CN112300229A - Method for purifying acarbose from acarbose fermentation liquor - Google Patents
Method for purifying acarbose from acarbose fermentation liquor Download PDFInfo
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- CN112300229A CN112300229A CN202011227432.8A CN202011227432A CN112300229A CN 112300229 A CN112300229 A CN 112300229A CN 202011227432 A CN202011227432 A CN 202011227432A CN 112300229 A CN112300229 A CN 112300229A
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- acarbose
- resin
- exchange resin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
Abstract
The invention belongs to the technical field of organic matter purification, and relates to a method for purifying acarbose from acarbose fermentation liquor, which comprises the following steps: (1) crude extraction: filtering acarbose fermentation liquor, desalting, adsorbing by cation exchange resin, and eluting the cation exchange resin by hydrochloric acid to obtain a crude extract; (2) chromatography: subjecting the crude extract to cation exchange resin chromatography, sequentially performing gradient elution with 5 times column volume of 0.01mol/L, 0.02mol/L and 0.03mol/L hydrochloric acid, and collecting 0.03mol/L hydrochloric acid eluate; (3) fine extraction; adsorbing the retained chromatographic solution with XR19 macroporous adsorbent resin, and eluting with methanol solution to obtain refined solution; (4) freeze-drying: and (4) freeze-drying the refined solution to obtain the acarbose. The method can obtain the purity of more than 98 percent and the refining yield of more than 95 percent, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of organic matter purification, in particular to a method for purifying acarbose from acarbose fermentation liquor.
Background
Acarbose is a novel oral hypoglycemic agent. Competitively inhibits the glycoside hydrolase in the intestinal tract. Reducing the decomposition of polysaccharide and sucrose into glucose and correspondingly slowing the absorption of sugar, thereby having the function of reducing the blood sugar after meals. Generally, it is used singly or in combination with other oral hypoglycemic agents or insulin. Can be used in combination with diet for treating insulin-dependent diabetes mellitus or non-dependent diabetes mellitus.
Acarbose is generally prepared by seed flask culture, seed tank culture and fermenter culture. After fermentation culture, a plurality of impurities exist in the solution, and the difficulty is that how to extract high-purity acarbose. In the past, ion exchange resin is adopted for purification, the content of the obtained chromatography liquid after freeze-drying is only about 85 percent, and the requirement of more than 95 percent specified by pharmacopoeia cannot be met.
Therefore, it is necessary to develop a new purification method to avoid the above problems.
Disclosure of Invention
The main object of the present invention is to provide a process for the purification of acarbose from an acarbose fermentation broth, which enables the high-purity acarbose to be obtained in high yield.
The invention realizes the purpose through the following technical scheme: a process for purifying acarbose from an acarbose fermentation broth comprising the steps of:
(1) crude extraction: filtering acarbose fermentation liquor, desalting, adsorbing by cation exchange resin, and eluting the cation exchange resin by hydrochloric acid to obtain a crude extract;
(2) chromatography: subjecting the crude extract to cation exchange resin chromatography, sequentially performing gradient elution with 5 times column volume of 0.01mol/L, 0.02mol/L and 0.03mol/L hydrochloric acid, and collecting 0.03mol/L hydrochloric acid eluate;
(3) fine extraction; adsorbing the retained chromatographic solution with XR19 macroporous adsorbent resin, and eluting with methanol solution to obtain refined solution;
(4) freeze-drying: and (4) freeze-drying the refined solution to obtain the acarbose.
Specifically, the cation exchange resin in the step (1) adopts CT151 cation exchange resin, and the loading capacity is 6-8% of the volume of the resin; elution was performed with 10 column volumes of hydrochloric acid, pH 1.6.
Specifically, the desalting in the step (1) is performed by an IR120 cation exchange resin and an IRA67 anion exchange resin, and the loading capacity is 10-15 times of the resin volume.
Specifically, the cation exchange resin in the step (2) is UBK530 cation exchange resin, and the loading capacity is 3-5% of the volume of the resin.
Specifically, the macroporous adsorption resin in the step (3) has an adsorption flow rate of 1.0BV/h, and the loading capacity is 2-3% of the volume of the resin.
Further, the step (3) adopts 5 times of resin volume and methanol solution with 20 percent of volume percentage concentration to elute at the flow rate of 1.0 BV/h.
Adopt above-mentioned technical scheme beneficial effect to be:
the method can obtain the purity of more than 98 percent and the refining yield of more than 95 percent, and is suitable for industrial production.
Detailed Description
The invention relates to a method for purifying acarbose from acarbose fermentation liquor, which comprises the following steps:
(1) crude extraction: filtering acarbose fermentation liquor, desalting, adsorbing by cation exchange resin, and eluting the cation exchange resin by hydrochloric acid to obtain a crude extract;
(2) chromatography: subjecting the crude extract to cation exchange resin chromatography, sequentially performing gradient elution with 5 times column volume of 0.01mol/L, 0.02mol/L and 0.03mol/L hydrochloric acid, and collecting the final section of chromatographic solution;
(3) fine extraction; adsorbing the retained chromatographic solution with XR19 macroporous adsorbent resin, and eluting with methanol solution to obtain refined solution;
(4) freeze-drying: and (4) freeze-drying the refined solution to obtain the acarbose.
The present invention will be described in further detail with reference to specific examples.
Example 1:
(1) filtering the acarbose fermentation liquor by using a plate-and-frame filter, sequentially passing through IR120 cation exchange resin and then through IRA67 anion exchange resin for desalination, wherein the carrying capacity is 10 times of the resin volume; then adsorbing by CT151 cation exchange resin with loading capacity of 6% of resin volume, and eluting CT151 cation exchange resin with 10 times column volume of hydrochloric acid with pH1.6 to obtain crude extract.
(2) And (3) carrying out UBK530 cation exchange resin chromatography on the crude extract, carrying 3% of the resin volume, carrying out gradient elution by using hydrochloric acid of 0.01mol/L, 0.02mol/L and 0.03mol/L in 5 times of column volume in sequence, collecting the eluent of 0.03mol/L hydrochloric acid, and retaining.
(3) And (3) adsorbing the reserved chromatographic solution by XR19 macroporous adsorption resin, wherein the adsorption flow rate of the macroporous adsorption resin is 1.0BV/h, the loading capacity is 2% of the volume of the resin, and eluting by using methanol solution with the volume 5 times that of the resin and the volume percentage concentration of 20% to obtain refined solution. (refining yield 95.53%)
(4) The purified solution was lyophilized to give acarbose (purity 98.41%).
Example 2:
(1) filtering the acarbose fermentation liquor by using a plate-and-frame filter, sequentially passing through IR120 cation exchange resin and then through IRA67 anion exchange resin for desalination, wherein the carrying capacity is 15 times of the resin volume; then the crude extract is obtained by adsorbing with CT151 cation exchange resin with the loading capacity of 8% of the resin volume, and eluting with 10 times of column volume of hydrochloric acid with pH 1.6.
(2) And (3) carrying out UBK530 cation exchange resin chromatography on the crude extract, carrying the crude extract with the capacity of 3-5% of the volume of the resin, carrying out gradient elution by using hydrochloric acid of 0.01mol/L, 0.02mol/L and 0.03mol/L in 5 times of the volume of the column in sequence, collecting the eluent of hydrochloric acid of 0.03mol/L, and retaining the eluent.
(3) And (3) adsorbing the reserved chromatographic solution by XR19 macroporous adsorption resin, wherein the adsorption flow rate of the macroporous adsorption resin is 1.0BV/h, the loading capacity is 3% of the volume of the resin, and eluting by using methanol solution with the volume 5 times that of the resin and the volume percentage concentration of 20% to obtain refined solution. (refined yield 96.26%)
(4) The purified solution was lyophilized to give acarbose (purity 98.12%).
Example 3:
(1) filtering the acarbose fermentation liquor by using a plate-and-frame filter, sequentially passing through IR120 cation exchange resin and then through IRA67 anion exchange resin for desalination, wherein the carrying capacity is 12 times of the resin volume; then the crude extract is obtained by adsorbing with CT151 cation exchange resin with the loading capacity of 7% of the resin volume, and eluting with 10 times of column volume of hydrochloric acid with pH 1.6.
(2) And (3) carrying out UBK530 cation exchange resin chromatography on the crude extract, carrying 4% of the resin by volume, carrying out gradient elution by using hydrochloric acid of 0.01mol/L, 0.02mol/L and 0.03mol/L in 5 times of column volume in sequence, collecting the eluent of 0.03mol/L hydrochloric acid, and retaining.
(3) And (3) adsorbing the reserved chromatographic solution by XR19 macroporous adsorption resin, wherein the adsorption flow rate of the macroporous adsorption resin is 1.0BV/h, the loading capacity is 2.5 percent of the volume of the resin, and eluting by using methanol solution with the concentration of 20 percent by volume which is 5 times the volume of the resin to obtain refined solution. (refining yield 95.67%)
(4) The purified solution was lyophilized to give acarbose (purity 98.62%).
Example 4:
(1) filtering the acarbose fermentation liquor by using a plate-and-frame filter, sequentially passing through IR120 cation exchange resin and then through IRA67 anion exchange resin for desalination, wherein the carrying capacity is 11 times of the resin volume; then adsorbing by CT151 cation exchange resin with loading capacity of 6.5% of resin volume, and eluting CT151 cation exchange resin with 10 times column volume of hydrochloric acid with pH of 1.6 to obtain crude extract.
(2) And (3) carrying out UBK530 cation exchange resin chromatography on the crude extract, carrying the crude extract with the capacity of 4.5% of the resin volume, carrying out gradient elution by using hydrochloric acid of 0.01mol/L, 0.02mol/L and 0.03mol/L in 5 times of the column volume in sequence, collecting the eluent of 0.03mol/L hydrochloric acid, and keeping the eluent.
(3) And (3) adsorbing the reserved chromatographic solution by XR19 macroporous adsorption resin, wherein the adsorption flow rate of the macroporous adsorption resin is 1.0BV/h, the loading capacity is 2.8 percent of the volume of the resin, and eluting by using methanol solution with the concentration of 5 times the volume of the resin and 20 percent of the volume percentage to obtain refined solution. (refining yield 95.34%)
(4) The purified solution was lyophilized to give acarbose (purity 98.21%).
Example 5:
(1) filtering the acarbose fermentation liquor by using a plate-and-frame filter, sequentially passing through IR120 cation exchange resin and then through IRA67 anion exchange resin for desalination, wherein the carrying capacity is 14 times of the resin volume; then the crude extract is obtained by adsorbing with CT151 cation exchange resin with the loading capacity of 7.5% of the resin volume, and eluting with 10 times of column volume of hydrochloric acid with pH of 1.6.
(2) And (3) carrying out UBK530 cation exchange resin chromatography on the crude extract, carrying the crude extract with the capacity of 3.6% of the resin volume, carrying out gradient elution by using hydrochloric acid of 0.01mol/L, 0.02mol/L and 0.03mol/L in 5 times of the column volume in sequence, collecting the eluent of 0.03mol/L hydrochloric acid, and keeping the eluent.
(3) And (3) adsorbing the reserved chromatographic solution by XR19 macroporous adsorption resin, wherein the adsorption flow rate of the macroporous adsorption resin is 1.0BV/h, the loading capacity is 2.4% of the volume of the resin, and eluting by using methanol solution with the concentration of 5 times the volume of the resin and 20% of the volume percentage to obtain refined solution. (refined yield 96.10%)
(4) The purified solution was lyophilized to give acarbose (purity 98.97%).
In conclusion, the method can obtain the purity of more than 98 percent and the refining yield of more than 95 percent, and is suitable for industrial production.
The desalting in the step (1) can be realized by using ultrafiltration membranes, nanofiltration membranes, gel columns and the like.
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (6)
1. A process for purifying acarbose from an acarbose fermentation broth comprising the steps of:
(1) crude extraction: filtering acarbose fermentation liquor, desalting, adsorbing by cation exchange resin, and eluting the cation exchange resin by hydrochloric acid to obtain a crude extract;
(2) chromatography: subjecting the crude extract to cation exchange resin chromatography, sequentially performing gradient elution with 5 times column volume of 0.01mol/L, 0.02mol/L and 0.03mol/L hydrochloric acid, and collecting 0.03mol/L hydrochloric acid eluate;
(3) fine extraction; adsorbing the retained chromatographic solution with XR19 macroporous adsorbent resin, and eluting with methanol solution to obtain refined solution;
(4) freeze-drying: and (4) freeze-drying the refined solution to obtain the acarbose.
2. A process for the purification of acarbose from an acarbose fermentation broth according to claim 1, characterized in that: the cation exchange resin in the step (1) adopts CT151 cation exchange resin, and the loading capacity is 6-8% of the volume of the resin; elution was performed with 10 column volumes of hydrochloric acid, pH 1.6.
3. A process for the purification of acarbose from an acarbose fermentation broth according to claim 1, characterized in that: the desalination in the step (1) firstly passes through IR120 cation exchange resin and then passes through IRA67 anion exchange resin, and the loading capacity is 10-15 times of the resin volume.
4. A process for the purification of acarbose from an acarbose fermentation broth according to claim 1, characterized in that: and (3) adopting UBK530 cation exchange resin as the cation exchange resin in the step (2), wherein the loading capacity is 3-5% of the volume of the resin.
5. A process for the purification of acarbose from an acarbose fermentation broth according to claim 1, characterized in that: the macroporous adsorption resin in the step (3) has an adsorption flow rate of 1.0BV/h, and the loading capacity is 2-3% of the volume of the resin.
6. A process according to claim 5 for the purification of acarbose from an acarbose fermentation broth, characterized in that: and (3) eluting by using methanol solution with 5 times of resin volume and 20% volume percentage concentration at the elution flow rate of 1.0 BV/h.
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| CN202011227432.8A CN112300229A (en) | 2020-11-06 | 2020-11-06 | Method for purifying acarbose from acarbose fermentation liquor |
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| CN202011227432.8A CN112300229A (en) | 2020-11-06 | 2020-11-06 | Method for purifying acarbose from acarbose fermentation liquor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113336803A (en) * | 2021-06-03 | 2021-09-03 | 同舟纵横（厦门）流体技术有限公司 | Method for removing monosaccharide and disaccharide from acarbose and acarbose purification method |
Citations (8)
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|---|---|---|---|---|
| WO2003014135A1 (en) * | 2001-08-07 | 2003-02-20 | Biogal Gyogyszergyar Rt | Method for purification of acarbose |
| CN1414003A (en) * | 2002-07-31 | 2003-04-30 | 浙江德清东立生物发展有限公司 | Method for preparing high-purity acarbose |
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2020
- 2020-11-06 CN CN202011227432.8A patent/CN112300229A/en active Pending
Patent Citations (8)
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Non-Patent Citations (1)
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| 夏焕章 主编: "《发酵工艺学》", 31 August 2015 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113336803A (en) * | 2021-06-03 | 2021-09-03 | 同舟纵横（厦门）流体技术有限公司 | Method for removing monosaccharide and disaccharide from acarbose and acarbose purification method |
| CN113336803B (en) * | 2021-06-03 | 2023-11-21 | 同舟纵横（厦门）流体技术有限公司 | Method for removing mono-disaccharide from acarbose and acarbose purification method |
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Application publication date: 20210202 |