CN103570778A - Preparation method of high-purity Rebaudioside A - Google Patents
Preparation method of high-purity Rebaudioside A Download PDFInfo
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- CN103570778A CN103570778A CN201310532269.XA CN201310532269A CN103570778A CN 103570778 A CN103570778 A CN 103570778A CN 201310532269 A CN201310532269 A CN 201310532269A CN 103570778 A CN103570778 A CN 103570778A
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Abstract
The invention discloses a preparation method of high-purity Rebaudioside A, which is implemented by taking sulfoacid-based chromatographic packing as a stationary phase and taking an organic solvent aqueous solution as a moving phase through the steps of chromatographic column balancing, sample feeding and eluting, so that high-purity Rebaudioside A is obtained. According to the invention, the adopted sulfoacid-based chromatographic packing made of a rigid inorganic medium skeleton material is stable in performance, so that obtained products are high in purity, therefore, the method is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method of high purity content rebaudioside-A, belong to food chemistry technical field.
Background technology
Stevioside is a kind of natural sweeteners, is the mixture of diterpene glucoside.Wherein content rebaudioside-A (RA) is acknowledged as the best substitute of sucrose and other synthetic sweetening agents.Western developed country after 2008, particularly U.S. FDA give RA(purity >=97%) GRAS (It is generally accepted safe material) status amplifies rapidly the demand of high purity RA on Hou， world market.
At present, the preparation method who discloses highly purified RA has recrystallization method and chromatography.Recrystallization method be utilize each glucosides of stevioside organic solvent as methyl alcohol in solubleness difference and repeatedly crystalline go out highly purified RA, this method operation is loaded down with trivial details, yield is lower, solvent consumption is large and cost is higher.
People (the Preparative purification of rebaudioside A from aqueous extracts using chromatography:a process idea such as Dominik Bergs, J. Verbr. Lebensm., 2012 (7): 295 – 303) adopt 3 step chromatogram series systems for RA, its final step treating process adopts primary amine groups (NH (CH
2)
2nH
2) silica matrix chromatogram packing, eluent is 80% acetonitrile solution, finally obtains purity to be
96.8%rA, this step yield is
97.5%.
US Patent No. 20120083593 discloses the process for purification of a kind of RA.Its core is to adopt primary amine groups (NH (CH
2)
2nH
2) resin, this resin belongs to weak base anion-exchange resin, the skeleton of resin is polystyrene-divinylbenzene or polymethacrylate, with water-miscible organic solvent, as the aqueous acetone solution wash-out of the combination of methyl alcohol, Virahol, acetone, acetonitrile or 5%-40%, through crystallization, obtain the RA component of purity > 98%.
Above-mentioned two kinds of methods all adopt primary amine groups filler, but the shortcoming such as primary amine groups filler exists facile hydrolysis, and the filler life-span is short, thereby finally can cause the raising of product cost.
Summary of the invention
The object of the present invention is to provide a kind of preparation method who is suitable for the content rebaudioside-A of suitability for industrialized production, to overcome primary amine groups filler facile hydrolysis, poor stability, short deficiency of filler life-span.
The present invention is achieved through the following technical solutions:
A preparation method for content rebaudioside-A, it comprises the following steps:
(1) chromatographic column balance: pass into moving phase balance chromatographic column in chromatographic column;
(2) loading: by the dissolving crude product that contains content rebaudioside-A in moving phase, pump in chromatographic column or pass through content rebaudioside-A crude product moving phase solution and chromatograph packing material mix, dried chromatograph packing material is put into the column cap of chromatographic column, described chromatograph packing material is sulfonic group or Phenylsulfonic acid base chromatograph packing material;
(3) wash-out: with ultraviolet or ELSD detector monitors, be eluted to content rebaudioside-A with moving phase and go out peak from chromatographic column and finish.
The skeleton of described sulfonic group or Phenylsulfonic acid base chromatograph packing material is silica gel, aluminum oxide or zirconium dioxide, is preferably silica gel, and functional group is sulfonic group-SO
3 -r or Phenylsulfonic acid base-phe-SO
3 -r, R is H
+, Na
+, K
+, Ca
2+or Mg
2+.
Described sulfonic group or Phenylsulfonic acid base chromatograph packing material are commercial Sepra
tMsCX, ZORBAX 300SCX or Intersil CX.
In above-mentioned preparation method, moving phase is methanol aqueous solution, aqueous ethanolic solution, isopropanol water solution, aqueous acetone solution or acetonitrile solution, in step (1) and (2), the moving phase when moving phase of balance chromatographic column and loading is the aqueous acetone solution of volume ratio 85-95%.
In step (3), moving phase during wash-out is isocratic elution or multistage isocratic elution or linear gradient elution.If employing isocratic elution, moving phase is the aqueous acetone solution of volume ratio 85-95%; If adopt multistage isocratic elution, first adopt the aqueous acetone solution of volume ratio 85-95% to be eluted to rebaudioside C and go out peak and finish, then switch to aqueous acetone solution or the pure water wash-out lower than volume ratio 85%; If employing linear gradient elution, moving phase concentration during loading is to pure water solution.
The flow velocity of above-mentioned chromatographic column balance, loading, each step of wash-out is 50-600cm/hr, is preferably 200-250cm/hr.
Advantage of the present invention and positively effect:
1, US Patent No. 20120083593 adopts superpolymer framework materials, easy swelling and to cause post to be pressed unstable, and even moving phase is difficult to flow, and the present invention adopts rigid inorganic medium framework material, can overcome its deficiency;
2, the people's such as US Patent No. 20120083593 and Dominik Bergs scheme all adopts the primary amine groups filler of poor stability, facile hydrolysis, and the filler life-span is short, and the present invention adopts stable sulfonic group to overcome its deficiency;
3, the content rebaudioside-A purity that the inventive method obtains is high, is suitable for suitability for industrialized production.
Embodiment
Following examples are used for further illustrating the present invention, but the mode described in embodiment that do not represent is to implement unique channel of the present invention, does not also mean that any limitation of the invention.
Embodiment 1 isocratic elution
60% purity RA is so kind as to give by Qufu Sheng Ren pharmaceutical Co. Ltd, with 90% (v/v) acetone-water solution preparation, becomes 10g/L sample solution standby.By Sepra
tMsCX(Phenomenex) silica matrix sulfonic group chromatograph packing material homogenate method packs 10*250 mm stainless steel chromatogram post into, dress column pressure 100 bar.Then use 100ml 90% (v/v) the acetone-water solution all identical therewith with the following step flow velocity of 225cm/hr() stand-by after flow velocity balance chromatographic column.
First pump into the above-mentioned RA sample solution preparing of 22.7 mL, then with 90% acetone-water eluant solution substep, collect elutriant and amount to 290 mL.After HPLC detects, merge the component that RA purity is higher, obtain RA purity 98.09% product 13.28g, yield is 97.50%.
Embodiment 2 gradient elutions
The preparation of sample solution is with embodiment 1, and the filling of chromatographic column and balance are with embodiment 2.
First pump into the above-mentioned RA sample solution preparing of 22.7 mL, then with 90% acetone-water eluant solution and with HPLC, monitor, when RA starts to spill, moving phase switches to pure water, collects pure water elutriant 25ml, obtain RA purity 97.32% product 13.35g, yield is 98.02%.
Embodiment 3
Adopt and embodiment 1 and 2 similar approach, different is to adopt commercialization chromatographic column ZORBAX 300SCX (Agilent Technologies) or Intersil CX (GL Sciences), moving phase is that acetonitrile solution obtains RA purity and is all greater than 97%, and yield is all greater than 97%.
Claims (10)
1. a preparation method for content rebaudioside-A, is characterized in that, comprises the following steps:
(1) chromatographic column balance: pass into moving phase balance chromatographic column in chromatographic column;
(2) loading: the dissolving crude product that contains content rebaudioside-A, in moving phase, is pumped in chromatographic column or by by content rebaudioside-A crude product moving phase solution and chromatograph packing material mix, dried chromatograph packing material is put into the column cap of chromatographic column; Chromatograph packing material is sulfonic group or Phenylsulfonic acid base chromatograph packing material;
(3) wash-out: with ultraviolet or ELSD detector monitors, be eluted to content rebaudioside-A with moving phase and go out peak from chromatographic column and finish.
2. the preparation method of content rebaudioside-A according to claim 1, is characterized in that: the skeleton of described sulfonic group or Phenylsulfonic acid base chromatograph packing material is silica gel, aluminum oxide or zirconium dioxide, and functional group is sulfonic group-SO
3 -r or Phenylsulfonic acid base-phe-SO
3 -r, R is H
+, Na
+, K
+, Ca
2+or Mg
2+.
3. the preparation method of content rebaudioside-A according to claim 2, is characterized in that: the skeleton of sulfonic group or Phenylsulfonic acid base chromatograph packing material is silica gel.
4. the preparation method of content rebaudioside-A according to claim 2, is characterized in that: sulfonic group or Phenylsulfonic acid base chromatograph packing material are commercial Sepra
tMsCX, ZORBAX 300SCX or Intersil CX.
5. the preparation method of content rebaudioside-A according to claim 2, is characterized in that: moving phase is methanol aqueous solution, aqueous ethanolic solution, isopropanol water solution, aqueous acetone solution or acetonitrile solution.
6. the preparation method of content rebaudioside-A according to claim 2, is characterized in that: in step (1) and (2), the moving phase when moving phase of balance chromatographic column and loading is the aqueous acetone solution of volume ratio 85-95%.
7. the preparation method of content rebaudioside-A according to claim 2, is characterized in that: in step (3), moving phase during wash-out is isocratic elution or multistage isocratic elution or linear gradient elution.
8. the preparation method of content rebaudioside-A according to claim 7, is characterized in that: the moving phase of isocratic elution is the aqueous acetone solution of volume ratio 85-95%.
9. the preparation method of content rebaudioside-A according to claim 7, it is characterized in that: the multistage is during isocratic elution, first adopt the aqueous acetone solution of volume ratio 85-95% to be eluted to rebaudioside C and go out peak and finish, then switch to aqueous acetone solution or the pure water wash-out lower than volume ratio 85%.
10. the preparation method of content rebaudioside-A according to claim 7, is characterized in that: moving phase concentration when linear gradient elution is loading is to pure water solution.
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| CN103570778B CN103570778B (en) | 2017-07-28 |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5241275A (en) * | 1975-09-27 | 1977-03-30 | Ajinomoto Kk | Production of sweetening agent |
| WO2006038221A1 (en) * | 2004-10-04 | 2006-04-13 | Council Of Scientific And Industrial Research | Process for production of steviosides from stevia rebaudiana bertoni |
| US20070082102A1 (en) * | 2005-10-11 | 2007-04-12 | Stevian Biotechnology Corporation Sdn. Bhd | Sweetner and use |
| WO2009140394A1 (en) * | 2008-05-13 | 2009-11-19 | Cargill, Incorporated | Separation of rebaudioside a from stevia glycosides using chromatography |
| CN101941997A (en) * | 2006-12-15 | 2011-01-12 | 成都华高药业有限公司 | Stevia rebaudiana Bertoni extract and extraction method thereof and extraction method of rebaudioside A |
| JP2011051909A (en) * | 2009-08-31 | 2011-03-17 | Sanei Gen Ffi Inc | Method for purifying rebaudioside a |
| WO2012042508A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and purification of stevioside and rebaudioside a |
-
2013
- 2013-11-04 CN CN201310532269.XA patent/CN103570778B/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5241275A (en) * | 1975-09-27 | 1977-03-30 | Ajinomoto Kk | Production of sweetening agent |
| WO2006038221A1 (en) * | 2004-10-04 | 2006-04-13 | Council Of Scientific And Industrial Research | Process for production of steviosides from stevia rebaudiana bertoni |
| US20070082102A1 (en) * | 2005-10-11 | 2007-04-12 | Stevian Biotechnology Corporation Sdn. Bhd | Sweetner and use |
| CN101941997A (en) * | 2006-12-15 | 2011-01-12 | 成都华高药业有限公司 | Stevia rebaudiana Bertoni extract and extraction method thereof and extraction method of rebaudioside A |
| WO2009140394A1 (en) * | 2008-05-13 | 2009-11-19 | Cargill, Incorporated | Separation of rebaudioside a from stevia glycosides using chromatography |
| JP2011051909A (en) * | 2009-08-31 | 2011-03-17 | Sanei Gen Ffi Inc | Method for purifying rebaudioside a |
| WO2012042508A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and purification of stevioside and rebaudioside a |
Non-Patent Citations (4)
| Title |
|---|
| XIN-YI HUANG 等: "Preparative isolation and purification of steviol glycosides from Stevia rebaudiana Bertoni using high-speed counter-current chromatography", 《SEPARATION AND PURIFICATION TECHNOLOGY》, 31 December 2010 (2010-12-31), pages 220 - 224 * |
| 刘英超 等主编: "《中枢神经系统疾病蛋白质组学》", 30 September 2010, article "第二节 高效液相色谱法", pages: 113-115 * |
| 刘超 等: "高效液相色谱法测定甜叶菊糖中的甜菊苷和莱鲍迪苷A", 《分析试验室》, vol. 26, no. 7, 31 July 2007 (2007-07-31), pages 23 - 26 * |
| 张全香 等: "接枝大孔吸附树脂HPD-T01纯化甜菊总昔的研究", 《第16届反应性高分子学术讨论会》, 31 July 2012 (2012-07-31), pages 97 - 98 * |
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