CN106317136B - A method of separating ɑ-arbutin from ɑ-arbutin fermentation liquid - Google Patents
A method of separating ɑ-arbutin from ɑ-arbutin fermentation liquid Download PDFInfo
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- CN106317136B CN106317136B CN201510345857.1A CN201510345857A CN106317136B CN 106317136 B CN106317136 B CN 106317136B CN 201510345857 A CN201510345857 A CN 201510345857A CN 106317136 B CN106317136 B CN 106317136B
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Abstract
The present invention relates to a kind of to separate ɑ-arbutin method from ɑ-arbutin fermentation liquid, excessively complicated, at high cost and undesirable effect technical problem which solve existing methods, it includes the following steps: to filter ɑ-arbutin fermentation liquid by hollow-fibre membrane, removes thallus, obtains filtrate；By filtrate tune pH to acidity, solution is then collected into efflux by moderately polar macroporous absorbent resin；By the too low polar macroporous adsorption resin of efflux, it is rinsed with water pillar later, then is eluted with ethanol solution, obtains eluent；By eluent is vacuum dried be concentrated into crystallization and be precipitated when, place, ɑ-arbutin is precipitated in crystalline form；Crystallization organic solvent is hanged, is filtered, and with organic solvent washing, manufactured goods can be obtained.The present invention can be used for separating ɑ-arbutin from ɑ-arbutin fermentation liquid.
Description
Technical field
It is specifically a kind of that ɑ-arbutin is separated from ɑ-arbutin fermentation liquid the present invention relates to biological chemical field
Method.
Background technique
Arbutin is a kind of quinhydrones glycosides compound, is the inhibitor of tyrosinase, can block the conjunction of DOPA and DOPA quinone
At so that the generation of melanin is effectively inhibited, with whitening function.What is contained in natural extract is β-arbutin, but ɑ-bear
The whitening effect of fruit glycosides is 10 times of β-arbutin or more, validity and highly-safe in β-arbutin.ɑ-arbutin chemistry closes
At more difficult, easily formation ɑ-arbutin and β-arbutin mixture, separating difficulty are big.Therefore more and more research trends in
With microbial method or enzymatic clarification ɑ-arbutin.
The Chinese invention patent application of Publication No. CN1635139A discloses a kind of method of fermenting and producing ɑ-arbutin,
It using xanthomonas campestris, ferments using quinhydrones and sucrose as substrate, fermentation liquid passes through low pole macroporous absorption tree respectively
Rouge AB-8 and polar macroporous adsorption resin S-8 column are separated, and then obtain sterling by vacuum drying.
The Chinese invention patent application of Publication No. CN102329838A is disclosed a kind of converted with Aspergillus niger strain and produced
ɑ-arbutin method, using quinhydrones and maltose as fermenting substrate, fermentation liquid is through Amberlite XAD-16 or D101 macropore
It adsorbs resin to extract, obtains ɑ-arbutin.
The above two pieces patent application is all briefly touched upon for isolating and purifying process, and no matter finally obtained product is from color
Or all up for improving in purity.
In addition, bioanalysis is all to do substrate using quinhydrones, it is remaining all there is a small amount of quinhydrones after fermentation, and quinhydrones is
A kind of known skin sensitizer, disables in skin care item, if therefore ɑ-arbutin cannot be effective during isolating and purifying
Quinhydrones is removed, will have a direct impact on product quality, to even result in product unqualified.
The Chinese invention patent application of Publication No. CN103483401A proposes one kind and removes from ɑ-arbutin fermentation liquid
The method for removing quinhydrones repeatedly extract and be stripped with tributyl phosphate, and need to introduce inorganic salts.This method makes arbutin
Purifying process more complicates.
Summary of the invention
The present invention is exactly to provide to solve the technical problem that existing method is excessively complicated, at high cost and undesirable effect
A method of it separating ɑ-arbutin from ɑ-arbutin fermentation liquid, ɑ-arbutin separative efficiency and product matter can be improved
Amount.
The present invention provides a kind of separation ɑ-arbutin method from ɑ-arbutin fermentation liquid, it is characterized in that including following step
It is rapid: (1) ɑ-arbutin fermentation liquid to be filtered by hollow-fibre membrane, remove thallus, obtain filtrate；It (2) will be obtained in step (1)
Then solution is collected efflux by moderately polar macroporous absorbent resin to 3-5 by filtrate tune pH；(3) by step (2)
Obtained in the too low polar macroporous adsorption resin of efflux, be rinsed with water pillar later, then eluted with ethanol solution, must elute
Liquid；(4) by eluent obtained in step (3) it is vacuum dried be concentrated into crystallization be precipitated when, place, ɑ-arbutin is with crystalline
Formula is precipitated；(5) ɑ-black bearberry glycosidal crystalline organic solvent formed in step (4) hang, filtering, and with organic solvent washing,
White ɑ-black bearberry glycoside product can be obtained.
Preferably, the hollow-fibre membrane in step (1) is the hollow-fibre membrane that molecular cut off is 6000-60000.
Preferably, in step (2), moderately polar macroporous absorbent resin refers to Amberlite XAD-6, XAD-7, XAD-
7HP or XAD-8 resin.
Preferably, the low polar macroporous absorbent resin in step (3), refers to H103, H107 or H1020 resin, the second
The concentration of volume percent of alcoholic solution is 10%-15%.
Preferably, the organic solvent in step (5) is acetone.
In the present invention alpha-arbutin to isolate and purify process relatively easy, can thoroughly remove quinhydrones, gained alpha-arbutin
Product color degree is good (referring to Fig. 1), purity is high.With HPLC external standard method detect the purity of gained arbutin up to 99% or more (referring to
Fig. 2), HPLC testing conditions are as follows: pillar: C18 column (4.6*150mm, 3.5um)；Mobile phase: 90% contains the water of 1/1000 formic acid,
10% methanol；Flow velocity: 0.3mL/min；Column temperature: 45 DEG C.
The alpha-arbutin product that the method obtains through the invention through nuclear magnetic spectrum verifying really for alpha-arbutin (referring to
Fig. 3 A and Fig. 3 B), coupling constant J=3.6Hz at alpha-arbutin 5.34ppm is different from the coupling constant J=of β-arbutin herein
7.8, and do not see impurity peaks from nuclear magnetic spectrum.
Detailed description of the invention
Fig. 1 is present invention gained arbutin product characteristics photo；
Fig. 2 is the HPLC map of present invention gained arbutin；
Fig. 3 A is that the nuclear magnetic spectrum hydrogen of gained arbutin is composed；
Fig. 3 B is that the nuclear magnetic spectrum carbon of gained arbutin is composed.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without this hair described in claims should will not be limited
It is bright.Used reagent is bought from open market in the embodiment of the present invention.ɑ-arbutin fermentation liquid refers to use in the present invention
Quinhydrones does substrate microbial fermentation and prepares ɑ-arbutin tunning.
Embodiment 1
4L ɑ-arbutin fermentation liquid is removed into bacterium by doughnut membrane filtration system (molecular cut off of film is 60000)
Then body, the hydrochloric acid tune pH 3.0 of filtered clear liquid 1.0mol/L pass through Amberlite macroporous absorbent resin XAD-7
(1.5 liters), efflux continue through macroporous absorbent resin H103 (1.5 liters), rinse H103 resin column with the water of 3 times of column volumes,
It is eluted, is collected containing ɑ-arbutin eluent, vacuum is dense with the ethanol solution of the concentration of volume percent 10% of 4 times of column volumes afterwards
When being reduced to most of crystallization precipitation, placed in 4 DEG C of environment 1 hour, more crystal are precipitated at this time, with the acetone being pre-chilled in advance
Crystallization is hanged, is filtered, and obtain ɑ-arbutin sterling with a small amount of acetone washing.
Embodiment 2
4L ɑ-arbutin fermentation liquid is removed into bacterium by doughnut membrane filtration system (molecular cut off of film is 30000)
Then body, the hydrochloric acid tune pH 4.0 of filtered clear liquid 1.0mol/L pass through Amberlite macroporous absorbent resin XAD-8
(1.5 liters), efflux continue through macroporous absorbent resin H103 (1.5 liters), rinse H103 resin column 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, collects containing ɑ-arbutin eluent, be concentrated in vacuo to
It when most of crystallization is precipitated, is placed in 4 DEG C of environment 1 hour, more crystal are precipitated at this time, will be tied with the acetone being pre-chilled in advance
Crystalline substance hangs, and filters, and obtain ɑ-arbutin sterling with a small amount of acetone washing.
Embodiment 3
4L ɑ-arbutin fermentation liquid is removed into bacterium by doughnut membrane filtration system (molecular cut off of film is 60000)
Then body, the hydrochloric acid tune pH 5 of filtered clear liquid 1.0mol/L pass through Amberlite macroporous absorbent resin XAD-7 (1.5
Rise), efflux continues through macroporous absorbent resin H107 (1.5 liters), H107 resin column is rinsed with the water of 3 times of column volumes, afterwards with 4
The ethanol elution of the concentration of volume percent 15% of times column volume is collected containing ɑ-arbutin eluent, is concentrated in vacuo to big portion
It when crystallization being divided to be precipitated, is placed in 4 DEG C of environment 1 hour, more crystal are precipitated at this time, are hanged crystallization with the acetone being pre-chilled in advance
It rises, filtering, and obtains ɑ-arbutin sterling with a small amount of acetone washing.
Embodiment 4
4L ɑ-arbutin fermentation liquid is removed into bacterium by doughnut membrane filtration system (molecular cut off of film is 6000)
Then body, the hydrochloric acid tune pH 4.0 of filtered clear liquid 1.0mol/L pass through Amberlite macroporous absorbent resin XAD-6
(1.5 liters), efflux continue through macroporous absorbent resin H103 (1.5 liters), rinse H103 resin column 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, collects containing ɑ-arbutin eluent, be concentrated in vacuo to
It when most of crystallization is precipitated, is placed in 4 DEG C of environment 1 hour, more crystal are precipitated at this time, will be tied with the acetone being pre-chilled in advance
Crystalline substance hangs, and filters, and obtain ɑ-arbutin sterling with a small amount of acetone washing.
Claims (5)
1. a kind of separate ɑ-arbutin method from ɑ-arbutin fermentation liquid, it is characterized in that including the following steps:
(1) ɑ-arbutin fermentation liquid is filtered by hollow-fibre membrane, removes thallus, obtains filtrate；
(2) by filtrate tune pH to 3-5 obtained in step (1), then solution is received by moderately polar macroporous absorbent resin
Collect efflux；
(3) by the too low polar macroporous adsorption resin of efflux obtained in step (2), it is rinsed with water pillar later, then use ethyl alcohol
Solution elution, obtains eluent；
(4) by eluent obtained in step (3) it is vacuum dried be concentrated into crystallization be precipitated when, place, ɑ-arbutin is to crystallize
Form is precipitated；
(5) ɑ-black bearberry glycosidal crystalline organic solvent formed in step (4) hang, filtering, and with organic solvent washing
Obtain ɑ-black bearberry glycoside product.
2. according to claim 1 separate ɑ-arbutin method from ɑ-arbutin fermentation liquid, it is characterised in that described
Hollow-fibre membrane in step (1) is the hollow-fibre membrane that molecular cut off is 6000-60000.
3. according to claim 1 separate ɑ-arbutin method from ɑ-arbutin fermentation liquid, it is characterised in that described
In step (2), moderately polar macroporous absorbent resin refers to Amberlite XAD-6, XAD-7, XAD-7HP or XAD-8 resin.
4. according to claim 1 separate ɑ-arbutin method from ɑ-arbutin fermentation liquid, it is characterised in that described
Low polar macroporous absorbent resin in step (3), refers to H103, H107 or H1020 resin, the volume basis of the ethanol solution
Specific concentration is 10%-15%.
5. according to claim 1 separate ɑ-arbutin method from ɑ-arbutin fermentation liquid, it is characterised in that described
Organic solvent in step (5) is acetone.
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| CN108329363B (en) * | 2018-01-30 | 2020-10-27 | 安徽华恒生物科技股份有限公司 | Method for removing hydroquinone in alpha-arbutin conversion solution |
| CN108558964B (en) * | 2018-04-18 | 2020-06-16 | 山东众山生物科技有限公司 | Purification method of α -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 |
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 |
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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 |
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| 刘春巧.《发酵法生产α-熊果苷及其分离纯化》.《北京化工大学硕士学位论文》.2010,20-36. * |
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