CN103570778B - A kind of preparation method of high-purity content rebaudioside-A - Google Patents
A kind of preparation method of high-purity content rebaudioside-A Download PDFInfo
- Publication number
- CN103570778B CN103570778B CN201310532269.XA CN201310532269A CN103570778B CN 103570778 B CN103570778 B CN 103570778B CN 201310532269 A CN201310532269 A CN 201310532269A CN 103570778 B CN103570778 B CN 103570778B
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- content rebaudioside
- preparation
- solution
- elution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention discloses a kind of preparation method of high-purity content rebaudioside-A, it uses sulfonic group chromatograph packing material as stationary phase, and mobile phase is aqueous solutions of organic solvent, by chromatogram column equilibration, loading, elution and obtain high-purity content rebaudioside-A.The present invention is suitable for industrialized production using the sulfonic group chromatograph packing material of rigid inorganic medium framework material, its performance stabilization, obtained product purity height.
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 sweetener, is the mixture of diterpene glucoside.Wherein content rebaudioside-A（RA）It is acknowledged as
The best substitute of sucrose and other synthesis sweetening agents.Western developed country after 2008, particularly U.S. FDA give RA
（Purity >=97%）Behind GRAS (generally accepted safe material) status, high-purity RA demand is amplified rapidly on world market.
At present, it has been disclosed that the RA of high-purity preparation method has recrystallization method and chromatography.Recrystallization method is to utilize stevia rebaudianum
Each glucosides of sugar in organic solvent such as methanol the difference of solubility and repeatedly crystallize the RA for preparing high-purity, the method process is numerous
Trivial, yield is relatively low, solvent consumption is big and cost is higher.
Dominik Bergs et al.（Preparative purification of rebaudioside A from
aqueous extracts using chromatography: a process idea, J. Verbr. Lebensm.,
2012(7):295–303）RA is prepared using 3 step chromatographic tandems, its final step subtractive process uses primary amine groups（-NH(CH2)2NH2）Silica matrix chromatogram packing, eluant, eluent is 80% acetonitrile solution, finally gives the RA that purity is 96.8%, the step yield
For 97.5%.
United States Patent (USP) US20120083593 discloses a kind of RA process for purification.Its core is to use primary amine groups（-NH
(CH2)2NH2）Resin, the resin belongs to weak-base anion-exchange resin, and the skeleton of resin is polystyrene-divinyl base
Benzene or polymethacrylates, with the third of water-miscible organic solvent such as methanol, isopropanol, acetone, the combination of acetonitrile or 5%-40%
The ketone aqueous solution is eluted, the crystallized RA components for obtaining purity ＞ 98%.
Above two method uses primary amine based filler, but primary amine based filler is the shortcomings of have facile hydrolysis, filler short life,
Thus eventually result in the raising of product cost.
The content of the invention
It is an object of the invention to provide a kind of preparation method for the content rebaudioside-A for being suitable for industrialized production, to overcome
Primary amine based filler facile hydrolysis, stability are poor, filler short life deficiency.
The present invention is to be achieved through the following technical solutions：
A kind of preparation method of content rebaudioside-A, it comprises the following steps：
（1）Chromatogram column equilibration：Flowing is passed through into chromatographic column to balance each other chromatographic column；
（2）Loading：By the dissolving crude product containing content rebaudioside-A in mobile phase, it is pumped into chromatographic column or by by Lai Baodi
Glucoside A crude product mobile phase solution and chromatograph packing material mix, dried chromatograph packing material are put into the column cap of chromatographic column, the chromatogram is filled out
Expect for sulfonic group or benzene sulfonic acid base chromatograph packing material；
（3）Elution：With ultraviolet or ELSD detector monitors, content rebaudioside-A is eluted to from chromatographic column appearance knot with mobile phase
Beam.
The skeleton of the sulfonic group or benzene sulfonic acid base chromatograph packing material is silica gel, aluminum oxide or zirconium dioxide, preferably silica gel,
Functional group is sulfonic group-SO3 -R or benzene sulfonic acid base-phe-SO3 -R, R are H+、Na+、K+、Ca2+Or Mg2+。
The sulfonic group or the Sepra that benzene sulfonic acid base chromatograph packing material is commercializationTMSCX, ZORBAX 300SCX or
Intersil CX。
In above-mentioned preparation method, mobile phase is methanol aqueous solution, ethanol water, isopropanol water solution, aqueous acetone solution
Or acetonitrile solution, step（1）With（2）In, mobile phase when balancing the mobile phase and loading of chromatographic column is volume ratio 85-
95% aqueous acetone solution.
Step（3）In, mobile phase during elution is isocratic elution or multistage isocratic elution or linear gradient elution.If adopting
With isocratic elution, mobile phase is volume ratio 85-95% aqueous acetone solution；According to multistage isocratic elution, first using volume ratio
85-95% aqueous acetone solution, which is eluted to rebaudioside C appearances, to be terminated, then switch to less than volume ratio 85% aqueous acetone solution or
Pure water is eluted；According to linear gradient elution, then flowing phase concentration during loading is to pure water solution.
Above-mentioned chromatogram column equilibration, loading, the flow velocity of each step of elution are 50-600cm/hr, preferably 200-250cm/hr.
Advantages of the present invention and good effect：
1st, United States Patent (USP) US20120083593 uses high polymer framework material, is easily swelled and causes post pressure unstable, even
Mobile phase is difficult to flow, and the present invention uses rigid inorganic medium framework material, and it can be overcome not enough；
2nd, United States Patent (USP) US20120083593 and Dominik Bergs et al. scheme are using stability is poor, facile hydrolysis
Primary amine based filler, filler short life, the present invention use stable sulfonic group to overcome its deficiency；
3rd, the content rebaudioside-A purity that the inventive method is obtained is high, is suitable for industrialized production.
Embodiment
Following examples are used to further illustrate the present invention, but do not indicate that mode described in embodiment is to implement the present invention
Unique channel, does not mean that any limitation of the invention yet.
The isocratic elution of embodiment 1
60% purity RA is given by Qufu Sheng Ren pharmaceutical Co. Ltds, and 10g/L is configured to 90% (v/v) acetone-water solution
Sample solution is standby.By SepraTMSCX（Phenomenex）Silica matrix sulfonic group chromatograph packing material homogenate method loads 10*250
Mm stainless steel chromatographic columns, dress column pressure 100 bar.Then with 100ml 90% (v/v) acetone-water solution with 225cm/hr（Below
Each step flow velocity is identical with this）It is stand-by after flow velocity balance chromatographic column.
The above-mentioned RA sample solutions prepared of 22.7 mL are first pumped into, are then received with the elution of 90% acetone-water solution and substep
Collection eluent amounts to 290 mL.Merge the higher component of RA purity after being detected through HPLC, obtain the product 13.28g of RA purity 98.09%,
Yield is 97.50%.
The gradient elution of embodiment 2
The preparation be the same as Example 1 of sample solution, filling and the balance be the same as Example 2 of chromatographic column.
The above-mentioned RA sample solutions prepared of 22.7 mL are first pumped into, is then eluted with 90% acetone-water solution and uses HPLC
Monitoring, when RA starts to spill, mobile phase switches to pure water, collects pure water eluent 25ml, obtains the product of RA purity 97.32%
13.35g, yield is 98.02%.
Embodiment 3
Using with Examples 1 and 2 similar approach, unlike using commercialization chromatographic column ZORBAX 300SCX
(Agilent Technologies) or Intersil CX (GL Sciences), it is equal that mobile phase obtains RA purity for acetonitrile solution
More than 97%, yield is all higher than 97%.
Claims (4)
1. a kind of preparation method of content rebaudioside-A, it is characterised in that comprise the following steps：
（1）Chromatogram column equilibration：It is passed through flowing into chromatographic column to balance each other chromatographic column, mobile phase is volume ratio 85-95% acetone
The aqueous solution；
（2）Loading：By the dissolving crude product containing content rebaudioside-A in mobile phase, it is pumped into chromatographic column or by the way that content rebaudioside-A is thick
Product mobile phase solution and chromatograph packing material mix, dried chromatograph packing material are put into the column cap of chromatographic column；Chromatograph packing material is commodity
The Sepra of changeTMSCX, ZORBAX 300SCX or Intersil CX；Mobile phase is volume ratio 85-95% aqueous acetone solution；
（3）Elution：With ultraviolet or ELSD detector monitors, with mobile phase isocratic elution or multistage isocratic elution or linear gradient
Content rebaudioside-A is eluted to from chromatographic column appearance to terminate.
2. the preparation method of content rebaudioside-A according to claim 1, it is characterised in that：The mobile phase of isocratic elution is body
Aqueous acetone solution of the product than 85-95%.
3. the preparation method of content rebaudioside-A according to claim 1, it is characterised in that：During multistage isocratic elution, first adopt
Rebaudioside C appearances are eluted to volume ratio 85-95% aqueous acetone solution to terminate, then switch to the acetone less than volume ratio 85%
The aqueous solution or pure water elution.
4. the preparation method of content rebaudioside-A according to claim 1, it is characterised in that：When linear gradient elution is loading
Flowing phase concentration to pure water solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310532269.XA CN103570778B (en) | 2013-11-04 | 2013-11-04 | A kind of preparation method of high-purity content rebaudioside-A |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310532269.XA CN103570778B (en) | 2013-11-04 | 2013-11-04 | A kind of preparation method of high-purity content rebaudioside-A |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103570778A CN103570778A (en) | 2014-02-12 |
| CN103570778B true CN103570778B (en) | 2017-07-28 |
Family
ID=50043554
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310532269.XA Expired - Fee Related CN103570778B (en) | 2013-11-04 | 2013-11-04 | A kind of preparation method of high-purity content rebaudioside-A |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103570778B (en) |
Citations (6)
| 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 |
| WO2012042508A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and purification of stevioside and rebaudioside a |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5570162B2 (en) * | 2009-08-31 | 2014-08-13 | 三栄源エフ・エフ・アイ株式会社 | Purification method for rebaudioside A |
-
2013
- 2013-11-04 CN CN201310532269.XA patent/CN103570778B/en not_active Expired - Fee Related
Patent Citations (6)
| 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 |
| WO2012042508A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and purification of stevioside and rebaudioside a |
Non-Patent Citations (3)
| Title |
|---|
| Preparative isolation and purification of steviol glycosides from Stevia rebaudiana Bertoni using high-speed counter-current chromatography;Xin-Yi Huang 等;《Separation and Purification Technology》;20101231;第220-224页 * |
| 接枝大孔吸附树脂HPD-T01纯化甜菊总昔的研究;张全香 等;《第16届反应性高分子学术讨论会》;20120731;第97-98页 * |
| 高效液相色谱法测定甜叶菊糖中的甜菊苷和莱鲍迪苷A;刘超 等;《分析试验室》;20070731;第26卷(第7期);第23-26页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103570778A (en) | 2014-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sang et al. | Deep eutectic solvent-based extraction coupled with green two-dimensional HPLC-DAD-ESI-MS/MS for the determination of anthocyanins from Lycium ruthenicum Murr. fruit | |
| Zhang et al. | Separation and purification of neohesperidin from the albedo of Citrus reticulata cv. Suavissima by combination of macroporous resin and high‐speed counter‐current chromatography | |
| CN108690101A (en) | For being enriched with the method for being derived from rebaudioside B and/or rebaudioside D in the glycoside composition of STEVIA REBAUDIANA | |
| CN101792822B (en) | Method for separating and purifying xylose and arabinose from hemicellulose acid hydrolysis liquid | |
| CN102617673B (en) | Method for separating and purifying naringin and neohesperidin from white skin layer of citrus grandis | |
| CN101407535B (en) | Preparation of high-purity Momordica grosvenori mogroside V | |
| Liu et al. | Offline preparative 2-D polar-copolymerized reversed-phase chromatography× zwitterionic hydrophilic interaction chromatography for effective purification of polar compounds from Caulis Polygoni Multiflori | |
| JP6646052B2 (en) | Methods for adjusting the composition of chromatography products | |
| CN103570778B (en) | A kind of preparation method of high-purity content rebaudioside-A | |
| Dang et al. | Large-scale preparative isolation of bergenin standard substance from Saxifraga atrata using polyamide coupled with MCI GEL® CHP20P as stationary phases in medium pressure chromatography | |
| Liu et al. | Recovery of steviol glycosides from industrial stevia by-product via crystallization and reversed-phase chromatography | |
| Hubert et al. | Intensified separation of steviol glycosides from a crude aqueous extract of Stevia rebaudiana leaves using centrifugal partition chromatography | |
| Song et al. | A novel bioreactor for highly efficient biotransformation of resveratrol from polydatin with high-speed counter-current chromatography | |
| Sun et al. | Dual-tautomerism separation method based on asymmetric transformation: Gram-scale preparation of high-purity punicalagin from pomegranate peel wastes | |
| Yang et al. | An effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography for enrichment and separation of three minor components with similar polarity from Dracocephalum tanguticum | |
| CN107778338A (en) | A kind of rebaudioside C isolation and purification methods | |
| CN105820213B (en) | The method for efficiently separating purifying knob not Kangding | |
| CN103265521A (en) | Preparation method of demethylated beilidifolin | |
| CN103613622B (en) | A kind of method of isolated and purified content rebaudioside-A | |
| Wu et al. | Isolation, identification and pharmacokinetic analysis of fructosyl puerarins from enzymatic glycosylation | |
| CN115536715A (en) | Method for extracting stevioside RD | |
| CN103610800A (en) | Method for separating and purifying seven flavonoid compounds from tangerine oil vacuole layer | |
| Chen et al. | Purification and preparation of rebaudioside A from steviol glycosides using one-dimensional hydrophilic interaction chromatography | |
| CN101070335A (en) | Process for preparing high-purity gentiopicrin | |
| CN107722078B (en) | Method for extracting, separating and purifying phlorizin by n-propanol-potassium sodium tartrate aqueous two-phase system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170728 Termination date: 20171104 |