
- the water content of the dried leaves of the Stevia plant is 1 to 11% by weight.
- the dried leaf of a stevia plant means a leaf having a reduced water content by drying the fresh leaf of the stevia plant.
- the water content of the dried leaves of the Stevia plant is preferably 1 to 10% by weight, more preferably 2 to 8% by weight, and particularly preferably 3 to 4% by weight.
- the dried leaves of Stevia plants are not particularly limited as long as they contain steviol glycosides, but preferably have a higher content of RevD or rebaudioside M (RebM) than the dried leaves of natural Stevia plants. ..
- the dried leaves of such a stevia plant can be obtained, for example, by the method described in International Publication No. 2019/074089.
- the dried leaves of a steviol plant preferable to be used in the production method of the present invention contain 5.0 to 25 g of steviol glycoside in 100 g of dried leaves when the water content is 3 to 4% by weight.
- the dried leaves in another embodiment have 6.0 to 24 g, 7.0 to 23 g, 8.0 to 22 g, 9.0 to 21 g, 10 to 100 g in 100 g of dried leaves when the water content is 3 to 4% by weight. It may contain 20 g or 11-19 g of steviol glycosides.
- the dried leaves of the steviol plant used in the production method of the present invention contain 8 to 17 g of steviol glycosides in 100 g of the dried leaves when the water content is 3 to 4% by weight.
- the preferred dried leaf in this embodiment contains 0.5 g or more of RebD in 100 g of the dried leaf when the water content is 3 to 4% by weight.
- the preferable dried leaf used in another embodiment contains 0.19 g or more of RebM in 100 g of the dried leaf when the water content is 3 to 4% by weight.
- a preferable dried leaf used in another embodiment contains 3.0 g or more of rebaudioside A (RebA) in 100 g of the dried leaf when the water content is 3 to 4% by weight.
- the dried leaves have a RebD of 0.6 g or more, 0.7 g or more, 0.8 g or more, 1.0 g or more in 100 g of the dried leaves when the water content is 3 to 4% by weight.
- It may be present in an amount of 1 g or more or 4.2 g or more, and may be present in an amount of 6.0 g or less, 5.5 g or less, or 5.0 g or less, for example.
- the dried leaves have RebM 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 per 100 g of dried leaves.
- the dried leaves have RebA of 3.0 g or more, 3.5 g or more, 4.0 g or more, 4.5 g or more, 5 in 100 g of the dried leaves when the water content is 3 to 4% by weight.
- 0.0g or more 5.5g or more, 6.0g or more, 6.5g or more, 7.0g or more, 7.5g or more, 8.0g or more, 8.5g or more, 9.0g or more, 9.5g or more, 10g It may be present in an amount of 11 g or more, 12 g or more, 13 g or more, or 14 g or more, and may be present in an amount of 17 g or less, 16 g or less, or 15 g or less, for example.
- the dried leaves may contain 0.5 to 0.9 g or 0.6 to 0.85 g of RebD in 100 g of the dried leaves when the water content is 3 to 4% by weight.
- RebM is 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 in 100 g of dried leaves having a water content of 3 to 4% by weight.
- RebA is 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 in 100 g of dried leaves having a water content of 3 to 4% by weight. Included in any amount of ⁇ 15g, 5 ⁇ 14g, 5 ⁇ 13g, 5 ⁇ 12g, 6 ⁇ 17g, 6 ⁇ 16g, 6 ⁇ 15g, 6 ⁇ 14g, 6 ⁇ 13g, 6 ⁇ 12g, 6 ⁇ 11g. May be.
- Extraction of steviol glycosides from dried leaves can be performed using a solvent such as water, alcohol, or a mixed solution thereof.
- Preferred extraction solvents include ion-exchanged water, pure water (for example, milliQ water), and an aqueous ethanol solution.
- the dried leaves may or may not be crushed.
- the extraction process may be performed using a kneader extractor (for example, SKN-R100, manufactured by Sanyu Kikai Co., Ltd.) or the like.
- Steviol glycosides can be extracted more efficiently by heating an aqueous solvent during extraction.
- the temperature at the time of extraction is, 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. It may be, preferably 45 to 70 ° C.
- Extraction may be performed not only once but multiple times. By performing multiple extractions, more steviol glycosides contained in the leaves are extracted. From the viewpoint of efficiency, it is preferable that the extraction is performed twice.
- a clear liquid can be obtained by solid-liquid separation treatment of the obtained extract.
- the solid-liquid separation treatment is not particularly limited as long as the solid and liquid are sufficiently separated, and 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 clear liquid may be obtained by performing a second solid-liquid separation treatment after the first solid-liquid separation treatment.
- a treatment liquid can be obtained by adding a coagulant to the clarification liquid obtained by the solid-liquid separation treatment.
- the flocculant is not particularly limited, and a known inorganic flocculant or organic polymer flocculant can be used.
- the flocculant is aluminum sulfate, polyaluminum chloride, iron (III) chloride or a hydrate thereof, a synthetic polymer flocculant (polyacrylamide high polymer, partial hydrolyzate of polyacrylamide, etc.). ), Arginic acid, chitin, chitosan, one or more selected from calcium hydroxide.
- the flocculant may be used in combination of two or more kinds, and may be used, for example, in a combination of calcium hydroxide and iron chloride, or in a combination of calcium hydroxide, iron chloride and chitosan.
- the amount of the coagulant added is not particularly limited as long as it causes coagulation for each coagulant, but for example, it should be added in an amount of 3.0 to 50% by weight with respect to the soluble solid content contained in the clarified solution. Can be done.
- calcium hydroxide can be added in an amount corresponding to 10 to 30% by weight of the solid content in the clarification solution, preferably in an amount of 12 to 28% by weight, more preferably in an amount of 14 to 25% by weight. can do.
- iron (III) chloride hexahydrate it can be added in an amount corresponding to 15 to 40% by weight of the solid content in the clarified solution, preferably 18 to 38% by weight, and more preferably 20 to 35% by weight.
- chitosan solution it can be added in an amount of% by weight.
- it can be added in an amount corresponding to 3.0 to 10% by weight of the solid content in the clarified solution, preferably 4.0 to 8.0% by weight. %, More preferably 4.5-7.0% by weight.
- the pH at the time of the coagulation treatment is not particularly limited, and can be appropriately selected so that the coagulation is optimized depending on the type of coagulant. In one aspect of the present invention, even if the pH of the clarified solution during the coagulation treatment is 2.0 to 13, 3.0 to 13, 4.0 to 13, 5.0 to 13 or 6.0 to 13. Good.
- the temperature of the coagulation treatment is not particularly limited, and it may be carried out at room temperature (about 25 ° C.) without heating or cooling.
- the agglutination contained in the treatment liquid may be removed before the resin purification treatment described later.
- the agglomerates can be removed by any method such as filtration.
- Centrifugation can also be mentioned as a method for removing agglomerates in addition to the above methods.
- the treatment liquid obtained by the coagulation treatment is treated with a hydrophobic porous resin.
- Steviol glycosides are amphipathic with hydrophilic and hydrophobic groups in their molecular structure and have a molecular weight of around 1,000. It is also known that it is stable at pH 2.5 to 9.0 and does not ionize even if it is acidic or basic.
- the treatment liquid that has undergone the aggregation treatment contains a large amount of components other than steviol glycosides.
- such components include components with different molecular weights from steviol glycosides, such as iron ions, and components that are ionized, such as amino acids, and these components are made hydrophobically porous. It is considered that it can be removed by treatment with a quality resin.
- the steviol glycoside having a hydrophobic steviol skeleton is hydrophobically bound to the synthetic resin and captured.
- highly hydrophilic impurities do not bind to the resin and are transferred to the through fraction and removed. Therefore, the treatment liquid that has undergone the aggregation treatment is added to the column packed with the above resin, and then washed with water. The purity of steviol glycosides is thought to improve. Further, since the bond between the steviol glycoside and the functional group of the synthetic resin is dissociated by the low-polarity solvent, there is an advantage that the steviol glycoside can be finally recovered in a high yield.
- the porous resin used in one aspect of the crude purification method of the present invention is not particularly limited as long as it is a porous resin having a low affinity for water, and for example, a copolymer of styrene and divinylbenzene, polyethylene, polypropylene, etc. , Polystyrene, poly (meth) acrylonitrile, polyamide and one or more hydrophobic resins selected from polycarbonate are preferred.
- the copolymer of styrene and divinylbenzene has not been subjected to an ion exchange group introduction treatment (that is, it does not have an ion exchange group).
- styrene and divinylbenzene are copolymerized to form a three-dimensional network structure, and then an ion exchange group is introduced into the resin. "Not done” means that such processing has not been done.
- the hydrophobic porous resin has a hydrophobic group, and the hydrophobic group is selected from an aryl group, an alkyl group, an alkylsilyl group, an ester group and an epoxy group1 Includes one or more. If one or more hydrophobic groups selected from these are contained, other hydrophobic groups may be further contained.
- Examples of the aryl group include a phenyl group, a benzyl group, a tolyl group, a xsilyl group and the like
- examples of the alkyl group include an alkyl group of C1 to 20, for example, a methyl group, an ethyl group, a propyl group, a butyl group and a pentyl group.
- examples include a group and an octadecyl group.
- the most frequent pore radius of the hydrophobic porous resin is 10 to 200 ⁇ . In a preferred embodiment, the most frequent pore radii are 10-150 ⁇ , 15-100 ⁇ , or 20-80 ⁇ . It is considered that having such pore characteristics allows the steviol glycoside to be efficiently adsorbed to the pores and efficiently separated from other components.
- the treatment liquid may be treated with an anion exchange resin before the treatment with the hydrophobic porous resin.
- an anion exchange resin By pre-treating with an anion exchange resin, components bound to the hydrophobic resin such as dyes and catechins can be effectively removed.
- an anion exchange resin is not particularly limited, and examples thereof include a basic anion exchange resin.
- a basic anion exchange resin a 1st to 2nd secondary amino group is introduced as a functional group.
- a weakly basic anion exchange resin or a strongly basic anion exchange resin having a quaternary ammonium group for example, a trimethylammonium group or a dimethylethanolammonium group
- quaternary ammonium group for example, a trimethylammonium group or a dimethylethanolammonium group
- the solution obtained from the resin purification treatment may be further concentrated to remove the aqueous solvent.
- Such treatment is not particularly limited, and examples thereof include a method of evaporating the aqueous solvent by heating and a method of removing the aqueous solvent by drying under reduced pressure.
- spray drying may be performed.
- the temperature conditions for spray drying are usually such that the inlet temperature is about 120 to 200 ° C. and the outlet temperature is about 80 to 120 ° C.
- the crude product used in the production method of the present invention contains a total steviol glycoside in an amount of 50 to 95% by mass based on the total weight of the crude product, and is preferably 55 to 95, 60 to 95. 70-95, 80-95, 90-95, 50-90, 50-80, 50-70, 50-65, 50-60, 55-90, 55-80, 55-70, 55-60, 60- It contains a total steviol glycoside in any amount (% by mass) of 70, 60-80, 50-94, 55-94, 60-94.
- the total steviol glycoside preferably consists of seven types of steviol glycosides, RebA, RebB, RebC, RebD, stevioside, RebF and RevM.
- the total steviol glycoside may be abbreviated as TSG.
- the crude product contains at least RevA and RevD.
- the content (% by mass) of RebA in the crude product is 5 to 70, 15 to 70, 20 to 70, 25 to 70, 35 to 70, 45 to 70, 55 to 70, 65 to 70, It is any of 5 to 65, 5 to 45, 5 to 25, 15 to 65, 25 to 45, 40 to 75, 45 to 65, and the RebD content (mass%) is 1 to 70, 2 to.
- the content of RevA in the crude product is 1 to 30% by mass, and the content of RevD is any of the above.
- the solvent is placed in an arbitrary container, for example, a crystal can provided with a stirring blade, and the crude product is mixed in the solvent to prepare a crystallization solution.
- the supersaturation degree (10 ° C.) of RebD is set to a specific range while the supersaturation degree (10 ° C.) of RebA is set to a specific range so that RebA does not crystallize.
- the crude product is mixed in the solvent in an amount such that such supersaturation is achieved.
- the degree of supersaturation (10 ° C.) means the degree of supersaturation when 10 ° C. is used as a reference temperature. There is no intention to require cooling at 10 ° C.
- the degree of supersaturation is expressed by the following formula.
- ⁇ (CC * ) / C *
- ⁇ indicates the degree of supersaturation
- C indicates the ratio (ppm) of the amount of the substance added to the solvent
- C * indicates the saturated dissolution concentration (ppm).
- C is not simply "concentration of the substance” but “ratio of the amount of the substance added to the solvent", assuming that the added substance is not completely dissolved and is in a suspended state. That's it.
- the ratio of the added amount to the solvent is used as the standard of the degree of supersaturation regardless of the presence or absence of dissolution of the added substance and the degree thereof.
- a seed crystal may be used, but the amount of the seed crystal added is not included in the calculation of the degree of supersaturation.
- the saturated solubility is measured by the following method. That is, 100 ml of a solvent having a predetermined methanol or ethanol concentration (w / w) is placed in a container (capacity 100 ml or 200 ml, preferably 200 ml), the liquid temperature is set to 10 ° C. in advance with a water bath, and then the mixture is stirred at 200 rpm. Add an excess amount (the amount until suspension) of the target substance. After 24 hours, the filtrate is obtained with a 0.45 um membrane filter, and the amount of the target substance (RebA or RevD) contained in the filtrate is measured by LCMS. As the LCMS, preferably, LCMS8050 manufactured by Shimadzu Corporation is used.
- the degree of supersaturation (10 ° C.) of RebD is 10 or more, 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. 10-40, 10-20, 15-200, 35-200, 55-200, 75-200, 95-200, 115-200, 135-200, 155-200, 175-200, 15-180, 15 ⁇ 160, 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, 100-120 Is it?
- the degree of supersaturation (10 ° C.) of RebD is 10-350, 20-350, 50-350, 10-300, 20-300, 50-300, 10-205 or 20-205.
- the degree of supersaturation (10 ° C.) of RebA is 18 or less, including 0 or less. In one aspect of the invention, the degree of supersaturation of RebA (10 ° C.) is greater than 0 and 18 or less. More preferably, 3-18, 3-13, 3-8, 5-18, 7-18, 9-18, 11-18, 13-18, 15-18, 17-18, 5-13, 7 It is any of ⁇ 11, 5 ⁇ 15, 7 ⁇ 15, 9 ⁇ 15, 11 ⁇ 15, 13 ⁇ 15.
- the degree of supersaturation (10 ° C.) of RebA is 16 or less, -1 to 16 or 0 to 16.
- ⁇ D / ⁇ A is any of 4 to 1,000,000, 5 to 1,000,000, 4 to 750,000, and 5 to 750,000.
- the solvent used to prepare the crystallization solution is a solvent of methanol or ethanol.
- An aqueous solution of methanol or an aqueous solution of ethanol is preferable. More preferably, it is an aqueous solution of ethanol.
- an ethanol solvent is used instead of a methanol solvent.
- “Use an ethanol solvent without using a methanol solvent” means that the methanol concentration in the solvent is preferably 1 mg / L or less, more preferably 0.5 mg / L or less, and particularly preferably 0.1 mg / L. It means that it is as follows. The lower limit of the methanol concentration is 0 mg / L.
- Ethanol has a lower environmental load than methanol, and in addition, it has a lower effect on the human body.
- ventilation equipment and ventilation capacity based on laws and regulations and special health examinations for employees are obligatory at the manufacturing site, but ethanol does not have such an obligation, which is a great advantage for industrial use. is there.
- the amount of the crude product added is appropriately set so that the degree of supersaturation is within the above range, but is preferably 1 to 30% by mass, more preferably 1 to 25, 1 to 20, 1 per solvent. ⁇ 15,1 ⁇ 10, 5 ⁇ 30, 5 ⁇ 25, 5 ⁇ 20, 5 ⁇ 15, 5 ⁇ 10, 10 ⁇ 30, 15 ⁇ 30, 20 ⁇ 30, 25 ⁇ 29, 10 ⁇ 25, 15 ⁇ 20
- the amount is mixed so as to be one of the amounts (% by mass) of.
- the amount of the crude product added is 1 to 28% by mass, 1 to 6% by mass, 6 to 25% by mass or 6 to 28% by mass per solvent.
- the amount of the crude product added is appropriately set so that the degree of supersaturation is within the above range, but preferably, the crude product has a RebD of 0.15 to 3.50, 0.30 to 0.30 to solvent. 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 3.00, 0.60 to 3.00, 0.75 to 3.00, 0.90 to 3.00, 1.
- the crude product is added in an amount such that RebD per solvent is in any amount (% by mass) of 0.20 to 3.50 and 0.25 to 3.50.
- the concentration (mass%) of the solvent containing methanol or ethanol is preferably 99.9% by mass or less, and more preferably 92% by mass or less.
- the concentration (% by mass) of the solvent containing methanol or ethanol is either 73% by mass or more and less than 95% by mass, or 73 to 92% by mass.
- the liquid temperature (° C.) of the solvent when the crude product is mixed with the solvent is 40 to 80, 42 to 80, 44 to 80, 46 to 80, 48 to 80, 50 to 80, 52 to 80, 57 to 80, 62-80, 67-80, 72-80, 40-75, 40-70, 40-65, 40-60, 40-55, 40-50, 42-75, 44-70, 46-65, 48- It is preferably maintained at any of 60 and 50 to 55.
- the crude product when the crude product is mixed with the solvent, it is preferable to mix the seed crystals together. It is preferable to use RebD as the seed crystal.
- the particle size of the seed crystal is not particularly limited.
- the solution temperature is supersaturated from the viewpoint of avoiding dissolution of the seed crystals.
- the seed crystal addition rate Cs represented by the following formula is 0.050 to 10.000, 0.075 to 10.000, 0.100 to 10.000, 0.200 to 10.000, 0. 300 to 10.000, 0.400 to 10.000, 0.500 to 10.000, 0.600 to 10.000, 0.700 to 10.000, 0.800 to 10.000, 0.900 to 10.000, 1.000 to 10.000, 2.000 to 10.000, 3.000 to 10.000, 4.000 to 10.000, 5.000 to 10.000, 6.000 to 10.
- Cs Ws / W th
- Ws seeding Akiraryou (unit: g) of the, W th are assumed Yield (unit: g) of indicating the.
- W th (CC * ) / 1,000,000 ⁇ amount of solvent
- C is the ratio (ppm) of the amount of RebD added to the solvent.
- C * indicates saturated solubility (unit: ppm).
- the unit of solvent amount is ml.
- C does not include the amount of seed crystals.
- the crystallization solution is cooled with stirring by an arbitrary crystallization device, for example, an auxiliary crystal machine such as a cylindrical rotary auxiliary crystal machine, to precipitate RebD.
- an auxiliary crystal machine such as a cylindrical rotary auxiliary crystal machine
- a suitable cooling rate is 0.002 ° C./min or higher, more preferably 0.002 to 1.37 ° C./min, and particularly 0.002 to 1.37,0.
- the cooling rate (° C./min) is 0.02 to 0.2.
- the stirring cooling time (unit: time) may be appropriately set according to other conditions such as the presence or absence of seed crystals, but from the viewpoint of improving the yield, 1 to 48, 6 to 48, 11 to 48, 16 ⁇ 48, 21 ⁇ 48, 26 ⁇ 48, 31 ⁇ 48, 36 ⁇ 48, 41 ⁇ 48, 46 ⁇ 48, 1 ⁇ 43, 1 ⁇ 38, 1 ⁇ 33, 1 ⁇ 28, 1 ⁇ 23, 1 ⁇ 18 Any of 1 to 13, 1 to 8, 1 to 3, 6 to 43, 11 to 38, 16 to 33, and 21 to 28 is preferable.
- the stirring cooling time (unit: hours) is 6 to 24 hours, 12 hours or more, or 12 to 24 hours.
- the cooling temperature (° C.) in the precipitation step is appropriately determined depending on other conditions such as the presence or absence of seed crystals, but is 3 to 40, 6 to 40, 9 to 40, 12 to 40, 15 to 40, 18 to 40. , 21-40, 24-40, 27-40, 30-40, 33-40, 20-40, 35 ° C or lower, 3-35, 6-35, 9-35, 12-35, 15-35, 18 It is preferable to cool to any of ⁇ 35, 21 to 35, 24 to 35, 27 to 35, 30 to 35, and 25 to 35.
- the cooling temperature is 9 to 20 ° C., 9 to 17 ° C., 4 to 20 ° C. or 4 to 17 ° C.
- the temperature (° C.) at the start of cooling is 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. Any of 40, 40 to 80, 40 to 70, 40 to 60 is preferable.
- the stirring speed (rpm) is 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. , 205-600, 255-600, 305-600, 355-600, 405-600, 455-600, 505-600, 555-600, 50-600, 200-600, 350-600, 500-600, 100 Any of ⁇ 450, 100 to 300, 100 to 150, 200 to 500, and 300 to 400 is preferable.
- the solid composition containing the precipitated RebD and the liquid phase are separated 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 the liquid are sufficiently separated, and examples thereof include treatment with a centrifuge, treatment with a membrane filter, and treatment with a mesh. After separation and before drying, if necessary, the surface may be washed by spraying a small amount on the RebD crystal with the same solvent used for crystallization or the like.
- the mother liquor after the solid-liquid separation treatment may be used for crystallization of other components such as RebA and RebM.
- the above-mentioned crude purification step, crystallization solution preparation step, precipitation step and post-step are performed once to complete the production. May be (single crystallization).
- the process from the preparation of the solution for crystallization to the post-process may be repeated a plurality of times (multiple crystallization). From the viewpoint of the balance between yield and purity, double crystallization is preferable.
- ⁇ Twice crystallization> When performing double crystallization, the above-mentioned crude purification step to precipitation step are carried out, and the precipitated rebaudioside D is separated and dried to obtain a primary crystallized product, and then the above-mentioned crude product. Instead, use methanol or ethanol to prepare the primary crystallized product 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 primary crystallization solution is prepared by mixing in a solvent containing the mixture, and instead of the above-mentioned crystallization solution, the primary crystallization solution is cooled with stirring to precipitate RebD, and the precipitated RebD is separated. Then, by drying, a RebD-containing crystallized product can be obtained.
- the procedure and conditions for the double crystallization may be the same as the crystallization procedure and crystallization conditions described above. In that case, the "crude refined product" in the above description shall be read as "primary crystallized product".
- the purity can be further increased by lowering the concentration of the solvent containing methanol or ethanol used for preparing the crystallization solution as compared with the case of the single crystallization.
- the degree of supersaturation of RebA may be set lower than that in the first crystallization.
- crystallization may be performed many times, but sufficiently high-purity purification is possible even with a small number of crystallizations of 2 times or less. Therefore, there are various advantages such as reduction of shortage, shortening of cycle time, and energy saving.
- the RebD-containing crystallized product obtained by the method of the present invention (hereinafter, may be referred to as the RevD-containing crystallized product of the present invention) contains a large amount of RevD.
- 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, 35-79, 35-69, 35-59, 35-95, 45-95, 55-95, 65-95, 75-95, 85-95, 40-99, 50-99, 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- It is one of 95, 70-95, 80-95, 90-95, 40-85, 40-75, 40-65, 40-55,
- the RebD-containing crystallized product of the present invention has a low RebA content.
- the ratio of RebA to TSG (% by mass) is 5 to 80, 5 to 50, 10 to 50, 20 to 50, 30 under the condition that the ratio of RebD is in any of the above numerical ranges.
- 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, 5 to 10 is there.
- the ratio (% by mass) of RebA to TSG is 3.5-80, 3.5-35, 3.5-30 or 3.5-12.
- RevD can be crystallized in high yield.
- the ratio (mass%) of the crystallized RebD to the RevD contained in the crude product is 70 to 99, 75 to 99, 80 to 99, 85. ⁇ 99, 90-99, 70-94, 70-89, 70-84, 70-79, 70-74, 75-94, 80-89, 50-99, 55-99, 60-99, 65-99 , 50-94, 50-89, 50-84, 50-79, 50-74, 50-69, 55-94, 60-89, 65-84.
- the ratio (mass%) of the crystallized RebD to the RevD contained in the crude product is 39 to 85, more preferably 39 to. 82.
- the recovery rate of RebD (sometimes referred to as total yield) from leaf extraction to post-crystallization is also high, preferably 35 to 90, 45 to 90, 55 to 90, 65 to 90, or. It is 75 to 90% by mass.
- the proportion (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, It is 50 to 60, 60 to 80, 60 to 90, 60 to 70 or 70 to 80.
- the proportion (% by 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.
- the amount of methanol contained in the RebD-containing crystallized product is small, preferably less than 10 ppm, and particularly preferably 5 ppm. Less than, most preferably 2 ppm or less.
- the methanol content can be measured by headspace GCMS. The lower limit is preferably 1 ppm.
- the lower limit of the amount of methanol contained in the RebD-containing crystallized product is 0 ppm.
- the methanol content ratio of methanol to ethanol in the RebD-containing crystallized product MeOH / EtOH is also a small value. It is preferably 0.00010 to 0.00080, more preferably 0.00010 to 0.00070, particularly preferably 0.00010 to 0.00050, and most preferably 0. It is .00010 to 0.00030.
- the RebD-containing crystallized product of the present invention may be used as a sweetening composition.
- a sweetening composition may contain a sweetening agent other than the steviol glycoside in addition to the RevD-containing crystallized product of the present invention.
- sweeteners include high fructose corn syrup, sugar, high fructose corn syrup, glucose, malt sugar, high fructose corn syrup, sugar alcohol, oligosaccharide, honey, sugar cane juice (brown sugar honey), water candy, Luo Han Guo powder, Luo Han Guo extract, Natural sweeteners such as licorice powder, licorice extract, somatococcus daneri seed powder, somatococcus daneri seed extract, sucrose, and artificial sweeteners such as acesulfam potassium, sucrose, neotheme, aspartame, and saccharin can be mentioned. ..
- natural sweeteners are preferably used from the viewpoint of refreshingness, ease of drinking, natural taste, and imparting an appropriate richness
- fructose, glucose, maltose, sucrose, and sugar are particularly preferably used. Only one kind of these sweetness components may be used, or a plurality of kinds may be used.
- foods and drinks, fragrances and pharmaceuticals containing the RevD-containing crystallized product of the present invention (“food and drink of the present invention”, “fragrance of the present invention” and “the present invention, respectively” in the present specification. Also referred to as "medicine of invention”) is provided.
- the food and drink, flavors and pharmaceuticals of the present invention are not particularly limited as long as they contain the RebD-containing crystallized product of the present invention.
- the food and drink means a beverage and a food
- the food and drink is a beverage in a preferable correspondence. Therefore, in certain embodiments, the present invention provides a novel beverage or food product.
- the total amount (mass ppm) of steviol glycosides contained in the foods and drinks, fragrances and pharmaceuticals of the present invention varies depending on the specific foods and drinks, but in the case of beverages, it is preferably about 1 to 800 mass ppm.
- the food and drink, flavors and pharmaceuticals of the present invention may further contain sweeteners other than steviol glycosides.
- sweeteners include high fructose corn syrup, sugar, high fructose corn syrup, high fructose corn syrup, malt sugar, sucrose, high fructose corn syrup, sugar alcohol, oligosaccharide, honey, sugar cane juice (brown sugar honey), water candy, Rakan fruit powder, Natural sweeteners such as Rakan fruit extract, licorice powder, licorice extract, high fructose corn syrup seed powder, high fructose corn syrup seed extract, and artificial sweeteners such as acesulfam potassium, sucrose, neotame, aspartame, and saccharin. Be done.
- natural sweeteners are preferably used from the viewpoint of refreshingness, ease of drinking, natural taste, and imparting an appropriate richness
- fructose, glucose, maltose, sucrose, and sugar are particularly preferably used. Only one kind of these sweetness components may be used, or a plurality of kinds may be used.
- Examples of the food of the present invention are not particularly limited, but are confectionery, bread making, flour, noodles, cooked rice, processed agricultural / forest foods, processed livestock products, processed marine products, milk / dairy products, etc. Examples include fats and oils, processed fats and oils, seasonings or other food materials.
- beverage of the present invention are not particularly limited, but for example, carbonated beverages, non-carbonated beverages, alcoholic beverages, non-alcoholic beverages, beer-taste beverages such as beer and non-alcoholic beer, coffee beverages, tea beverages, etc.
- examples include cocoa beverages, nutritional beverages, and functional beverages.
- TSG means seven types of RevA, RevB, RevC, RevD, Stevioside, RevF, and RevM.
- the filter used for solid-liquid separation was a 0.45 um membrane filter manufactured by Advantech. Then, the RebD crystals were washed with 99% by mass ethanol. The washed crystals were dried at 50 ° C.
- a crude product having the following composition (unit: mass%) was prepared.
- the RebD purity per TSG in this crude product was 1.8% by mass.
- the TSG content in the crude product was 59.7% by mass.
- the following amount of crude product was dissolved in the following concentration and amount of ethanol solvent (78 ° C.). Then, it was cooled under the following temperature profile and stirring conditions, and RebD crystals were crystallized.
- a separable round bottom flask (manufactured by Tokyo Rika Kikai Co., Ltd.) was used for crystallization.
- ethanol 99.9% by mass grade of undenatured alcohol was used, and water was appropriately added thereto 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 supersaturation degree (10 ° C.) ⁇ A of RebA was 5.10 (when using 90.50% by mass ethanol solvent).
- RebD had a supersaturation degree (10 ° C.) of ⁇ D of 24.37 (when using 90.50% by mass ethanol solvent) and ⁇ D / ⁇ A of 4.78 (when using 90.50% by mass ethanol solvent).
- RebD crystals were obtained by solid-liquid separation.
- the filter used for solid-liquid separation was a 0.45 um membrane filter manufactured by Advantech.
- the RebD crystals were washed with 99% by mass ethanol.
- the washed RebD crystals were dried at 50 ° C. to obtain RebD crystals.
- the glycoside ratio of the obtained RebD crystallized product and the filtrate was analyzed by LCMS (manufactured by Shimadzu Corporation, LCMS8050). The results are shown in FIGS. 3 and 4.
- the cooling rate has a great influence on the appearance of crystal nuclei and crystal growth.
- the cooling rate is high, when crystal growth occurs from the initial state of a small number of nuclei, supersaturation cannot be completely consumed, and nucleation is often predominant.
- the temperature drop is slow, crystal growth becomes dominant.
- the crystallization time tends to be long, depending on the consumption rate of supersaturation.
- the effect of cooling rate on TSG composition and yield was investigated.
- the secondary crystallization was carried out under the condition of achieving a purity of about 90% (that is, the ethanol concentration in the secondary crystallization was 70% by mass).
- the yield in the case was about 43% by mass.
- the clarified liquid after solid-liquid separation was subjected to agglomeration, resin purification, and evaporation concentration steps. As a result, a crude product having the composition (unit: mass%) shown in the table below was obtained.
- the RebD ratio per TSG in the crude product was 3.8% by mass.
- the following amount of crude product was dissolved in the following concentration and amount of ethanol solvent (40 ° C.).
- the addition ratio of RebD to the solvent was 0.22% by mass.
- it was cooled under the following temperature profile and stirring conditions, and RebA crystals were crystallized.
- a separable round bottom flask (manufactured by Tokyo Rika Kikai Co., Ltd.) was used for crystallization.
- As ethanol 99.9% by mass grade of undenatured alcohol was used.
- 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 Advantech.
- the RebA crystals were washed with 99% by mass ethanol.
- the washed RebA crystals were dried at 50 ° C.
- the primary crystallized product was analyzed using Shimadzu LCMS8050.
- Table 11 shows the ratio (unit: mass%) of each steviol glycoside to TSG of the primary crystallized product. Yield information (unit: mass%) is shown in Table 12.
- the RebA purity with respect to TSG was 98.6% and the RebA yield was 41.8%. As a result, about 11.5% by mass of the RebD yield was mixed in the RebA crystal.
- RebD crystallization was attempted using RebD seed crystals from the mother liquor of primary crystallization. As shown in the table below, the RebD purity with respect to TSG was 94% by mass or more.
- the clarified liquid after solid-liquid separation was subjected to agglomeration, resin purification, and evaporation concentration steps.
- a crude product having the composition (unit: mass%) shown in the table below was obtained.
- the RebD ratio per TSG in the crude product was 4.6% by mass.
- the proportion of TSG in the crude product was 54.0% by mass.
- the following amount of crude product (indicated 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 supersaturation degree of RebA (10 ° C.) was 9.98
- the supersaturation degree of RebD (10 ° C.) was 57. It was 64.
- a separable round bottom flask (manufactured by Tokyo Rika Kikai Co., Ltd.) was used for crystallization.
- Example 9 An extraction operation was performed using dried leaves of Stevia plants, and the obtained extract was subjected to a solid-liquid separation operation.
- the ratio (mass%) of each steviol glycoside per TSG was as follows.
- the content of TSG in the dried leaves used in this experimental example was 11.8 g per 100 g.
- the clarified liquid after solid-liquid separation was subjected to agglomeration, 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.
- An unmodified alcohol 99.9% by mass grade was used, and water was appropriately added thereto to adjust the following concentration and amount of ethanol.
- An ethanol solvent was put into a crystallization vessel with a stirrer (capacity: 1 liter) and heated. When the temperature of the ethanol solvent reached about 55 ° C., the crude product was put into a container. The temperature was lowered while stirring at about 350 rpm. At this time, the cooling time (that is, 11 hours) -1 hour (that is, 10 hours) was uniformly cooled. After completion of crystallization, solid-liquid separation was performed.
- the filter used for solid-liquid separation was a 0.45 um membrane filter manufactured by Advantech. The obtained crystals were dried at 60-70 ° C.
- the unit process yield (ratio of crystallized RebD to RevD contained in the crude product before crystallization) was 80.0% by mass.
- the TSG purity (ratio of TSG to crystallized product) was 63.2% by mass, and the RebD purity (ratio of RebD to TSG) was 35.8% by mass.
- Evaporation Concentration Ethanol was removed while evaporating and concentrating the solution using a centrifugal thin film vacuum evaporator Evapol (manufactured by Okawara Seisakusho). Water remained even after the evaporative concentration treatment, and the composition was liquid.
- the obtained steviol glycoside composition was spray-dried to obtain a crude product.
- the composition (unit: mass%) of the crude product after spray drying is as follows.
- the table below shows the purity, supersaturation, and RebA unit process yield and RebD unit process yield of various components in the crude products used in Experimental Examples 10 to 12.
- RebA purity and RebD purity are measured by LCMS8050 manufactured by Shimadzu Co., Ltd. and expressed as a ratio (w / w) to TSG.
- the formula for calculating the degree of supersaturation is as described above.
- the saturated solubilities of RebA and RebD (90% by mass EtOH, 10 ° C.) were as follows.
- 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 for crystallization.
- the RebD secondary crystallized product obtained in Experimental Example 12 was used as Sample No. It was set to 5.
- Sample No. Sample No. 4 was obtained by further drying No. 4 at 60 to 70 ° C. for 3 days. It was set to 6.
- Sample No. Sample No. 5 was obtained by further drying No. 5 at 60 to 70 ° C. for 3 days. It was set to 7.
- Example 14 1. Extraction / Solid-Liquid Separation 15 times the amount (mass) of dried Stevia leaves (moisture content: 9 to 11% by weight) was heated to 60 to 65 ° C., and the dried Stevia leaves were immersed in the water. Then, in a stirring tank (volume: 400 L), extraction was performed for 60 minutes while stirring at 75 rpm using a stirring blade (radius 27 cm ⁇ 2 sheets ⁇ 2 stages). Next, a mixture of dried Stevia leaves and water was filtered through a 100-mesh nylon mesh set in a 95 cm diameter Büchner funnel, diatomaceous soil (Selite 503) was added, filtered with a filter press, and a precision filter membrane (precise filtration membrane).
- a primary extract was obtained by solid-liquid separation by filtering through a pore size (pore size: 10 ⁇ m).
- the filtered leaves were extracted again under the same conditions and solid-liquid separated to give a clear secondary extract, which was added to the primary extract to give a clarified solution.
- Evaporation Concentration Ethanol was removed while evaporating and concentrating the solution using a centrifugal thin film vacuum evaporator Evapol (manufactured by Okawara Seisakusho). Then, using an evaporator, concentration was carried out in two steps to remove ethanol. Water remained even after the evaporative concentration treatment, and the composition was liquid.
- the obtained steviol glycoside composition was spray-dried to obtain a crude product.
- the composition (unit: mass%) of the crude product after spray drying is as follows.
- the ratio of each steviol glycoside per TSG in the secondary crystallized product is as shown in the table below.
- the unit process yield of RebD up to the secondary crystallization was 98.9% by mass.
- the proportion of TSG in the secondary crystallized product was 95.9% by mass.
- Secondary crystallization was performed in the same manner as for primary crystallization, except that the conditions in the table below were adopted.
- Secondary crystallization was performed in the same manner as for primary crystallization, except that the conditions in the table below were adopted.
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Abstract
Description
[1]ステビア植物からの抽出物を粗精製して得られた粗精製物を用いる、レバウディオサイドD含有晶析物の製造方法であって、
前記粗精製物における総ステビオール配糖体含有量が50~95質量%であり、且つ、前記粗精製物が少なくともレバウディオサイドAとレバウディオサイドDとを含有しており、
前記粗精製物を、レバウディオサイドDの10℃での過飽和度が10以上であり且つレバウディオサイドAの10℃での過飽和度が18以下になるように、メタノールまたはエタノールを含む溶媒中に混合して、晶析用溶液を調整する工程、並びに、
前記晶析用溶液を、攪拌しながら冷却し、レバウディオサイドDを析出させる工程を含むことを特徴とする、レバウディオサイドD含有晶析物の製造方法。
[2]前記総ステビオール配糖体が、レバウディオサイドA、レバウディオサイドB、レバウディオサイドC、レバウディオサイドD、ステビオシド、レバウディオサイドFおよびレバウディオサイドMである、上記[1]に記載の製造方法。
[3]前記粗精製物におけるレバウディオサイドAの含有量が5~70質量%であり、かつ、レバウディオサイドDの含有量が2~70質量%である、上記[1]または[2]に記載の製造方法。
[4]前記溶媒における前記メタノールまたはエタノールの濃度が99.9質量%以下である、上記[1]~[3]のいずれかに記載の製造方法。
[5]前記粗精製物を混合する際に、前記溶媒が40~80℃の温度に維持されている、上記[1]~[4]のいずれかに記載の製造方法。
[6]種晶として、レバウディオサイドDを使用する、上記[1]~[5]のいずれかに記載の製造方法。
[7]前記晶析用溶液を、攪拌しながら35℃以下の温度となるまで冷却し、レバウディオサイドDを析出させる、上記[1]~[6]のいずれかに記載の製造方法。
[8]前記晶析用溶液を、攪拌しながら0.002~1.37℃/分の速度で冷却し、レバウディオサイドDを析出させる、上記[1]~[7]のいずれかに記載の製造方法。
[9]前記晶析用溶液を、1~48時間の間攪拌しながら冷却し、レバウディオサイドDを析出させる、上記[1]~[8]のいずれかに記載の製造方法。
[10]前記粗精製物が、
ステビア植物の乾燥葉を溶媒を用いて抽出して抽出物を得る、抽出工程、
前記抽出物を固液分離処理することで清澄液を得る、固液分離工程、
前記清澄液に凝集剤を添加して凝集させ、処理液を得る、凝集工程、
前記処理液を疎水性多孔質樹脂で処理する、樹脂精製工程、および
樹脂精製後の溶液を濃縮する、濃縮工程
を含む方法により得られたものである、上記[1]~[9]のいずれかに記載の製造方法。
[11]さらに、析出したレバウディオサイドDと液相とを分離し、分離後のレバウディオサイドDを乾燥する工程を含む、上記[1]~[10]のいずれかに記載の製造方法。
[12]さらに、
析出したレバウディオサイドDを分離し、乾燥して、一次晶析物を得る工程、
該一次晶析物を、レバウディオサイドDの10℃での過飽和度が10以上であり且つレバウディオサイドAの10℃での過飽和度が18以下になるように、メタノールまたはエタノールを含む溶媒中に混合して、一次晶析物溶解液を調整する工程、
前記一次晶析物溶解液を、攪拌しながら冷却し、レバウディオサイドDを析出させる工程、並びに、
析出したレバウディオサイドDを分離し、乾燥する工程
を含む、上記[1]~[10]のいずれかに記載の製造方法。
[13]レバウディオサイドD含有晶析物における総ステビオール配糖体に対するレバウディオサイドDの割合が35~95質量%である、上記[1]~[12]のいずれかに記載の製造方法。
[14]レバウディオサイドD含有晶析物における総ステビオール配糖体に対するレバウディオサイドAの割合が10~50質量%である、上記[13]に記載の製造方法。
[15]晶析回数を一回とした場合の、粗精製物中に含まれていたレバウディオサイドDに対する晶析したレバウディオサイドDの割合が、70~99質量%である、上記[1]~[14]のいずれかに記載の製造方法。
[16]上記[1]~[15]のいずれかに記載の製造方法により製造された、レバウディオサイドD含有晶析物。
[17]上記[16]に記載のレバウディオサイドD含有晶析物を含む飲食品。
[18]飲料である、上記[17]に記載の飲食品。
本発明の製造方法には、ステビア植物の粗精製物を用いる。ステビア植物の粗精製物は市販品を用いてもよく、ステビア植物、特にステビア植物の葉からの抽出物を精製して得てもよい。また、粗精製物として、市販品や抽出物を粗精製して得たものに対し、さらにRebDを添加したものを用いてもよい。
ステビア植物からの抽出物を粗精製して粗精製物を得る場合、ステビア植物からの抽出物は、市販品を用いてもよく、ステビア植物、特にステビア植物の葉を抽出処理して得てもよい。粗精製物を得る好適な方法としては、
(A)ステビア植物の乾燥葉を溶媒を用いて抽出して抽出物を得る、抽出工程、
(B)抽出物を固液分離処理することで清澄液を得る、固液分離工程、
(C)清澄液に凝集剤を添加して凝集させ、処理液を得る、凝集工程、
(D)処理液を疎水性多孔質樹脂で処理する、樹脂精製工程、および
(E)樹脂精製処理後の溶液を濃縮する、濃縮工程、
を含む方法が挙げられる。以下、この方法を、本発明の粗精製方法と呼ぶことがある。各工程について以下に説明する。
本発明の粗精製方法の一態様には、ステビア植物の乾燥葉を水性溶媒を用いて抽出して抽出物(抽出液)を得ることが含まれる。本明細書において、ステビア植物の乾燥葉とは、ステビア植物の新鮮葉を乾燥させることにより含水量を減らしたものをいう。ステビア植物の乾燥葉の含水率は、好ましくは1~10重量%、より好ましくは、2~8重量%、特に好ましくは3~4重量%である。ステビア植物の乾燥葉はステビオール配糖体が含まれていれば特に限定されないが、好ましくはRebDまたはレバウディオサイドM(RebM)の含有量が天然のステビア植物の乾燥葉よりも多いものが好ましい。そのようなステビア植物の乾燥葉は、例えば、国際公開第2019/074089号公報等に記載の方法で得ることができる。
本発明の粗精製方法の一態様においては、得られた抽出液を固液分離処理することで清澄液を得ることができる。固液分離処理としては、固体と液体が十分に分離されれば特に限定されないが、例えば、遠心分離器による処理や、メッシュによる処理、フィルタープレスによる処理が挙げられる。
固液分離処理は、複数の手段を用いてもよく、例えば、第1の固液分離処理の後に第2の固液分離処理を行って清澄液を得てもよい。
本発明の粗精製方法の一態様においては、固液分離処理によって得られた清澄液に凝集剤を添加させることで処理液を得ることができる。凝集剤としては、特に限定されず、公知の無機凝集剤や有機高分子凝集剤を用いることができる。本発明の他の態様において、凝集剤は、硫酸アルミニウム、ポリ塩化アルミニウム、塩化鉄(III)またはその水和物、合成高分子凝集剤(ポリアクリルアミド高重合体やポリアクリルアミドの部分加水分解物など)、アルギン酸、キチン、キトサン、水酸化カルシウムから選択される1つ以上である。凝集剤としてこれらの凝集剤の1つ以上が含まれていればよく、他の凝集剤がさらに含まれていてもよい。凝集剤は2種以上のものを組み合わせて使用してもよく、例えば、水酸化カルシウムと塩化鉄の組合せ、または水酸化カルシウムと塩化鉄とキトサンとの組み合わせなどで使用してもよい。
本発明の粗精製方法の一態様においては、凝集処理によって得られた処理液を疎水性多孔質樹脂で処理する。ステビオール配糖体は分子構造中に親水基と疎水基を持つ両親媒性であり、分子量は1,000前後である。また、pH2.5~9.0において安定であり、酸性・塩基性でもイオン化はしないことが知られている。一方、凝集処理を経た処理液にはステビオール配糖体以外の成分も大量に含まれている。理論に拘束されるものではないが、そのような成分には、鉄イオンのように分子量がステビオール配糖体と異なる成分やアミノ酸のようにイオン化する成分があり、これらの成分を、疎水性多孔質樹脂の処理によって除去することができると考えられる。
樹脂精製処理を得た溶液は、更に濃縮処理を行って水性溶媒を除去してもよい。そのような処理は特に限定されないが、加熱によって水性溶媒を蒸発させることや、減圧乾燥によって水性溶媒を除去する方法などが挙げられる。
本発明の製造方法に用いられる粗精製物は、粗精製物の総重量に対して50~95質量%の量の総ステビオール配糖体を含み、好適には、55~95、60~95、70~95、80~95、90~95、50~90、50~80、50~70、50~65、50~60、55~90、55~80、55~70、55~60、60~70、60~80、50~94、55~94、60~94のいずれかの量(質量%)の総ステビオール配糖体を含む。
続いて、任意の容器、例えば、攪拌翼を備えた結晶缶に溶媒を入れ、粗精製物を溶媒中に混合し、晶析用溶液を調製する。このとき、RebAが結晶化しないように、RebAの過飽和度(10℃)を特定の範囲に設定しながら、RebDの過飽和度(10℃)を特定の範囲に設定する。そして、そのような過飽和度が達成されるような量で粗精製物を溶媒中に混合する。
なお、過飽和度(10℃)とは、10℃を基準温度とした場合の過飽和度を意味する。10℃の冷却を必須とする意図はない。
σ=(C-C*)/C*
式中、σは過飽和度を、Cは、溶媒に対する物質の添加量の占める割合(ppm)を、C*は飽和溶解濃度(ppm)を示す。
なお、本明細書において、Cを単に「物質の濃度」ではなく「溶媒に対する物質の添加量の占める割合」としたのは、添加した物質が完全溶解せず懸濁状態となる場合を想定してのことである。本明細書では、添加した物質の溶解の有無及びその程度によらず、添加量の溶媒に対する割合を過飽和度の基準とする。
また、本発明においては、種晶を使用する場合があるが、過飽和度の計算に、この種晶の添加量は含めないものとする。
Cs=Ws/Wth
式中、Wsは種晶量(単位:g)を、Wthは想定収量(単位:g)を示す。
Wth=(C-C*)/1,000,000×溶媒量
式中、Cは溶媒に対するRebDの添加量の占める割合(ppm)を、
C*は飽和溶解度(単位:ppm)を示す。
溶媒量の単位はmlである。
Cには種晶の量は含めない。
任意の結晶装置、例えば円筒回転式助晶機等の助晶機にて、晶析用溶液を攪拌しながら冷却し、RebDを析出させる。
析出したRebDを含む固体組成物と液相とを遠心分離や濾別等により分離し、分離後のRebDを乾燥する。分離手段は、固体と液体が十分に分離されれば特に限定されないが、例えば、遠心分離器による処理や、メンブレンフィルターによる処理、メッシュによる処理が挙げられる。分離後乾燥前に、必要に応じて、晶析に用いたのと同じ溶媒等でRebD結晶に少量噴霧して表面を洗浄してもよい。
固液分離処理後の母液は、RebA、RebM等の他の成分の晶析に利用してもよい。
二回晶析を行う場合、上述の粗精製工程~析出工程までを実施し、析出したレバウディオサイドDを分離し、乾燥して、一次晶析物を得た後、上述した粗精製物に代えて一次晶析物をレバウディオサイドDの10℃での過飽和度が10以上であり且つレバウディオサイドAの10℃での過飽和度が18以下になるように、メタノールまたはエタノールを含む溶媒中に混合して、一次晶析物溶解液を調製し、上述した晶析用溶液に代えて一次晶析物溶解液を攪拌しながら冷却し、RebDを析出させ、析出したRebDを分離し、乾燥することで、RebD含有晶析物を得ることができる。二回晶析における手順や条件は、前述した晶析手順や晶析条件と同様にしてよい。その場合、上述の説明における「粗精製物」を「一次晶析物」に読み替えるものとする。
本発明の方法により得られるRebD含有晶析物(以下、本発明のRebD含有晶析物と呼ぶことがある。)は、RebDを多く含有している。好適には、本発明のRebD含有晶析物におけるTSGに対するRebDの割合(質量%)は、35~99、45~99、55~99、65~99、75~99、85~99、35~89、35~79、35~69、35~59、35~95、45~95、55~95、65~95、75~95、85~95、40~99、50~99、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のいずれかである。
別の態様において、本発明のRebD含有晶析物におけるTSGの割合(質量%)は、好適には、50以上、60以上、70以上または75以上である。
核発生および結晶成長の現象において、過飽和度は非常に大きな影響を与える。そこで、ステビア葉の主成分であるRebAおよびターゲット成分であるRebDの各溶媒における溶解度を測定した。
具体的には、所定のエタノール濃度(w/w)の溶媒100mlを50mlまたは100mlガラス容器(セパラブル丸底フラスコ,東京理化器械株式会社)に入れ、ウォーターバス(PCC-7000、東京理化器械株式会社)であらかじめ所定の温度にした後、200rpmで攪拌(SPZ-100、東京理化器械株式会社)し、RebA(J-100、守田化学株式会社)またはRebD(Jining Renewal&Joint International、China)を過剰量(懸濁するまでの量)投入した。24時間後、0.45umメンブレンフィルターでろ液を獲得し、ろ液中に含まれるRebAまたはRebD量をLCMS(島津社製、LCMS8050)にて測定し、溶解度曲線を作成した。結果を図1に示す。図1によれば、10℃、90%エタノール溶媒におけるRebA及びRebDの飽和溶解濃度はそれぞれ、8,250ppmと99.3ppmであった。
本実験では、溶媒に対するRebDの割合がRebDの回収率に与える影響を確認した。
(晶析条件)
Initial Temp 45℃
End Temp 10℃
RebD Jining Renewal&
Joint Internation
al製
Agitation speed 100rpm
Cs=Ws/Wth
式中、Wsは種晶量(g)を、Wthは想定収量(g)を、Csは種晶添加率(単位無し)を示す。
Wth=(C-C*)/1,000,000×溶媒量
式中、Cは溶媒に対するRebDの添加量(種晶の量は除く)の占める割合(ppm)を、C*は飽和溶解度(ppm)を示す。溶媒量の単位はmlである。
次いで、固液分離を行って結晶を得た。固液分離に用いたフィルターは、アドバンテック製0.45umメンブレンフィルターであった。
その後、RebD結晶を99質量%エタノールにて洗浄した。洗浄後の結晶を50℃にて乾燥した。
本実験では、溶媒として用いるエタノールの濃度が、RebD晶析物のTSG(総ステビオール配糖体)組成および収量に与える影響を検討した。
次いで、下記温度プロファイルおよび攪拌条件にて冷却し、RebD結晶を晶析した。晶析には、セパラブル丸底フラスコ(東京理化器械株式会社製)を用いた。また、エタノールとしては、未変性アルコール99.9質量%グレードを用い、これに適宜水を添加してエタノール濃度を調整した。なお、溶媒に対するRebDの比率は0.25質量%、溶媒に対するTSGの比率は13.8質量%、RebAの過飽和度(10℃)σAは5.10(90.50質量%エタノール溶媒使用時)、RebDの過飽和度(10℃)σDは24.37(90.50質量%エタノール溶媒使用時)、σD/σAは4.78(90.50質量%エタノール溶媒使用時)であった。
なお、過飽和度の算出式は以下のとおりである。初期濃度(溶媒に対する物質の添加量の占める割合)に種晶量は含まない。
σ=(初期濃度-10℃での飽和溶解度)/(10℃での飽和溶解度)
その後、RebD結晶を99質量%エタノールにて洗浄した。洗浄後のRebD結晶を50℃にて乾燥し、RebD晶析物が得られた。
得られたRebD晶析物とろ液の配糖体比率をLCMS(島津社製、LCMS8050)にて分析した。結果を図3および図4に示す。
図4より、エタノール濃度が低い場合、結晶の析出が悪い傾向であったが、図3は、RebD純度はエタノール濃度にあまり影響を受けないことを示唆した。本実験で採用した溶媒に対するTSGの比率(13.8%)においては最適なエタノール溶媒濃度が存在することがわかった。
冷却速度は結晶核の出現および結晶成長に大きく影響を与える。例えば、冷却速度が速い場合に、初期の少ない核の状態から結晶成長する際、過飽和が消費しきれず、核発生が優位となることが多い。一方、温度降下が遅い場合、結晶成長が優位となる。しかし、その場合、過飽和の消費速度にもよるが晶析時間は長くなる傾向にある。
本実験では冷却速度のTSG組成および収量への影響を検討した。
晶析時間は結晶成長に大きな影響を与えると同時に精製工程全体のタイムサイクルに影響を与える。結果として、晶析時間はコストや製品特性(収量、純度等)にも影響を与える。そのため、本実験では、晶析時間のTSG組成および収量への影響を検討した。
本実験では、RebDの更なる高純度化を目指すべく、一次晶析物を用いて二次晶析の実施の是非を検討した。具体的には、実験例2~5の結果を参考に、一次晶析条件を下記表のとおりに決定した。そして、表3の条件に代えて下記表の条件を採用した点以外は、実験例3と同様にして一次晶析を行い、RebD一次晶析物を得た。
本実験例では、一次晶析でRebDではなくRebAを晶析させる場合について検討した。
次いで、下記温度プロファイルおよび攪拌条件にて冷却し、RebA結晶を晶析した。晶析には、セパラブル丸底フラスコ(東京理化器械株式会社製)を用いた。また、エタノールとしては、未変性アルコール99.9質量%グレードを用いた。
次いで、固液分離を行ってRebA結晶を得た。固液分離に用いたフィルターは、アドバンテック製0.45umメンブレンフィルターであった。
その後、RebA結晶を99質量%エタノールにて洗浄した。洗浄後のRebA結晶を50℃にて乾燥した。
本実験では、研究室で行った小規模実験の結果得られた最適条件を採用してRebD晶析を行った。
次いで、下記温度プロファイルおよび攪拌条件にて冷却し、晶析した。晶析には、セパラブル丸底フラスコ(東京理化器械株式会社製)を用いた。また、エタノールとしては、未変性アルコール99.9質量%グレードを用い、これに適宜水を添加してエタノール濃度を調整した。
次いで、固液分離を行って結晶を得た。固液分離に用いたフィルターは、アドバンテック製0.45umメンブレンフィルターであった。
その後、結晶を99質量%エタノールにて洗浄した。洗浄後の結晶を50℃にて乾燥した。
ステビア植物の乾燥葉を用いて抽出操作を行い、得られた抽出物を固液分離操作に供した。乾燥葉において、TSGあたりの各ステビオール配糖体の割合(質量%)は、下記のとおりであった。本実験例で用いた乾燥葉中のTSGの含有量は、100g当たり11.8gであった。
攪拌機付き晶析用容器(容量1リットル)に、エタノール溶媒を投入し、加熱した。
エタノール溶媒の温度が約55℃になったところで、容器に粗精製物を投入した。
約350rpmで攪拌しながら、温度を下げた。このとき、冷却時間(すなわち11時間)-1時間(すなわち、10時間)で均等に冷却した。
晶析終了後、固液分離を行った。固液分離に用いたフィルターは、アドバンテック製0.45umメンブレンフィルターであった。
得られた結晶を60-70℃で乾燥した。
本実験は、エタノール溶媒中に多くのRebAが存在する場合、すなわち、RebAの過飽和度を高く設定した場合のRebD晶析について検討したものである。
ステビア乾燥葉(含水率:3~4重量%)の15倍量(質量)のイオン交換水を60℃±5℃に加熱し、その水にステビア乾燥葉を浸した。その後、ニーダー抽出器(SKN-R100、三友機器株式会社製)で8rpmの撹拌棒で撹拌しながら60分間抽出を行った。次に、18メッシュと140メッシュのメッシュを通して濾過し、冷水を用いて熱交換器で冷却し、ろ液をディスク型遠心分離機(9150rpm(11601G)、24L/min)で固液分離して一次抽出液を得た。その間に、濾過後の葉を再び同じ条件下で抽出し、固液分離して透明な二次抽出液を得、一次抽出液に加えて、清澄液を得た。
清澄液中の可溶性固形分の16.16質量%に相当する量のCa(OH)2(Brix(可溶性固形分濃度)から計算)を清澄液に添加し、得られた混合液を15分間撹拌した。その後、清澄液中の可溶性固形分の28.28質量%に相当する量のFeCl3・6H2Oを添加し、混合液を30分間撹拌し、クエン酸でpHを7に調整した後、清澄液中の可溶性固形分(g)の5.63倍に相当する体積(mL)の0.5%(w/v)キトサン溶液を添加した。この混合液を3分間強く撹拌し、2分間弱く撹拌し、そして10分間放置した。その後、電気的に中性の凝固沈殿物を遠心分離によって除去した。その結果、清澄な処理液が得られた。
樹脂精製としては、(i)陰イオン交換樹脂を用いた精製と(ii)疎水性多孔質樹脂(イオン交換基の導入がなされていないもの)を用いた精製とを行った。
(i)陰イオン交換樹脂を用いた精製
カラムに高多孔質の塩基性陰イオン交換樹脂(三菱ケミカル社製)を充填し、そのカラムに凝集分離後の処理液を投入して精製を行った。凝集分離後の処理液をカラムに投入したのに次いでカラムの容積の2倍量のイオン交換水で押し出し、精製されたステビオール配糖体組成物を含む溶液を回収した。この精製により、処理液中の黒色不純物や着色成分が除去された。
カラムに疎水性多孔質樹脂(三菱ケミカル社製)を充填し、そのカラムに(i)陰イオン交換樹脂を用いた精製後の試料を投入して精製を行った。疎水性多孔質樹脂としては、スチレンとジビニルベンゼンとの共重合体であって、イオン交換基を持たないものであり、最頻度細孔半径が45Åのものを用いた。上記(i)の精製後の溶液をカラムに投入後、カラムの容積の3倍量の0.01Mクエン酸水溶液、カラムの容積の3倍量の0.01M水酸化ナトリウム水溶液を用いてカラムを洗浄した。その後、カラムの容積の4倍量の60%エタノール水溶液でステビオール配糖体組成物を溶出し、回収した。
遠心式薄膜真空蒸発装置エバポール(大川原製作所製)を用いて、溶液を蒸発濃縮しながらエタノールを除去した。蒸発濃縮処理後も水が残存しており、組成物は液状であった。
RebA純度、RebD純度は、島津社製、LCMS8050により測定し、TSGに対する割合(w/w)で表す。
過飽和度の計算式は前述のとおりである。過飽和度の算出にあたり、RebAとRebDの飽和溶解度(90質量%EtOH、10℃)は、それぞれ以下のとおりであった。
RebA=8270.5ppm
RebD=99.33ppm
RebA単位工程収率は、晶析に用いた原料中のRebA量に対する、晶析物中に含まれるRebA量の割合を示す。RebD単位工程収率も同様である。
本実験は、晶析用の溶媒としてエタノール溶媒を使用したときに、メタノール溶媒使用時に比べてRebD晶析物の組成に変化が起こるかを検討したものである。
1.抽出・固液分離
ステビア乾燥葉(含水率:9~11重量%)の15倍量(質量)の水を60~65℃に加熱し、その水にステビア乾燥葉を浸した。その後、攪拌槽(容積:400L)において攪拌羽(半径27cm×2枚×2段)を用いて75rpmで撹拌しながら60分間抽出を行った。次に、ステビア乾燥葉と水の混合物を、直径95cmのブフナー漏斗にセットした100メッシュのナイロン製メッシュに通して濾過し、珪藻土(セライト503)を加え、フィルタープレスでろ過し、精密濾過膜(孔径:10μm)でろ過することで固液分離して一次抽出液を得た。その間に、濾過後の葉を再び同じ条件下で抽出し、固液分離して透明な二次抽出液を得、一次抽出液に加えて、清澄液を得た。
清澄液中の可溶性固形分の16.16質量%に相当する量のCa(OH)2(Brix(可溶性固形分濃度)から計算)を清澄液に添加し、得られた混合液を15分間撹拌した。その後、清澄液中の可溶性固形分の28.28質量%に相当する量のFeCl3・6H2Oを添加し、混合液を30分間撹拌し、クエン酸でpHを7に調整した後、清澄液中の可溶性固形分(g)の5.63倍に相当する体積(mL)の0.5%(w/v)キトサン溶液を添加した。この混合液を3分間強く撹拌し、2分間弱く撹拌し、そして10分間放置した。その後、電気的に中性の凝固沈殿物を遠心分離によって除去した。その結果、清澄な処理液が得られた。
樹脂精製としては、(i)陰イオン交換樹脂を用いた精製と(ii)疎水性多孔質樹脂(イオン交換基の導入がなされていないもの)を用いた精製とを行った。
(i)陰イオン交換樹脂を用いた精製
カラムに高多孔質の塩基性陰イオン交換樹脂(三菱ケミカル社製)を充填し、そのカラムに凝集分離後の処理液を投入して精製を行った。凝集分離後の処理液をカラムに投入したのに次いでカラムの容積の2倍量のイオン交換水で押し出し、精製されたステビオール配糖体組成物を含む溶液を回収した。この精製により、処理液中の黒色不純物や着色成分が除去された。
カラムに疎水性多孔質樹脂(三菱ケミカル社製)を充填し、そのカラムに(i)陰イオン交換樹脂を用いた精製後の試料を投入して精製を行った。疎水性多孔質樹脂としては、スチレンとジビニルベンゼンとの共重合体であって、イオン交換基を持たないものであり、最頻度細孔半径が45Åのものを用いた。上記(i)の精製後の溶液をカラムに投入後、カラムの容積の3倍量の0.01Mクエン酸水溶液、カラムの容積の3倍量の0.01M水酸化ナトリウム水溶液を用いてカラムを洗浄した。その後、カラムの容積の6倍量の60%エタノール水溶液でステビオール配糖体組成物を溶出し、回収した。
遠心式薄膜真空蒸発装置エバポール(大川原製作所製)を用いて、溶液を蒸発濃縮しながらエタノールを除去した。次いで、エバポレーターを用い、2段階に分けて濃縮を実施しエタノールを除去した。蒸発濃縮処理後も水が残存しており、組成物は液状であった。
セパラブルフラスコに、エタノール溶媒を投入し、加熱した。
エタノール溶媒の温度が約71℃になったところで、容器に粗精製物と種晶を投入した。なお、RebDの過飽和度(10℃)は162.854、RebAの過飽和度(10℃)は0.793であった。
攪拌しながら、温度を下げた。
晶析終了後、固液分離を行った。固液分離に用いたフィルターは、アドバンテック製0.45umメンブレンフィルターであった。
得られた結晶を60-70℃で乾燥した。
なお、種晶添加率(Cs)は0.08であった。
Claims (18)
- ステビア植物からの抽出物を粗精製して得られた粗精製物を用いる、レバウディオサイドD含有晶析物の製造方法であって、
前記粗精製物における総ステビオール配糖体含有量が50~95質量%であり、且つ、前記粗精製物が少なくともレバウディオサイドAとレバウディオサイドDとを含有しており、
前記粗精製物を、レバウディオサイドDの10℃での過飽和度が10以上であり且つレバウディオサイドAの10℃での過飽和度が18以下になるように、メタノールまたはエタノールを含む溶媒中に混合して、晶析用溶液を調整する工程、並びに、
前記晶析用溶液を、攪拌しながら冷却し、レバウディオサイドDを析出させる工程を含むことを特徴とする、レバウディオサイドD含有晶析物の製造方法。 - 前記総ステビオール配糖体が、レバウディオサイドA、レバウディオサイドB、レバウディオサイドC、レバウディオサイドD、ステビオシド、レバウディオサイドFおよびレバウディオサイドMである、請求項1に記載の製造方法。
- 前記粗精製物におけるレバウディオサイドAの含有量が5~70質量%であり、かつ、レバウディオサイドDの含有量が2~70質量%である、請求項1または2に記載の製造方法。
- 前記溶媒における前記メタノールまたはエタノールの濃度が99.9質量%以下である、請求項1~3のいずれか一項に記載の製造方法。
- 前記粗精製物を混合する際に、前記溶媒が40~80℃の温度に維持されている、請求項1~4のいずれか一項に記載の製造方法。
- 種晶として、レバウディオサイドDを使用する、請求項1~5のいずれか一項に記載の製造方法。
- 前記晶析用溶液を、攪拌しながら35℃以下の温度となるまで冷却し、レバウディオサイドDを析出させる、請求項1~6のいずれか一項に記載の製造方法。
- 前記晶析用溶液を、攪拌しながら0.002~1.37℃/分の速度で冷却し、レバウディオサイドDを析出させる、請求項1~7のいずれか一項に記載の製造方法。
- 前記晶析用溶液を、1~48時間の間攪拌しながら冷却し、レバウディオサイドDを析出させる、請求項1~8のいずれか一項に記載の製造方法。
- 前記粗精製物が、
ステビア植物の乾燥葉を溶媒を用いて抽出して抽出物を得る、抽出工程、
前記抽出物を固液分離処理することで清澄液を得る、固液分離工程、
前記清澄液に凝集剤を添加して凝集させ、処理液を得る、凝集工程、
前記処理液を疎水性多孔質樹脂で処理する、樹脂精製工程、および
樹脂精製後の溶液を濃縮する、濃縮工程
を含む方法により得られたものである、請求項1~9のいずれか一項に記載の製造方法。 - さらに、析出したレバウディオサイドDと液相とを分離し、分離後のレバウディオサイドDを乾燥する工程を含む、請求項1~10のいずれか一項に記載の製造方法。
- さらに、
析出したレバウディオサイドDを分離し、乾燥して、一次晶析物を得る工程、
該一次晶析物を、レバウディオサイドDの10℃での過飽和度が10以上であり且つレバウディオサイドAの10℃での過飽和度が18以下になるように、メタノールまたはエタノールを含む溶媒中に混合して、一次晶析物溶解液を調整する工程、
前記一次晶析物溶解液を、攪拌しながら冷却し、レバウディオサイドDを析出させる工程、並びに、
析出したレバウディオサイドDを分離し、乾燥する工程
を含む、請求項1~10のいずれか一項に記載の製造方法。 - レバウディオサイドD含有晶析物における総ステビオール配糖体に対するレバウディオサイドDの割合が35~95質量%である、請求項1~12のいずれか一項に記載の製造方法。
- レバウディオサイドD含有晶析物における総ステビオール配糖体に対するレバウディオサイドAの割合が10~50質量%である、請求項13に記載の製造方法。
- 晶析回数を一回とした場合の、粗精製物中に含まれていたレバウディオサイドDに対する晶析したレバウディオサイドDの割合が、70~99質量%である、請求項1~14のいずれか一項に記載の製造方法。
- 請求項1~15のいずれか一項に記載の製造方法により製造された、レバウディオサイドD含有晶析物。
- 請求項16に記載のレバウディオサイドD含有晶析物を含む飲食品。
- 飲料である、請求項17に記載の飲食品。
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CN114437152A (zh) * | 2021-12-31 | 2022-05-06 | 蚌埠市华东生物科技有限公司 | 一种甜菊糖苷水提液的脱色除杂方法 |
CN114437152B (zh) * | 2021-12-31 | 2024-03-26 | 蚌埠市华东生物科技有限公司 | 一种甜菊糖苷水提液的脱色除杂方法 |
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JPWO2021085643A1 (ja) | 2021-12-23 |
BR112022008220A2 (pt) | 2022-07-12 |
EP4052771A1 (en) | 2022-09-07 |
CN114599663A (zh) | 2022-06-07 |
EP4052771A4 (en) | 2023-10-25 |
JP2022166190A (ja) | 2022-11-01 |
AU2020374113A1 (en) | 2022-05-26 |
US20230027601A1 (en) | 2023-01-26 |
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