JP2011051909A - Method for purifying rebaudioside a - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating and highly purifying rebaudioside A from a mixture containing rebaudioside A and stevioside, particularly an industrially applicable method. <P>SOLUTION: The method for purifying rebaudioside A from a composition containing rebaudioside A and stevioside uses a simulated moving-bed chromatography separation apparatus having at least four packed towers packed with a resin having a molecular sieve action. The resin used here is a styrene-based gel-type strong acidic cation exchange resin having a moisture content of 50-57 mass%. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for preparing rebaudioside A, which is a natural sweetening component contained in Stevia rebaudiana, in a more pure form. More specifically, the present invention relates to a method for separating and purifying rebaudioside A from a mixture containing rebaudioside A and stevioside.

Stevia (scientific name: Stevia rebaudiana) is an Asteraceae perennial plant originating in South America, including Paraguay, and is generally abbreviated as stevia. Sweet ingredients contained in Stevia include Stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Zulcoside A, Rubusoside, steviolbioside and the like are known, and among these sweetening components, stevioside, rebaudioside A and rebaudioside C, particularly stevioside and rebaudioside A, mainly occupy the components of stevia extract. .
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. In addition, Rebaudioside C has only 40 to 60 times the sweetness of sugar, and its sweetness has a drawback that it has a strong astringency. On the other hand, rebaudioside A has a sweetness 300 times that of sugar, and its sweetness is similar to sugar and has no lingering residue, and is extremely useful as a high-intensity sweetener.

  However, since most of the stevia-derived sweeteners currently used in the food industry are a mixture of the above-mentioned sweetening ingredients, the advantages of rebaudioside A are other sweetening ingredients such as stevioside and rebaudioside C. It disappears, and it becomes a sweetener with bitterness and astringency as a whole.

To deal with this drawback, a recrystallization method is generally used as a method for preparing rebaudioside A having good taste quality with high purity (see, for example, Patent Document 1), but this recrystallization method is applied. In order to obtain this, an extract containing rebaudioside A at a concentration as high as 70% or more must be obtained at the extraction stage.
Furthermore, the recrystallization has a problem of using a large amount of an organic solvent such as alcohol.

  Therefore, conventionally, there is a demand for a method for obtaining rebaudioside A with high purity even from a raw material having a low content of rebaudioside A. For example, as a method for purifying rebaudioside A from a mixture of stevioside, rebaudioside A, rebaudioside C, and zulcoside A, a gel filtration filler using a hydrophilic vinyl polymer as a stationary phase is used. A method using gel filtration chromatography using water as a mobile phase has been proposed (see, for example, Patent Document 2), but this method limits the amount of test sample that can be purified at one time. Since purification of Dioside A requires 16 hours or more, there is a problem that it cannot be processed in a large amount and is difficult to use on an industrial scale.

JP 52-62300 A JP-A-6-192283

  An object of the present invention is to provide a method for purifying rebaudioside A. More specifically, the present invention is a method capable of obtaining rebaudioside A, which is a natural sweetening component contained in stevia, in particular in a high purity form by separating it from stevioside, and on an industrial scale. The purpose is to provide a method that can be used in

  Conventionally, a simulated moving bed (SMB) is known as an industrial separation method. The simulated moving bed method is a method for separating the two components into two fractions from a solution (stock solution) containing two or more components according to the difference in the moving speed of each component. More specifically, the method connects in series four or more unit packed towers filled with an adsorbent having a selective adsorption ability with respect to a target component in two or more components contained in the stock solution, By passing the stock solution and the eluent containing two or more components through the packed bed constituting the endless circular circulation system by connecting the unit packed column in the most downstream part and the unit packed column in the most upstream part, An adsorption zone is formed in the circulation system of the packed bed in the order of affinity for the adsorbent, and a raw solution supply port, an eluent supply port, and an extraction port for the A component (a component moving at a high speed in the packed bed) contained in the raw solution. By moving the extraction port of the B component (component with a low moving speed in the packed bed) contained in the stock solution to the downstream side in the fluid circulation direction of the packed bed while maintaining a fixed positional relationship, Remove the packed bed (stationary phase) from the eluent ( Is moved in the opposite direction to the flow of Dosho), the target component (A component or B component) is a method for continuously separating (see Publication No. JP 2003-61700).

  In order to separate and purify rebaudioside A from a test sample containing rebaudioside A and stevioside using the simulated moving bed method based on the principle of molecular sieve (gel filtration), first, An attempt was made to separate rebaudioside A from a stevia extract containing rebaudioside A and stevioside using chromatography using an ion exchange resin as a stationary phase. Specifically, as an ion exchange resin, Diaion UBK510L (moisture content 61-65%, degree of crosslinking: 4%) manufactured by Mitsubishi Chemical Corporation, which is a styrenic gel-type strongly acidic cation exchange resin, Diaion Separation was attempted using UBK530 (water content 52-55.5%, crosslinking degree 6%) and Diaion UBK550 (crosslinking degree 8%). Incidentally, the higher the degree of crosslinking, the smaller the pore size of the resin.

  As a result, when Diaion UBK 550 (moisture content 46-49.5%, crosslinking degree 8%) was used as the stationary phase (filler), the resin could not be separated by passage, while the stationary phase ( When Diaion UBK 510L (moisture content 61-65%, degree of cross-linking: 4%) was used as the filler, although it eluted in the bed volume, it could not be separated (see Experimental Example 1).

In contrast, when Diaion UBK530 (moisture content 52-55.5%, crosslinking degree 6%) was used as the stationary phase (filler), it tended to elute into the void volume and pass through the resin. As compared with Rebaudioside A, elution of stevioside tended to be delayed. Originally, Diaion UBK530 is a resin used for the purification and separation of substances with a molecular weight of 500 or less. Rebaudioside A (C ₄₄ H ₇₀ O ₂₃ , molecular weight 967) and stevioside have a large molecular weight relative to the resin. It is considered that (C ₃₈ H ₆₀ O ₁₈ , molecular weight 805) cannot be separated and purified.

  However, although the present inventors tended to use the Diaion UBK530 to cause both of the rebaudioside A and stevioside substances to elute into the void volume and pass through the resin, surprisingly, there was a slight amount of resorption. We found that elution of stevioside tended to be delayed as compared to baudioside A, and found the possibility of separating both.

  Therefore, based on this finding, we attempted purification of rebaudioside A using a simulated moving bed method in which columns packed with the above-mentioned resin were connected. As a result, the elution from the simulated moving bed method was repeated. It was confirmed that rebaudioside A can be sequentially separated from stevioside and highly purified rebaudioside A can be obtained. For this reason, a styrenic gel-type strongly acidic cation exchange resin having a moisture content of more than 49.5% and less than 61%, specifically, a moisture content of 50 to 57% is used as the resin to be packed in the packed tower. It was found that by performing simulated moving bed chromatography separation, rebaudioside A can be separated from stevioside and purified to high purity.

  The present invention has been completed on the basis of such findings, and rebaudioside A is obtained from a method for purifying rebaudioside A having the following constitution, more specifically, a mixture containing rebaudioside A and stevioside. The present invention relates to a method for separation and purification.

Item 1. Rebaudioside A and stevioside are separated by using a simulated moving bed type chromatographic separation apparatus equipped with a simulated moving bed in which at least four packed towers packed with a resin having molecular sieve action are connected in series. A method for purifying baudioside A, comprising:
A step of using, as the resin, a styrenic gel-type strongly acidic cation exchange resin having a water content of 50 to 57% by weight, and (a) providing a test sample containing rebaudioside A and stevioside to the simulated moving layer ,
(B) recovering the eluent corresponding to the peak fraction from the simulated moving layer using the absorbance at an absorption wavelength of 210 nm as an index,
(C) The method as described above, further comprising the step of subjecting the eluent to the steps (a) and (b) at least once.

  Item 2. In the above step (c), the “weight ratio of Rebaudioside A to Stevioside (Reba A / Stev)” in the eluent recovered in Steps (a) and (b) is Item 2. The method according to Item 1, which is larger than the “weight ratio (Reba A / Stev) of rebaudioside A to stevioside” in the eluent immediately before being subjected to a) and (b).

  Item 3. Item 3. The method according to Item 1 or 2, further comprising a step of subjecting the recovered eluent to a reverse osmosis membrane concentration treatment after the step (b).

  Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the test sample containing rebaudioside A and stevioside is a stevia extract containing rebaudioside A and stevioside.

  Item 5. The rebaudio having a “weight ratio of Rebaudioside A to Stevioside (Reba A / Stev)” of 20 or more, preferably 30 or more, which is purified by the method according to any one of Items 1 to 4. Side A-containing composition.

  Item 6. Item 6. A sweetener comprising the rebaudioside A-containing composition according to item 5.

  According to the method of the present invention, rebaudioside A having the best taste quality among natural sweetening components derived from stevia can be industrially prepared efficiently and at low cost. More specifically, according to the method of the present invention, rebaudioside A can be efficiently separated and purified from a mixture of rebaudioside A and stevioside.

The conceptual diagram of the simulated moving bed type | formula chromatographic separation apparatus used by this invention is shown. (A) is equipped with a reverse osmosis membrane treatment machine (RO machine) downstream of five packed towers, and (B) is equipped with a reverse osmosis membrane treatment machine (RO machine) downstream of five packed towers. Indicates the device. In the figure, the meaning of each symbol is as follows. 1: raw material tank, 2: developing solvent tank, 3: simulated moving bed, 3-1: packed tower 1, 3-2: packed tower 2, 3-3: packed tower 3, 3-4: packed tower 4, 3 -5: packed tower 5, 4: UV measuring section, 5: purified liquid recovery tank, 6: developing solvent recovery tank, 7: reverse osmosis membrane concentrator (RO machine), 8: RO treatment liquid recovery tank, 9: circulation Liquid tank. In Experimental Example 1, Stevia sweetener (Stevilon TK, containing 52% stevioloside, 25% rebaudioside A) and styrenic cation exchange resin (Diaion UBK-550 (crosslinking degree 8, moisture content 46-49.5%): Mitsubishi The chromatogram of the result of having performed the gel filtration chromatography using Chemical Co., Ltd. is shown. In Experimental Example 1, Stevia sweetener (Stevilon TK, containing 52% stevioside, 25% rebaudioside) and styrenic anion exchange resin (Diaion WA-30 (water content 43-55%): Mitsubishi Chemical Corporation) The chromatogram of the result of having performed gel filtration chromatography using is shown. In Experimental Example 1, Stevia sweetener (Stevilon TK, stevioside 52%, rebaudioside 25% contained) and styrenic cation exchange resin (Diaion UBK-530 (crosslinking degree 6, moisture content 52-55.5%): Mitsubishi Chemical) The chromatogram of the result of having performed gel filtration chromatography using Co., Ltd. is shown. In Experimental Example 2, Stevia sweetener (containing Steviron TK, stevioside 52%, rebaudioside A 25%) was supplied to a simulated moving bed type chromatographic separation apparatus (FIG. 1 (A)) equipped with five packed towers. The result of the second separation operation, that is, the elution pattern of rebaudioside A (-■-) and stevioside (-◆-) In Experimental Example 2, the fraction recovered in the first separation operation (first elution fraction) was subjected to a simulated moving bed chromatographic separation apparatus (FIG. 1 (A)) equipped with five packed towers. The result of the second separation operation, that is, the elution pattern of rebaudioside A (-■-) and stevioside (-◆-) is shown. In Experimental Example 2, the fraction collected in the second separation operation (second elution fraction) was subjected to a simulated moving bed chromatographic separation apparatus (FIG. 1 (A)) equipped with five packed towers. The result of the third separation operation, that is, the elution pattern of rebaudioside A (-■-) and stevioside (-◆-) is shown. In Experimental Example 3, a stevia sweetener (stevilon TK, containing 52% stevioloside, 25% rebaudioside A, 12% rebaudioside C) and a simulated moving bed chromatographic separation apparatus equipped with five packed towers (FIG. 1 (B)) Shows the results of the first separation operation, that is, the elution pattern of rebaudioside A (-■-) and stevioside (-◆-) In Experimental Example 3, the fraction recovered in the first separation operation (first elution fraction) was subjected to a simulated moving bed chromatographic separation apparatus (FIG. 1 (B)) equipped with five packed towers. The result of the second separation operation, that is, the elution pattern of rebaudioside A (-■-) and stevioside (-◆-) In Experimental Example 3, the fraction collected by the second separation operation (second elution fraction) was subjected to a simulated moving bed chromatographic separation apparatus (FIG. 1 (B)) equipped with five packed towers. The result of the third separation operation, that is, the dissolution pattern of rebaudioside A (-■-) and stevioside (-◆-)

  The present invention is a method of using a simulated moving bed type chromatographic separation apparatus (hereinafter referred to as “SMB chromatographic separation apparatus”) for rebaudioside A purification.

  Hereinafter, the method of the present invention will be described with reference to the conceptual diagram of the SMB chromatographic separation apparatus described in FIGS. 1 (A) and 1 (B). However, the apparatus described in FIG. 1 is an embodiment of the SMB chromatographic separation apparatus used in the method of the present invention, and the method of the present invention is not limited to the apparatus.

As shown in FIG. 1A, the SMB chromatographic separation apparatus used in the method of the present invention comprises a “raw material tank 1 ” containing a raw material, a “developing solvent tank 2 ” containing a developing solvent, and a resin having a molecular sieving action. “Pseudo moving layer 3 ” formed by connecting five packed towers in series, “UV detecting unit 4 ” for measuring the absorbance of the eluent eluted from the pseudo moving layer 3, and the peak detected by the UV detecting unit 4 “Purified liquid collection tank 5 ” for collecting the eluent corresponding to the fraction (near wavelength 210 nm) and “Developing solvent collection tank 6 ” for collecting the eluate other than the above peak fraction (near wavelength 210 nm) are provided. . Since the eluent that does not contain rebaudioside A does not necessarily have to be recovered, “developing solvent recovery tank 6 ” in FIG. 1 is an optional part.

  Although FIG. 1 illustrates an embodiment in which five packed towers are connected in series, the packed tower constituting the “pseudo-moving bed 3” is at least four packed towers filled with a resin having a molecular sieve action. What is necessary is just to connect in series. Specifically, there are four types: a packed tower for extracting a portion rich in rebaudioside A, a packed column for extracting a portion rich in stevioside, a packed column for supplying raw materials, and a packed column for separating rebaudioside A and stevioside. The number of packed towers can be adjusted as appropriate as long as it has. It is desirable to have five or more packed towers.

Examples of the resin packed in the packed tower constituting the “pseudo moving layer 3 ” include a styrenic gel-type strongly acidic cation exchange resin having a molecular sieve action. Specifically, the resin is a linear chain obtained by suspension polymerization of styrene with one vinyl group on the benzene ring and divinylbenzene (DVB) with two vinyl groups on the benzene ring. Polystyrene, which is a polymer of the above, is crosslinked by DVB. A sulfonic acid, a quaternary ammonium salt or the like is introduced into this as a functional group. In the resin, the size of the molecular network (micropores) in the resin can be appropriately adjusted according to the amount of DVB used to bridge the polystyrenes during the suspension polymerization. When the amount of DVB is increased, the number of bridges increases and the mesh becomes denser. On the other hand, when DVB is less, the number of bridges decreases and the mesh becomes larger.

  Preferred examples include styrenic gel type strongly acidic cation exchange resins having a sulfate group as a functional group, and more preferred examples include gel type strongly acidic cation exchange resins made of sodium styrenesulfonate.

  It is known that the degree of crosslinking, ie, the degree of crosslinking, in such a gel type strongly acidic cation exchange resin is correlated with the water content of the resin (for example, “DIAION 1 Ion Exchange Resin / Synthetic Adsorbent Manual” (See page 48, Mitsubishi Chemical Corporation). As described above, the resin is produced by suspension polymerization using styrene and divinylbenzene as materials. If the amount of water during production is small, the crosslinking reaction of styrene and divinylbenzene proceeds, but the amount of moisture is large. This is because the cross-linking reaction is difficult to proceed due to moisture between them. Therefore, the degree of crosslinking in the ion exchange resin can be indicated by the water content in the resin. The “water content” (mass%) in the resin can be determined from the weight difference before and after the resin at room temperature is dried at 105 ° C. for 6 hours according to the following equation.

  Examples of the water content of the styrenic gel-type strongly acidic cation exchange resin used in the present invention include 50 to 57% by mass. Preferably, 52-55.5 mass% can be mentioned. The resin having the moisture content has a crosslinking degree of 5 to 7, preferably 5.5 to 6.5, more preferably 6 with 6 as the center.

  In addition, examples of the styrenic gel-type strongly acidic cation exchange resin used in the present invention include those having a resin particle size distribution of 200 to 240 μm (85% or more). Here, “200 to 240 μm (85% or more)” means that the diameter of 85% or more of the resin is in the range of 200 to 240 μm. The diameter of the particles can be measured using a sieve. In this case, specifically, the proportion of particles that pass through a sieve having a pore size of 240 μm and that do not pass through a sieve having a pore size of 199 μm is 85% or more of the total. The particle size distribution is 200 to 240 μm (85% or more).

  As such a resin, specifically, a commercially available product such as Diaion UBK-530 (crosslinking degree 6, water content 52 to 55.5%) (above, manufactured by Mitsubishi Chemical Corporation) can be used.

The “pseudo moving layer 3 ” is formed by connecting at least four packed columns (separation columns) packed with such a resin. The number of packed towers may be 4 or more, preferably 5 or more, and the upper limit is not limited, but can usually be about 10. Each packed layer may be filled with the same resin, but is not limited thereto, and may be filled with a different degree of crosslinking and / or particle size distribution as long as the above is true. The same resin is preferable.

  The size of the packed tower and the amount of resin to be packed can be appropriately set according to the amount of rebaudioside A to be purified, and are not particularly limited. If an example is given, for example, when 20 to 100 ml of stevia extract containing 10 to 20 g of rebaudioside A is used as a test sample, the total amount of resin used may be 3500 to 7000 ml. Such an amount of resin is divided into at least four packed columns (separation columns) and used.

  The developing solvent used for SMB is not particularly limited, but preferably includes a hydrous lower alcohol. Here, examples of the lower alcohol include alcohols having 1 to 6 carbon atoms, preferably alcohols having 1 to 3 carbon atoms. Ethanol is preferable. Examples of the alcohol concentration of the hydrous alcohol include 20 to 80% by volume, preferably 30 to 60% by volume, and more preferably 40 to 50% by volume.

  The raw material to be used for SMB, that is, the test sample may be one containing at least rebaudioside A and stevioside, and may further contain rebaudioside C. Preferably, the ratio of rebaudioside A contained in the test sample is 5 to 80% by weight, more preferably 10 to 30% by weight.

  Conventionally, the recrystallization method that has been generally used as a method for preparing rebaudioside A with high purity obtains an extract containing rebaudioside A at a high concentration of 70% or more before recrystallization. Had the drawback of having to do. However, if the purification method according to the present invention is used, rebaudioside A is highly separated and purified even from a test sample having a low content of 10 to 30% by weight of rebaudioside A. It has a very good advantage that

As for those containing rebaudioside A and stevioside, stevia sweetener containing stevia (Stevia rebaudiana Bertoni) extract or stevia extract containing rebaudioside A and stevioside prepared from this Can be mentioned.
For example, as stevia sweeteners available on the market, “Steviron TK” manufactured by Morita Chemical Co., Ltd., “Stevia DIC CP” manufactured by DIC Lifetech Co., Ltd., “Stevia ST-AB” manufactured by Ikeda Sakuka K.K. And “Stevia Fin H” manufactured by Nippon Paper Chemical Co., Ltd., “Pure Marmilon” manufactured by Maruzen Pharmaceutical Co., Ltd. and the like.

The method of the present invention can be carried out by performing the following steps (a) to (c) using the aforementioned SMB chromatographic separation apparatus:
(A) providing a test sample containing rebaudioside A and stevioside to the simulated moving layer;
(B) recovering the eluent corresponding to the peak fraction from the simulated moving layer using the absorbance at an absorption wavelength of 210 nm as an index,
(C) A step of subjecting the eluent to the steps (a) and (b) at least once again.

  This will be described with reference to FIG.

In FIG. 1A, the developing solvent supplied from the “developing solvent tank 2 ” circulates in the “pseudo moving layer 3 ”. Among them, the raw material (test sample) is added from the “raw material tank 1 ” to the upstream portion of the “pseudo moving bed 3 ”, that is, the upstream portion of the first packed tower 1 (reference numeral 3-1). The method starts (step (a)).

Thus, the test sample provided to the “pseudo moving bed 3 ” is mixed with the developing solvent in the pseudo moving bed 3 , that is, in the “packed tower 1 → packed tower 2 → packed tower 3 → packed tower 4 → packed tower 5”. And is eluted from the downstream portion of the last packed column 5 (reference numeral 3-5). The eluted eluent is measured for absorbance at an absorption wavelength of 210 nm in the “UV measurement unit 4 ”. The absorption wavelength 210 nm corresponds to the maximum absorption wavelength of rebaudioside A. Here, using the absorbance as an index, the eluent corresponding to the peak fraction is recovered in the “purified liquid recovery tank 5 ” (step (b)). On the other hand, the eluent that is not so is recovered in the “developing solvent recovery tank 6 ”.

The solution recovered in the “purified liquid recovery tank 5 ” is then concentrated as necessary. Although the method used for such concentration is not particularly limited, it can be simply concentrated under reduced pressure. In this case, the alcohol and water contained in the developing solvent are evaporated by concentration under reduced pressure, and the alcohol concentration of the developing solvent changes. For this reason, after the concentration, the alcohol concentration is adjusted to be the same as the developing solvent supplied to the “pseudo moving layer 3 ”.

The solution thus prepared is sent to the “raw material tank 1 ” and again supplied to the “pseudo moving layer 3 ” (step (a) in step (c)). Specifically, it is added to the upstream portion of the upstream portion of the "simulated moving layer 3 'to above, i.e. the first packed column 1 (reference numeral 3-1)," packed column of the "simulated moving layer 3" 1 → packing tower 2 → packing tower 3 → packing tower 4 → packing tower 5 ”, and then the eluent eluted from the packed tower 5 (reference numeral 3-5) is measured in the“ UV measuring section 4 ”. Using the absorbance at an absorption wavelength of 210 nm as an index, the eluent corresponding to the peak fraction is recovered in the “purified liquid recovery tank 5 ” (step (b) in step (c)).

  The solution thus recovered can be concentrated again as necessary until the rebaudioside A is separated and purified, and subjected to the above-described steps (a) and (b).

  The eluent thus obtained, that is, the eluent that was supplied to the steps (a) and (b) in step (c) and collected as the peak fraction, was “rebaudioside A and It is characterized in that the “weight ratio with stevioside” (hereinafter referred to as “Reba A / Stev”) is larger than “Reba A / Stev” in the eluent immediately before being subjected to the step (a). That is, every time step (a) and step (b) are performed in step (c), the ratio (weight) of rebaudioside A contained in the eluent becomes larger than the ratio of stevioside according to the number of times. The “Reba A / Stev” becomes larger than “Reba A / Stev” in the eluent immediately before being subjected to the step (a).

  In this case, an index for separation and purification of rebaudioside A is not particularly limited, and for example, “Reba A / Stev” in the eluent may be 20 or more. Preferably it is 30 or more, more preferably 35 or more.

In the above, the concentration under reduced pressure is exemplified for the concentration of the solution recovered in the “purified liquid recovery tank 5 ”, but a reverse osmosis membrane can be used instead. FIG. 1B shows a conceptual diagram of a simulated moving bed chromatographic separation apparatus having a concentration step using such a reverse osmosis membrane (RO). According to the concentration by the reverse osmosis membrane (RO), since the alcohol concentration in the solution does not change, the obtained concentrated liquid can be supplied to the raw material tank 1 again without adjusting the alcohol concentration.

  Here, as the membrane material used for the reverse osmosis membrane, components such as rebaudioside A, stevioside, and rebaudioside C contained in the sample do not permeate, and a solvent such as water or alcohol is selectively used. Any material that permeates may be used, and natural, synthetic, and semi-synthetic materials are not particularly limited. Examples thereof include cellulose, cellulose di-acetate or tri-acetate, polyamide, polysulfone, polystyrene, polyimide, polyacrylonitrile and the like. As an example, "NTR-729HR" manufactured by Nitto Denko Co., Ltd., which is a low-pressure RO membrane with a high permeate flow rate with a NaCl rejection of 90% or more, using a synthetic polymer composite membrane (PVA) as a reverse osmosis membrane. In addition, “ES10-D” manufactured by Nitto Denko Corporation, which is an ultra-low pressure RO membrane with a high permeate flow rate with a NaCl rejection of 99.5% or more, can be exemplified.

In this case, the solution recovered in the “purified liquid recovery tank 5 ” is supplied to the RO machine 7 where it is concentrated. The solution containing the rebaudioside A concentrated here is sent to the “raw material tank 1 ” and supplied again to the “pseudo moving layer 3 ”, and the “pseudo moving layer 3 ” in the “packing tower 1 → filling” It is purified through “column 2 → packed tower 3 → packed tower 4 → packed tower 5” (step (a) in step (c)). Next, the eluent eluted from the packed column 5 (reference numeral 3-5) is the “eluent” corresponding to the peak fraction, with the absorbance at the absorption wavelength of 210 nm measured by the “UV measuring section 4 ” as an index. It is recovered in the recovery tank 5 ”(step (b) in step (c)).

  The solution thus recovered is concentrated again as necessary until the rebaudioside A is separated and purified as described with reference to FIG. 1 (A), and the above (a) and (b). Provided to the process.

On the other hand, the solution that does not contain rebaudioside A and the like produced by concentration in the RO machine 7 is collected in the RO treatment liquid collection tank 8 . The solution is supplied to the circulating liquid tank 9 and repeatedly used as a developing solvent.

  In such an SMB chromatographic separation apparatus, the flow rate of the developing solvent varies depending on the size of the packed column and the amount of the test sample to be separated, but can usually be 5 to 30 ml / min, preferably 10 to 15 ml / min. .

  In the SMB chromatographic separation apparatus, the separation and purification of rebaudioside A is terminated with an indication that “Reba A / Stev” in the eluent is 80 or more, preferably 90 or more, more preferably 98 or more. Can do.

  The eluent thus obtained can be concentrated or dried as necessary and provided as a sweetener containing rebaudioside A with high purity.

  Hereinafter, the contents of the present invention will be specifically described using the following experimental examples. However, these experimental examples are only one aspect of the present invention, and the present invention is not limited to these examples. Unless otherwise specified, “%” means “mass%”.

Experimental example 1
The gel filtration method is a method of separating a target product from components having different molecular weights according to the pore size of a network structure formed by a stationary phase (resin), and the components having a high molecular weight are eluted.

  Using a stevia sweetener containing rebaudioside A and stevioside as raw materials, gel filtration chromatography is performed using the following three types of resins ((a), (b), (c)) as stationary phases, and gel filtration is used. Exploring the possibility of separation.

(1) Raw material solution Stevia sweetener containing rebaudioside A and stevioside (Stevilon TK: stevioside 52%, rebaudioside A 25% included: Morita Chemical Co., Ltd.) 1g of ethanol containing 25% ethanol by volume Use what was dissolved in.

(2) Gel filtration chromatography (i) Apparatus: A glass column having a diameter of 50 mm and a length of 50 cm is filled with 700 ml of the following resin, and the same five are connected in series.
(Ii) Eluent: Hydrous ethanol with an ethanol concentration of 55% by volume (iii) Stationary phase: Use one of the following packing materials packed in a glass column (column diameter 450 mm x 600 mm) (500 ml)
(a) Gel-type strongly acidic cation exchange resin of sodium styrenesulfonate (Diaion UBK-550 (crosslinking degree 8, water content 46-49.5%): Mitsubishi Chemical Corporation)
(b) Styrenic dimethylamine porous weakly basic anion exchange resin (Diaion WA-30: (water content 43-55%) Mitsubishi Chemical Corporation)
(c) Sodium styrenesulfonate gel-type strongly acidic cation exchange resin (Diaion UBK-530 (crosslinking degree 6, moisture content 52-55.5%): Mitsubishi Chemical Corporation)
(Iv) Flow rate: About 10 ml / min with a metering pump
(V) Detection method: Flow cell detector (UV210nm).

(3) Experiments and results FIG. 2 shows gel filtration chromatography using the resin (a) (Diaion UBK-550) as a filler. As can be seen from FIG. 2, both rebaudioside A and stevioside were eluted with the void volume of the resin (Diaion UBK-550). This indicates that all of these components pass through voids (void volumes) without entering the pore diameter of the resin. Moreover, since both peaks are sharp, it was confirmed that neither rebaudioside A nor stevioside adsorbs to the resin.

  FIG. 3 shows gel filtration chromatography using the resin (b) (Diaion WA-30) as a filler. Usually, the porous ion exchange resin has a larger pore size than the gel ion exchange resin. As can be seen from FIG. 3, both rebaudioside A and stevioside were eluted with the bed volume of the resin (Diaion WA-30). This indicates that all of these components fall within the pore size of the resin. However, since both peaks are broad, it was confirmed that both rebaudioside A and stevioside have adsorptivity to the resin. However, such a resin could not separate both components.

  Although not shown in the examples, sodium styrenesulfonate gel-type strongly acidic cation exchange resin (Diaion UBK-510L (crosslinking degree 4, water content 61-65%): Mitsubishi Chemical Corporation) is used as the resin. Even when used, it was not possible to separate both components of rebaudioside A and stevioside.

  FIG. 4 shows gel filtration chromatography using the resin (c) (Diaion UBK-530) as a filler. As can be seen from FIG. 4, as with Diaion UBK-550, both Rebaudioside A and Stevioside were eluted with the void volume of the resin (Diaion UBK-530). This indicates that these components pass through the voids without entering the pore diameter of the resin. However, although it was a little, elution of rebaudioside A tended to be delayed more than elution of stevioside, and it was considered that both components could be separated by the resin.

Experimental example 2
From the results of Experimental Example 1, the resin (Diaion UBK-530) of (c), that is, the gel-type strongly acidic cation exchange resin of sodium styrenesulfonate, was used as the stationary phase (filler) of the simulated moving bed chromatographic separation apparatus. And rebaudioside A was purified by the following method.

(1) Raw material solution to be purified Stevia sweetener containing rebaudioside A and stevioside (containing stevio TK: stevioside 52%, rebaudioside A 25%: Morita Chemical Co., Ltd.) 1.5 g was dissolved in 10 ml of water. I used something.

(2) SMB conditions
(a) Eluent: Hydrous ethanol with an ethanol concentration of 55 vol%
(b) Stationary phase (filler): Sodium styrenesulfonate gel type strongly acidic cation exchange resin (Diaion UBK530 (crosslinking degree 6, moisture content 52-55.5%): Mitsubishi Chemical Corporation)
(c) Packing tower (column): Five columns made of glass (column diameter 450 mm × 600 mm) packed with the above packing material are used (total amount of packing material: 3500 ml).
(d) Raw material flow rate: 9.4ml / min
(e) Eluent flow rate: 9.4ml / min
(f) Column temperature: Room temperature 24 ° C
(g) Cycle time: 480 minutes.

(3) Experiments and Results Hereinafter, the separation operation will be described with reference to the schematic diagram of the simulated moving bed chromatographic separation apparatus shown in FIG. 1 (A) and FIGS.

(i) First separation operation (see Fig. 5)
First, using the simulated moving bed type chromatographic separation apparatus (FIG. 1 (A)) provided with the above five packed towers 3-1 to 3-5 from the “raw material tank 1 ”, the above raw material liquid is divided into two cycles. Treated as a cycle. FIG. 5 shows the elution pattern of rebaudioside A (-■-) and stevioside (-♦-) in such a separation operation (two cycles). In the elution pattern of the first circulation (0 to 240 minutes), although the ratio of rebaudioside A / stevioside is high in the 140 to 145 minutes of the beginning of elution (140 minutes: 4, 143 minutes: 2, 145 minutes: 1 ), It is reversed from 145 minutes to 240 minutes thereafter, and the ratio is low (160 minutes: 0.32, 170 minutes: 0.31).

  Subsequently, the elution fraction from 142 minutes to 170 minutes was collected and subjected to the second circulation treatment. That is, 142 to 170 minutes of the liquid eluted from the packed tower 5 (reference numeral 3-5) was returned to the packed tower 1 (reference numeral 3-1), and the second circulation treatment was performed. The other part (170 minutes to 300 minutes) was collected in a developing solution recovery tank, and a new developing solvent (55% EtOH) was added to the developing solvent tank. In the second cycle elution (300 to 480 minutes), the rebaudioside A / stevioside ratio was higher in the 300 to 350 minutes than in the first cycle. Recovered. This purified solution was then concentrated in vacuo on a rotary evaporator, diluted with water-containing alcohol (55% EtOH), and then used for purification in the next second separation operation.

(ii) Second separation operation (see Fig. 6)
A simulated moving bed chromatographic separation apparatus equipped with the above five packed towers (Fig. 1) was used to recover 40 ml of the first purified solution collected and concentrated in the first separation operation and adjusted with hydrous alcohol (55% EtOH). (A)) was supplied to the stock solution supply port (upstream portion of packed tower 1 (reference numeral 3-1)) and eluted from the downstream portion of packed tower 5 (reference numeral 3-5). Next, the elution fraction of 138 to 170 minutes is returned to packed column 1 (reference numeral 3-1) and circulated twice, and the elution fraction of 290 to 314 minutes of the second circulation is recovered in “purified liquid recovery tank 5”. The other parts were collected in the developing solution collection tank. The recovered purified solution was then concentrated in vacuo on a rotary evaporator, diluted with water-containing alcohol (55% EtOH), and used for purification in the second separation operation (second elution fraction). The elution pattern of rebaudioside A (-■-) and stevioside (-◆-) in such a separation operation is shown in FIG.

(iii) Third separation operation (see Fig. 7)
A simulated moving bed chromatographic separation apparatus (figure 1) was prepared by using 15 ml of the second purified solution recovered and concentrated in the second separation operation and adjusted with hydrous alcohol (55% EtOH). (A)) The raw solution supply port (upstream part of packed tower 1 (symbol 3-1)) is eluted from the downstream part of packed tower 5 (symbol 3-5) and filled with an elution fraction of 138 to 150 minutes Returned to the tower 1 (reference numeral 3-1) and circulated twice (the spot of the graph is not described), the fractions 276 to 314 in the second circulation were collected in the “purified liquid collection tank” (the third elution fraction) ). The elution pattern of rebaudioside A (-■-) and stevioside (-◆-) in such a separation operation is shown in FIG.

  The ratio of rebaudioside A and stevioside contained in the first elution fraction, the second elution fraction and the third elution fraction obtained above was analyzed by HPLC under the following conditions. The weight ratio (RebaA / Stev) between A (RebaA) and stevioside (Stev) was determined.

<HPLC conditions>
Apparatus: JASCO HPLC2000 series
Stationary phase: Asahipak NH2P-50 manufactured by Shodex
Mobile phase: CH3CN: H ₂ O = 80:20 Isocratic flow rate: 1 ml / min
Detection wavelength 210nm.

  As a result, in the first separation operation, the weight ratio (RebaA / Stev) of rebaudioside A (RebaA) to stevioside (Stev) of steviron TK (raw material) was 0.48. The ratio increases to 0.94 for the minute, further increases to 1.44 for the second elution fraction, and further to 21.0 for the third elution fraction. As the separation operation is repeated, the ratio of rebaudioside A increases, and rebaudioside increases. It was confirmed that A was sequentially purified.

  In FIG. 1 (A), a stevia sweetener (Stevilon TK: stevioside 52%) may be used in the same manner as described above even if a simulated moving bed chromatographic separation apparatus having four packed towers instead of five packed towers is used. , rebaudioside A 25% content: Morita Chemical Co., Ltd.) confirmed that rebaudioside A can be purified.

Experimental example 3
According to Experimental Example 2, it was found that rebaudioside A and stevioside were separated slightly by using resin UBK530, and it was convinced that rebaudioside A could be gradually purified by repeating the above operation. However, since this operation is a batch process with two cycles as one cycle, it is difficult to perform on an industrial scale. Therefore, it was examined to perform this operation continuously. If the rebaudioside A / stevioside ratio is constant, it is known that the elution pattern is the same. Therefore, a flow UV meter is installed in the portion where the eluate flows, and the start of elution is detected and recovery is started. Thereafter, the valve is switched by a timer signal after a certain period of time, and the recovery of the purified liquid (FIG. 1 (B): purified liquid recovery tank 5) and the non-purified liquid (circulating liquid recovery tank 6) is performed by recovering to another place. It becomes possible.

  The developing solvent circulating in the packed towers 1 to 5 cannot be used for circulation because the components are tailing, and the recovered liquid (purified liquid recovery section and non-purified liquid recovery section) is new. It is necessary to add a developing solvent. In order to solve this problem, an RO machine (RO concentrator) was incorporated into the circulation. The RO machine is characterized in that low-molecular components such as water and alcohol are permeated and components such as rebaudioside A are not permeated. For this reason, since the liquid which permeate | transmitted RO machine 7 becomes equivalent to the developing solvent before use, a developing solvent becomes unnecessary and it becomes possible to continue circulating the same solvent by RO machine introduction. Further, since the RO concentrated solution has no change in the hydrous alcohol concentration, the alcohol adjustment performed in Example 1 is not necessary. Therefore, when the RO concentrated solution reaches a certain concentration, it can be returned to the raw material tank as it is. Thus, the batch system used in Experimental Example 2 could be converted into a batch continuous system by introducing the RO machine and controlling the computer. This system is shown in FIG.

  Using the resin (Diaion UBK-530) of (c) as the stationary phase (packing agent) of the simulated moving bed chromatographic separation device, that is, the gel type strongly acidic cation exchange resin of sodium styrenesulfonate, by the following method Rebaudioside A was purified.

(1) Raw material solution to be purified Stevia sweetener containing rebaudioside A and stevioside (Stevilon TK: stevioside 52%, rebaudioside A 25% contained: Morita Chemical Co., Ltd.) 20g dissolved in 40ml water It was used.

(2) SMB conditions
(a) Eluent: Hydrous ethanol with an ethanol concentration of 50% by volume
(b) Stationary phase (filler): Sodium styrenesulfonate gel type strongly acidic cation exchange resin (Diaion UBK530 (crosslinking degree 6, moisture content 52-55.5%): Mitsubishi Chemical Corporation)
(c) Packing tower (column): Five columns made of glass (column diameter 450 mm × 600 mm) packed with the above packing material are used (total amount of packing material: 3500 ml).
(d) Raw material flow rate: 9.0ml / min
(e) Eluent flow rate: 9.0ml / min
(f) Column temperature: Room temperature 24 ° C
(g) Cycle time: 480 minutes
(h) Detection: UV absorbance at 210 nm is measured.

(3) Experiments and Results Hereinafter, the separation operation will be described with reference to the schematic diagram of the simulated moving bed chromatographic separation apparatus shown in FIG. 1 (B) and FIGS.

(i) First separation operation (see Fig. 8)
First, the above raw material liquid is circulated from the “raw material tank 1” using a simulated moving bed chromatographic separation apparatus (FIG. 1 (B)) having the above five packed towers (reference numerals 3-1 to 3-5). Was treated as one cycle (FIG. 8). That is, in the first circulation, the elution fraction from 142 minutes to 180 minutes was returned to the packed column 1 (reference numeral 3-1), and the elution fraction from 180 to 300 minutes was sent to the circulating liquid recovery tank 6 without being circulated. Further, the elution fraction of 300 to 350 minutes was sent to the purified liquid collection tank 5, and the elution fraction of 350 to 480 minutes was sent to the circulating liquid collection tank 6. Hydrous alcohol (50% EtOH) was used as the developing solvent at the start of the separation, but after that, the recovered liquid sent to the circulating liquid recovery tank 6 and the purified liquid recovery tank 5 was used as a reverse osmosis membrane device (RO machine) 7 ( The permeate treated with NTR-729HR (manufactured by Nitto Denko) was sent to the RO treatment liquid recovery tank 8 and further sent to the circulating liquid tank 2 to be used as a developing solvent. The RO membrane treatment concentrated liquid in the purified liquid recovery tank 5 was sent to the raw material tank 1 as a raw material for the next second separation operation. The rebaudioside A / stevioside ratio of the purified liquid obtained by the first separation operation increased from 0.48 of the raw material to 1.05. FIG. 8 shows the total elution pattern of rebaudioside A (-■-) and stevioside (-◆-) in this operation.

(ii) Second separation operation (see Fig. 9)
A raw liquid obtained by concentrating the purified liquid obtained in the first separation operation on the RO membrane is used as a simulated moving bed chromatograph equipped with the above five packed towers (reference numerals 3-1 to 3-5) from the “raw material tank 1 ”. Using the separator (FIG. 1 (B)), two cycles were again processed as one cycle. That is, in the first circulation, the elution fraction of 142 to 190 minutes was returned to the packed column 1 (reference numeral 3-1), and the elution fraction of 190 to 310 minutes was sent to the circulating liquid recovery tank 6 without being circulated. Further, the elution fraction of 310 to 360 minutes was sent to the purified liquid collection tank 5, and the elution fraction of 360 to 480 minutes was sent to the circulating liquid collection tank 6 . Hydrous alcohol (50% EtOH) was used as the developing solvent at the start of the separation, but the permeate obtained by treating the recovered liquid sent to the circulating liquid recovery tank 6 and the purified liquid recovery tank 5 with the RO membrane was used as the RO treatment liquid. It was sent to the recovery tank 8 and further sent to the circulating fluid tank 6 to be used as a developing solvent. Further, the RO membrane treatment concentrated liquid in the purified liquid recovery tank 5 was sent to the raw material tank 1 as a raw material for the next third separation operation. By the second separation operation, the rebaudioside A / stevioside ratio of the purified liquid was increased from 1.05 to 2.52 of the raw material. FIG. 9 shows the entire dissolution pattern of rebaudioside A (-■-) and stevioside (-◆-) in this operation.

(iii) Third separation operation (see FIG. 10)
A raw liquid obtained by concentrating the purified liquid obtained in the second separation operation on the RO membrane is a simulated moving bed type equipped with the five packed towers (reference numerals 3-1 to 3-5) from the “raw material tank 1”. Using the chromatographic separation apparatus (FIG. 1 (B)), two cycles were again processed as one cycle. That is, in the first circulation, the elution fraction of 142 to 190 minutes was returned to the packed column 1 (reference numeral 3-1), and the elution fraction of 190 to 310 minutes was sent to the circulating liquid recovery tank 6 without being circulated. Further, the elution fraction for 310 to 380 minutes was sent to the purified liquid collection tank 5, and the elution fraction for 380 to 480 minutes was sent to the circulating liquid collection tank 6 . Hydrous alcohol (50% EtOH) was used as the developing solvent at the start of the separation, but after that, the permeate treated with the RO membrane was recycled to the circulating fluid recovery tank 6 and the purified fluid recovery tank 5 with RO treatment. It was sent to the liquid recovery tank 6 and further sent to the circulating liquid tank 9 to be used as a developing solvent. By the third separation operation, the rebaudioside A / stevioside ratio of the purified solution (RO membrane treatment concentrate) increased from 2.52 to 34.2. FIG. 10 shows the total dissolution pattern of rebaudioside A (-■-) and stevioside (-◆-) in this operation.

  The ratio of rebaudioside A and stevioside contained in the RO membrane treatment concentrate obtained by the first separation operation, the second separation operation and the third separation operation obtained above, Analysis was performed in the same manner as in Experimental Example 2, and the weight ratio (RebaA / Stev) between rebaudioside A (RebaA) and stevioside (Stev) was determined.

  As a result, in the first separation operation, the weight ratio (RebaA / Stev) of rebaudioside A (RebaA) and stevioside (Stev) of steviron TK (raw material) was 0.48. The separation operation increased to 1.05, the second separation operation increased to 2.52, and the third separation operation increased to 34.2. As the separation operation was repeated, the ratio of rebaudioside A increased. It was confirmed that baudioside A was sequentially purified.

  In FIG. 1 (B), a stevia sweetener (Stevilon TK: stevioside 52%) may be used in the same manner as described above even if a simulated moving bed chromatographic separation apparatus having four packed columns instead of five packed columns is used. , rebaudioside A 25% content: Morita Chemical Co., Ltd.) confirmed that rebaudioside A can be purified.

Claims (6)

Rebaudioside A and stevioside are separated by using a simulated moving bed type chromatographic separation apparatus equipped with a simulated moving bed in which at least four packed towers packed with a resin having molecular sieve action are connected in series. A method for purifying baudioside A, comprising:
A step of using, as the resin, a styrenic gel-type strongly acidic cation exchange resin having a water content of 50 to 57% by mass, and (a) providing a test sample containing rebaudioside A and stevioside to the simulated moving layer ,
(B) recovering the eluent corresponding to the peak fraction from the simulated moving layer using the absorbance at an absorption wavelength of 210 nm as an index,
(C) The method as described above, further comprising the step of subjecting the eluent to the steps (a) and (b) at least once. In the above step (c), the “weight ratio of Rebaudioside A to Stevioside (Reba A / Stev)” in the eluent recovered in Steps (a) and (b) is The method according to claim 1, characterized in that it is greater than the "weight ratio (Reba A / Stev) between rebaudioside A and stevioside" in the eluent immediately before being subjected to a) and (b). . The method according to claim 1 or 2, further comprising a step of subjecting the recovered eluent to a reverse osmosis membrane concentration treatment after the step (b). The method according to any one of claims 1 to 3, wherein the test sample containing rebaudioside A and stevioside is a stevia extract containing rebaudioside A and stevioside. The rebau produced by the method according to any one of claims 1 to 4, wherein the "weight ratio of rebaudioside A to stevioside (Reba A / Stev)" is 20 or more. Dioside A-containing composition. A sweetener comprising the rebaudioside A-containing composition according to claim 5.

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