JPH06192283A - Method for production pure rebaudioside a - Google Patents

Abstract

PURPOSE:To separate rebaudioside A from a sweet component of stevia. CONSTITUTION:Rebaudioside A in a sweet component contained in Stevia rebaudiana Bertoni, a plant belonging to Compositae, is separated by a gel filtration method. A hydrophilic vinyl polymer is used as a filtration gel in the gel filtration.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the plant Asteraceae Stevia
The present invention relates to a method for producing pure rebaudioside A, which is a natural sweetener contained in the above-ground part of Rebaudiana beltney, for example, leaves.

[0002]

BACKGROUND OF THE INVENTION Scientific name Stevia rebaudiana Bertney ( Stevia rebaudiana Bert
oni ) is a perennial plant of the Asteraceae family originating in Paraguay, South America, and is generally abbreviated as stevia. As the sweet component contained in stevia, stevioside (C
₃₈ H ₆₀ O ₁₈ , molecular weight 804), rebaudioside A (C ₄₄ H ₇₀ O ₂₃ , molecular weight 966), rebaudioside C (C ₄₄ H ₇₀ O ₂₂ , molecular weight 950),
Zulcoside A (C ₃₈ H ₆₀ O ₁₇ , molecular weight 789)
Etc. are known. Among these sweetness components, stevioside has a sweetness 200 times that of sugar, and its sweetness is relatively similar to sugar, but has a residual taste such as bitterness. Rebaudioside C and Zulcoside A have the disadvantage that they have a sweetness 40 to 60 times that of sugar, and the sweetness is astringent. In contrast, rebaudioside A has a sweetness 300 times that of sugar, and its sweetness is mellow and has no residual taste similar to sugar.

[0003]

However, most of the sweeteners extracted from Stevia which are currently used in the food industry are a mixture of the above-mentioned sweetening ingredients, and therefore rebaudioside A
The advantage of is eliminated by other sweetening ingredients, especially rebaudioside C and zulcoside A, and the sweetener has bitterness and astringency as a whole.

Recently, a method of separating rebaudioside A, which has a good taste quality, from other sweet components by recrystallization or the like (Japanese Patent Laid-Open No. 62-62300) has been researched against this drawback. Rebaudioside A by actual recrystallization
Rebaudioside C behaves in a similar manner to rebaudioside A during the separation of erythrocytes, it is necessary to repeat recrystallization several times to isolate only rebaudioside A. The yield of Side A is drastically reduced and the cost becomes very high. Further, there are many problems in recrystallization, such as using a large amount of an organic solvent such as alcohol.

Therefore, in the field of foods, pharmaceuticals and the like, the present inventor has recently come to widely use the gel filtration method in the separation and isolation of active ingredients such as proteins, sugars, enzymes and nucleic acids. I focused on that. In gel filtration,
Materials are basically separated according to their molecular size. That is, molecules larger than the network of the swollen gel do not enter inside the gel particles, so they migrate through the column through the space between the gel particles and other gel particles and are first eluted from the carrier bed. . The small molecules also enter the gel particles, but the extent thereof varies depending on the size of the molecules of the sample substance, and as a result, the mixture containing solutions having different molecular weights is separated and eluted in the order of the size of the molecules. Thus, the separation of substances in gel filtration is mainly performed by the molecular sieving action, but other factors such as adsorption and ion exchange also have some influence.

As a filler for gel filtration currently used in the fields of foods, pharmaceuticals, etc., cellulose, agarose, dextran, etc. are used as raw materials, but when this is applied to stevia, it produces a stevia sweetening component and impurities. However, the rebaudioside A and other stevia sweetening components cannot be separated. That is, for the separation of the stevia sweet component and impurities, a dialysis method using a translucent one of a cellophane membrane, a collodion membrane or a fibrin acetate membrane, an ultrafiltration method using a collodion membrane or a hydrated gel membrane, and various carrier gels. A method for purifying stevioside, characterized in that the stevia extract is treated using any of the gel filtration methods, and the colored substance in the extract is separated by the molecular sieving effect (JP-B-57-267).
38), but it is impossible to isolate rebaudioside A from other sweetening ingredients by such means.

[0007]

Means for Solving the Problems The present inventor has focused on synthetic polymer gels as a filler for gel filtration, instead of those made of cellulose, agarose, dextran, etc.
From gels made of polyacrylamide, polyvinyl, polystyrene, etc., we found a gel made of a porous and hydrophilic vinyl polymer that does not strongly adsorb Stevia sweetening ingredients and other ingredients, and use this gel as a filtering agent. Thus, the rebaudioside A was successfully separated from the other sweetening components in the mixture of sweetening components contained in the Asteraceae plant Stevia rebaudiana Bertney, and the present invention was completed.

According to the gel filtration method of the present invention, a solution containing stevia sweetener containing rebaudioside A is injected into a column filled with a gel filtration agent made of a porous and hydrophilic vinyl polymer, and a developing solvent is used. By flowing water or an organic solvent such as ethanol or methanol at a constant flow rate,
Each Stevia sweetening ingredient is developed and eluted. UV eluate
Rebaudioside A measured with a detector (210 nm)
Is a method for producing pure rebaudioside A by separating and other sweet components.

The solution of the sweetening component contained in Stevia which is the starting material used in the present invention is a solution of Stevia leaves extracted with water or an organic solvent, a solution of crudely refined or highly refined powder, and rebaudioside A. Any solution may be used as long as it is a solution containing stevia and other sweet components. Preferably, a solution having a high rebaudioside A content, for example, a solution having a rebaudioside A content increased to some extent by recrystallization, is more preferable in terms of production efficiency.

Therefore, the solution containing the stevia sweetening ingredient of the present invention is a solution or extract of a glycoside containing steviol such as stevioside, rebaudioside A, rebaudioside C, and zulcocide A as an aglycone. The solution may contain an organic solvent, such as ethanol, if necessary.

The filler for gel filtration used in the present invention is made of a porous vinyl polymer having a large number of hydrophilic hydroxyl groups, and does not strongly adsorb Stevia sweetness component and other components in the solution or extract. It is a carrier, and for example, commercially available Toyopearl (trade name, manufactured by Toso Corporation) is desirable. The above Toyopearl has excellent strength, so that it can be used at a high flow rate and has excellent pressure resistance. The molecular weight fractionation range of the filler is that the molecular weight of the stevia sweetening ingredient is 789 to 966. There is 100-1
It should be around 0000. Fillers with a fractionation range of less than 100 or more than 10,000 are not suitable because they lack the ability to separate Stevia sweetness components by the difference in molecular weight. The particle size of the filler must be in the range of 5 to 300 μm when swollen in an aqueous solution. With a filler having a particle size of less than 5 μm, a very high pressure is applied when the Stevia solution is passed,
Physical damage to the filler occurs, making it impossible to separate the stevia sweetening ingredient. In addition, the machines and equipment need to withstand high pressure, which is not preferable because of enormous equipment investment. The particle size is 3
Fillers larger than 00 μm are not suitable because the Stevia sweetness component cannot be separated. However, more preferably in consideration of the separation ability of rebaudioside A and other sweetening components, 2
It is preferable that the particle diameter is uniform in the range of 0 to 100 μm.

The concentration of the Stevia sweetener solution used for gel filtration may be from 0.1 to 70% by weight. However, if it is less than 0.1%, the production efficiency will be high and the cost will be high.
If the content exceeds 100%, the Stevia extract or powder solution is solidified and is not suitable for gel filtration.

Water is most suitable as the developing solvent in terms of economic efficiency, but depending on the case such as putrefaction prevention, organic solvent such as 1 to 50% of ethanol or methanol may be used to separate rebaudioside A. It has no effect. However, 5
Organic solvents exceeding 0% are not suitable because they chemically deteriorate the filler. Further, the flow rate of the developing solvent must be SV (flow rate per hour with respect to the volume of the filler) of 0.05 to 3.0, and the separation capacity and the production efficiency of rebaudioside A and other sweetening components. The upper SV of 0.1 to 0.5 is more preferable. If the SV is less than 0.05, it takes a long time to separate the stevia sweetness component, which is not suitable for a commercial base.
At a flow rate exceeding SV3.0, the Stevia sweetness component is not separated and is inappropriate.

The rebaudioside A thus obtained is a pure product containing no other sweetening ingredients and impurities,
It can be used as a sweetener as it is. Also, if necessary,
This can be concentrated to give a syrup-like sweetener, or can be dried and powdered to give a powder-like sweetener. It goes without saying that the rebaudioside A sweetener isolated in the present invention can be used as a sweetening source for general foods, diet foods, medicines, quasi drugs, cigarettes, feeds and the like.

The method of the present invention and the rebaudioside A obtained by the present invention will be specifically described below with reference to examples. (Example-1) Powder refined stevia sweetener (composition: stevioside: 22.0%, rebaudioside A: 55.
2%, rebaudioside C: 7.1%, zulcocide A: 2.0%) 1 g, water 15 ml (Stevia concentration 6.6)
Toyopearl HW which is a gel filtration filler made of porous and hydrophilic vinyl polymer
-40 (trade name, manufactured by Tosoh Corporation, molecular weight fractionation range 100-
The glass column (size: φ22 × L1000) filled with 390 ml of 10000, particle diameter 20 to 40 μm was injected. Water was used as a developing solvent, and the solution was passed through at a flow rate of SV0.2, and the eluate was measured by a UV detector (210 nm). A peak was obtained (see Figure 1). The components of each peak were identified by high performance liquid chromatography. The peak at 920 minutes was rebaudioside A, the peak at 1070 was a mixture of stevioside and rebaudioside C, and the peak at 1260 was zulcocide A. Yes (see Figures 2-4). Fractions of each component were collected and dried under reduced pressure to obtain rebaudioside A: 550 mg, a mixture of stevioside and rebaudioside C: 290 mg, and zulcoside A: 30 mg. Further, the obtained rebaudioside A was analyzed by applying it to a nuclear magnetic resonance (NMR) device, and it was found to be pure rebaudioside A (see FIGS. 5 to 6).

(Example-2) Powder-purified stevia sweetener (composition: stevioside: 55.1%, rebaudioside A: 21.9%, rebaudioside C: 7.1)
%, Zulucoside A: 3.0%), 95 ml of methanol (50 ml) was added, and the mixture was allowed to stand at 4 ° C. to obtain 1.9 g of stevioside crystals. When 2.1 g of a solid product obtained by drying the crystal mother liquor under reduced pressure was analyzed by high performance liquid chromatography, rebaudioside A: 42.5%, stevioside: 14.0%, Baudioside C: 13.5%, Zulcoside A: 6.0
% Was included. 1.5 g of this solid is added to 15 ml of water
(Stevia concentration was 10.00%), and gel filtration was performed using the same glass column and packing material as in Example-1. 10% methanol was used as a developing solvent, the solution was passed at a flow rate of SV 0.2, and the eluate was measured by a UV detection scab (210 nm). As a result, the stevia sweetening component peak was obtained at the same retention time as in Example-1. After confirming each peak by high performance liquid chromatography, each fraction was dried under reduced pressure. Rebaudioside A: 63
0 mg, a mixture of stevioside and rebaudioside C: 405 mg, and zulcoside A: 85 mg were obtained.

(Example-3) Strength and taste quality test of sweetener Rebaudioside A of the product of the present invention obtained in Example-1
(Invention product No. 1) and the commercially available Stevia sweeteners, Control product No. 1 and Control No. 2, were dried at 105 ° C. for 4 hours to remove water, and then the invention product No. 1 was 0.045%, control product N.
Regarding o1 and No2, the difference between the strength of sweetness and the quality of sweetness of the 0.050% solution was found to be that of the product of the present invention and the control product No.
Comparison was made between 1 and the product of the present invention and control product No. 2 (Tables 2 to 3). Further, the rebaudioside A (invention product No. 2) obtained in Example-2 was compared with the control products No. 1 and No. 2 by the same method as described above. (Table-4
~ Table-5). The content of the sweetening component in each sample is shown in Table 1.

[0018]

The present invention has such a constitution, and it is speculated that the method of the present invention has not only the ordinary molecular sieving action but also the action of the stevia sweetening component repeating weak adsorption and desorption with respect to the gel filtration filler. By combining them, rebaudioside A could be isolated and purified from other sweetening components.

[0019]

[Effect] According to the method of the present invention, rebaudioside A, which has the best taste quality among Stevia sweetness components, can be industrially produced efficiently, at low cost, and safely.

[Brief description of drawings]

FIG. 1 Separation pattern of stevia sweetness components in gel filtration of Example 2.

FIG. 2 is a chromatogram of peak A in FIG. 1 measured by high performance liquid chromatography.

FIG. 3 is a chromatogram of peak B in FIG. 1 measured by high performance liquid chromatography.

FIG. 4 is a chromatogram of peak C in FIG. 1 measured by high performance liquid chromatography.

5 is an NMR chart of rebaudioside A in FIG.

6 is an NMR chart of rebaudioside A in FIG.

[Explanation of symbols]

 A Rebaudioside A B Mixture of Stevioside and Rebaudioside C C Zulcoside A B1 Stevioside B2 Rebaudioside C

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[Procedure amendment]

[Submission date] February 28, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

Water is most suitable as the developing solvent in terms of economic efficiency, but depending on the case such as putrefaction prevention, organic solvent such as 1 to 50% of ethanol or methanol may be used to separate rebaudioside A. It has no effect. However, 5
Organic solvents exceeding 0% are not suitable because they chemically deteriorate the filler. Further, the flow rate of the developing solvent must be SV (flow rate per hour with respect to the volume of the filler) of 0.05 to 3.0, and the separation capacity and the production efficiency of rebaudioside A and other sweetening components. The upper SV of 0.1 to 0.5 is more preferable. If the SV is less than 0.05, it takes a long time to separate the stevia sweetness component, which is not suitable for a commercial base.
At a flow rate exceeding SV3.0, the Stevia sweetness component is not separated and is inappropriate.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0015

[Correction method] Change

[Correction content]

The method of the present invention and the rebaudioside A obtained by the present invention will be specifically described below with reference to examples. (Example-1) Powder refined stevia sweetener (composition: stevioside: 22.0%, rebaudioside A: 55.
2%, rebaudioside C: 7.1%, zulcocide A: 2.0%) 1 g, water 15 ml (Stevia concentration 6.6)
Toyopearl HW which is a gel filtration filler made of porous and hydrophilic vinyl polymer
-40 (trade name, manufactured by Tosoh Corporation, molecular weight fractionation range 100 to
10000, the time of particle diameter swelling 20~40μm) 390m
l Filled glass column (size: φ22 × L1000
mm 2 ). Using water as a developing solvent, SVO.
Flow at a flow rate of 2 and the eluate is UV detector (210 nm)
When it was measured at, 920 minutes, 1070 minutes after deployment,
A stevia sweet component-like peak was obtained at the position of 1260 minutes (see FIG. 1). The components of each peak were identified by high performance liquid chromatography. The peak at 920 minutes was rebaudioside A, the peak at 1070 was a mixture of stevioside and rebaudioside C, and the peak at 1260 was zulcocide A. Yes (see Figures 2-4). Moreover, when each fraction of each component was collected and dried under reduced pressure, rebaudioside A: 550 mg,
Mixture of stevioside and rebaudioside C: 29
0 mg and Zlucoside A: 30 mg were obtained. Further, the obtained rebaudioside A was analyzed by applying it to a nuclear magnetic resonance (NMR) device, and it was found to be pure rebaudioside A (see FIGS. 5 to 6).

Claims (2)

[Claims] 1. Asteraceae plant Stevia rebaudiana
A method for producing pure rebaudioside A, which comprises subjecting a solution of a sweet component contained in Bertney to gel filtration to fractionally purify rebaudioside A. 2. The method for producing pure rebaudioside A according to claim 1, wherein a polyvinyl gel is used as a filler for gel filtration.