CN106317136A - Method for separating alpha-arbutin from alpha-arbutin broth - Google Patents
Method for separating alpha-arbutin from alpha-arbutin broth Download PDFInfo
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- CN106317136A CN106317136A CN201510345857.1A CN201510345857A CN106317136A CN 106317136 A CN106317136 A CN 106317136A CN 201510345857 A CN201510345857 A CN 201510345857A CN 106317136 A CN106317136 A CN 106317136A
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- arbutin
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Abstract
The invention relates to a method for separating alpha-arbutin from alpha-arbutin broth, which solves the technical problems of complex current methods, high cost and non-ideal effect. The method comprises the following steps: filtrating the alpha-arbutin broth through a hollow-fibre membrane, removing thalline to obtain a filtrate; adjusting the pH value of the filtrate to acidity, then passing a solution through macroporous adsorption resin with medium polarity, collecting an eluant; passing the eluant through the macroporous adsorption resin with low polarity, flushing the columns by water, using an ethanol solution for elution to obtain an eluate; performing vacuum drying on the eluate, concentrating the material to precipitate the crystals, standing the material, precipitating the alpha-arbutin by a crystallization form; suspending the crystals by an organic solvent, filtering the crystals, and washing the crystals by the organic solvent to obtain the product. The method can be used for separating alpha-arbutin from the alpha-arbutin broth.
Description
Technical field
The present invention relates to biological chemical field, a kind of method of separation-arbutin from-arbutin fermentation liquid.
Background technology
Arbutin is a kind of HQG compounds, is the inhibitor of tryrosinase, can block DOPA and the synthesis of DOPA quinone, thus effectively suppress melanic generation, have whitening function.Contain in natural extract is β-arbutin, but the whitening effect of-arbutin is that more than 10 times of β-arbutin, effectiveness and safety are higher than β-arbutin.The chemosynthesis of-arbutin is more difficult, is easily formed-arbutin and the mixture of β-arbutin, and separating difficulty is big.Therefore increasing research trend is in microbial method or enzymatic clarification-arbutin.
The Chinese invention patent application of Publication No. CN1635139A discloses a kind of method of fermenting and producing-arbutin, utilize xanthomonas campestris, ferment with hydroquinone and sucrose for substrate, fermentation liquid is separated by low pole macroporous adsorbent resin AB-8 and polar macroporous adsorption resin S-8 post respectively, then obtains sterling by vacuum drying.
The Chinese invention patent application of Publication No. CN102329838A discloses a kind of method converting production-arbutin with Aspergillus niger strain, using hydroquinone and maltose as fermenting substrate, fermentation liquor Amberlite XAD-16 or D101 macroporous adsorbent resin extract, and obtain-arbutin.
Above two pieces patent application all briefly touches upon for isolated and purified process, and the product finally given all awaits improving from color and luster or purity.
Additionally, bioanalysis is all to use hydroquinone to do substrate, a small amount of hydroquinone all can be had after fermentation ends remaining, and hydroquinone is skin sensitizer known to one, disable in skin care item, if therefore-arbutin isolated and purified during can not effectively remove hydroquinone, directly affecting product quality, to even result in product defective.
The Chinese invention patent application of Publication No. CN103483401A proposes a kind of method removing hydroquinone from-arbutin fermentation liquid, repeatedly extracts and back extraction with tributyl phosphate, and need to introduce inorganic salt.The method makes arbutin purifying process more complicate.
Summary of the invention
The present invention is contemplated to solve the technical problem that existing method is excessively complicated, cost is high and effect is undesirable, it is provided that a kind of method of separation-arbutin from-arbutin fermentation liquid, and it can improve-separation efficiency of arbutin and product quality.
The present invention provides a kind of method of separation-arbutin from-arbutin fermentation liquid, it is characterized in that comprising the steps: that (1) general-arbutin fermentation liquid, by doughnut membrane filtration, removes thalline, obtains filtrate；(2) adjust pH to acid the filtrate that obtain in step (1), then by the solution macroporous adsorbent resin by middle polarity, collect effluent；(3) the too low polar macroporous adsorption resin of effluent that will obtain in step (2), rinses pillar with water afterwards, then uses ethanol solution eluting, obtain eluent；(4) by vacuum dried for the eluent obtained in step (3) be concentrated into crystallization time, place ,-arbutin separates out in crystalline form；(5) by step (4) is formed-arbutin crystallization organic solvent hang, filtration, and with organic solvent washing, i.e. can get white-arbutin product.
Preferably, the hollow-fibre membrane in step (1) be molecular cut off be the hollow-fibre membrane of 6000-60000.
Preferably, in step (2), pH is adjusted to refer to adjust pH to 3-5 to acidity.
Preferably, in step (2), the macroporous adsorbent resin of middle polarity, refer to Amberlite XAD-6, XAD-7, XAD-7HP or XAD-8 resin.
Preferably, the macroporous adsorbent resin of the low polarity in step (3), refer to that H103, H107 or H1020 resin, the concentration of volume percent of described ethanol solution are 10%-15%.
Preferably, the organic solvent in step (5) is acetone.
In the present invention, the isolated and purified process of alpha-arbutin is relatively easy, it is possible to thoroughly remove hydroquinone, and gained alpha-arbutin product color degree is good (to be seenFigure 1), purity is high.Reach more than 99% by the purity of HPLC external standard method detection gained arbutin (to seeFigure 2), HPLC testing conditions is: pillar: C18 post (4.6*150mm, 3.5um)；Flowing phase: 90% water containing 1/1000 formic acid, 10% methanol；Flow velocity: 0.3mL/min；Column temperature: 45 DEG C.
The alpha-arbutin product obtained by the method for the invention is through nuclear-magnetismFigureSpectrum checking (sees for alpha-arbutin reallyFigure 3A andFigure 3B), coupling constant J=3.6Hz at alpha-arbutin 5.34ppm, it is different from β-arbutin coupling constant J=7.8 herein, and from nuclear-magnetismFigureImpurity peaks is not seen in spectrum.
Accompanying drawing explanation
Figure 1For gained arbutin product characteristics photo of the present invention；
Figure 2HPLC for gained arbutin of the present inventionFigureSpectrum；
Figure 3A is the nuclear-magnetism of gained arbutinFigureSpectrum hydrogen spectrum；
Figure 3B is the nuclear-magnetism of gained arbutinFigureSpectrum carbon spectrum.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that the content described by embodiment is merely to illustrate the present invention, and not should also without limitation onRightThe present invention described in claim.Reagent used in the embodiment of the present invention is all bought from open market.In the present invention-arbutin fermentation liquid refers to that doing substrate fermentable with hydroquinone prepares the-tunning of arbutin.
Embodiment 1
4L-arbutin fermentation liquid is removed thalline by doughnut membrane filtration system (molecular cut off of film is 60000), the hydrochloric acid of the clear liquid 1.0mol/L after filtration adjusts pH 3.0, then by Amberlite macroporous adsorbent resin XAD-7 (1.5 liters), effluent continues through macroporous adsorbent resin H103 (1.5 liters), H103 resin column is rinsed with the water of 3 times of column volumes, afterwards with the ethanol solution eluting of the concentration of volume percent 10% of 4 times of column volumes, collect the eluent containing-arbutin, when being concentrated in vacuo to major part crystallization, place 1 hour in 4 DEG C of environment, now more crystal separates out, with the acetone of prior pre-cooling, crystallization is hanged, filter, and obtain-arbutin sterling with a small amount of washing with acetone.
Embodiment 2
4L-arbutin fermentation liquid is removed thalline by doughnut membrane filtration system (molecular cut off of film is 30000), the hydrochloric acid of the clear liquid 1.0mol/L after filtration adjusts pH 4.0, then by Amberlite macroporous adsorbent resin XAD-8 (1.5 liters), effluent continues through macroporous adsorbent resin H103 (1.5 liters), H103 resin column is rinsed with the water of 3 times of column volumes, afterwards with the ethanol elution of the concentration of volume percent 10% of 4 times of column volumes, collect the eluent containing-arbutin, when being concentrated in vacuo to major part crystallization, place 1 hour in 4 DEG C of environment, now more crystal separates out, with the acetone of prior pre-cooling, crystallization is hanged, filter, and obtain-arbutin sterling with a small amount of washing with acetone.
Embodiment 3
4L-arbutin fermentation liquid is removed thalline by doughnut membrane filtration system (molecular cut off of film is 60000), the hydrochloric acid of the clear liquid 1.0mol/L after filtration adjusts pH 5, then by Amberlite macroporous adsorbent resin XAD-7 (1.5 liters), effluent continues through macroporous adsorbent resin H107 (1.5 liters), H107 resin column is rinsed with the water of 3 times of column volumes, afterwards with the ethanol elution of the concentration of volume percent 15% of 4 times of column volumes, collect the eluent containing-arbutin, when being concentrated in vacuo to major part crystallization, place 1 hour in 4 DEG C of environment, now more crystal separates out, with the acetone of prior pre-cooling, crystallization is hanged, filter, and obtain-arbutin sterling with a small amount of washing with acetone.
Embodiment 4
4L-arbutin fermentation liquid is removed thalline by doughnut membrane filtration system (molecular cut off of film is 6000), the hydrochloric acid of the clear liquid 1.0mol/L after filtration adjusts pH 4.0, then by Amberlite macroporous adsorbent resin XAD-6 (1.5 liters), effluent continues through macroporous adsorbent resin H103 (1.5 liters), H103 resin column is rinsed with the water of 3 times of column volumes, afterwards with the ethanol elution of the concentration of volume percent 15% of 4 times of column volumes, collect the eluent containing-arbutin, when being concentrated in vacuo to major part crystallization, place 1 hour in 4 DEG C of environment, now more crystal separates out, with the acetone of prior pre-cooling, crystallization is hanged, filter, and obtain-arbutin sterling with a small amount of washing with acetone.
Claims (6)
1. a method for separation-arbutin from-arbutin fermentation liquid, it is characterized in that including as
Lower step:
(1) general-arbutin fermentation liquid passes through doughnut membrane filtration, removes thalline, obtains filtrate；
(2) adjust pH to acid the filtrate obtained in step (1), then solution is passed through medium
The macroporous adsorbent resin of polarity, collects effluent；
(3) the too low polar macroporous adsorption resin of effluent that will obtain in step (2), uses afterwards
Water rinses pillar, then uses ethanol solution eluting, obtains eluent；
(4) by vacuum dried for the eluent obtained in step (3) be concentrated into crystallization time,
Placing ,-arbutin separates out in crystalline form；
(5) by step (4) is formed-arbutin crystallization organic solvent hang, filtration,
And with organic solvent washing, i.e. can get-arbutin product.
The side of separation-arbutin from-arbutin fermentation liquid the most according to claim 1
Method, it is characterised in that the hollow-fibre membrane in described step (1) be molecular cut off be 6000-60000
Hollow-fibre membrane.
The side of separation-arbutin from-arbutin fermentation liquid the most according to claim 1
Method, it is characterised in that in described step (2), adjusts pH to refer to adjust pH to 3-5 to acidity.
The side of separation-arbutin from-arbutin fermentation liquid the most according to claim 1
Method, it is characterised in that in described step (2), the macroporous adsorbent resin of middle polarity, refer to Amberlite
XAD-6, XAD-7, XAD-7HP or XAD-8 resin.
The side of separation-arbutin from-arbutin fermentation liquid the most according to claim 1
Method, it is characterised in that the macroporous adsorbent resin of the low polarity in described step (3), refer to H103,
H107 or H1020 resin, the concentration of volume percent of described ethanol solution is 10%-15%.
The side of separation-arbutin from-arbutin fermentation liquid the most according to claim 1
Method, it is characterised in that the organic solvent in described step (5) is acetone.
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| CN201510345857.1A CN106317136B (en) | 2015-06-19 | 2015-06-19 | A method of separating ɑ-arbutin from ɑ-arbutin fermentation liquid |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108329363A (en) * | 2018-01-30 | 2018-07-27 | 合肥华恒生物工程有限公司 | The minimizing technology of quinhydrones in alpha-arbutin conversion fluid |
| CN108558964A (en) * | 2018-04-18 | 2018-09-21 | 山东众山生物科技有限公司 | A kind of purification process of alpha-arbutin |
| CN114044797A (en) * | 2021-09-30 | 2022-02-15 | 安徽华恒生物科技股份有限公司 | Extraction method and application of alpha-arbutin |
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|---|---|---|---|---|
| CN1635139A (en) * | 2004-11-15 | 2005-07-06 | 刘春巧 | Process for preparing alpha-arbutin through fermentation |
| CN102093442A (en) * | 2011-01-13 | 2011-06-15 | 赵玉红 | Method for separating and purifying arbutin from blueberry |
| CN103773827A (en) * | 2012-10-19 | 2014-05-07 | 北京化工大学 | Method for improving output of alpha-arbutin |
| CN104558066A (en) * | 2014-12-26 | 2015-04-29 | 高俊丽 | Extraction method for arbutin |
| CN104672286A (en) * | 2015-03-09 | 2015-06-03 | 大兴安岭林格贝寒带生物科技股份有限公司 | Method for enriching purified alpha-arbutin from blueberry leaves |
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- 2015-06-19 CN CN201510345857.1A patent/CN106317136B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1635139A (en) * | 2004-11-15 | 2005-07-06 | 刘春巧 | Process for preparing alpha-arbutin through fermentation |
| CN102093442A (en) * | 2011-01-13 | 2011-06-15 | 赵玉红 | Method for separating and purifying arbutin from blueberry |
| CN103773827A (en) * | 2012-10-19 | 2014-05-07 | 北京化工大学 | Method for improving output of alpha-arbutin |
| CN104558066A (en) * | 2014-12-26 | 2015-04-29 | 高俊丽 | Extraction method for arbutin |
| CN104672286A (en) * | 2015-03-09 | 2015-06-03 | 大兴安岭林格贝寒带生物科技股份有限公司 | Method for enriching purified alpha-arbutin from blueberry leaves |
Non-Patent Citations (1)
| Title |
|---|
| 《北京化工大学硕士学位论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108329363A (en) * | 2018-01-30 | 2018-07-27 | 合肥华恒生物工程有限公司 | The minimizing technology of quinhydrones in alpha-arbutin conversion fluid |
| CN108558964A (en) * | 2018-04-18 | 2018-09-21 | 山东众山生物科技有限公司 | A kind of purification process of alpha-arbutin |
| CN114044797A (en) * | 2021-09-30 | 2022-02-15 | 安徽华恒生物科技股份有限公司 | Extraction method and application of alpha-arbutin |
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