CN108359698A - A method of based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin - Google Patents
A method of based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin Download PDFInfo
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- C12P19/48—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin the cyclohexyl radical being substituted by two or more nitrogen atoms, e.g. destomycin, neamin
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Abstract
A method of based on long chain branches cyclodextrin synthesis of glucose base beta cyclodextrin, belonging to branched cyclodextrin synthesis technical field.The present invention is that substrate acts on reverse reaction long chain branches cyclodextrin through Pullulanase by the Fructus Hordei Germinatus oligose and beta cyclodextrin of maltotetraose to seven sugar mixing, the first step first removes unreacted beta cyclodextrin through gel filtration chromatography, then glucoamylase is added in obtained chromatographic solution, and thoroughly hydrolysis obtains glucosyl group beta cyclodextrin and glucose, removal glucose is detached through chromatographic column again, the higher glucosyl group beta cyclodextrin product of purity is obtained after freeze-drying.The present invention preparation process and separation process all have it is simple for process, without using organic solvent and it is environmentally protective the advantages that, in the process without poisonous and harmful substance remain.
Description
Technical field
The present invention relates to a kind of methods based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin, belong to branch
Cyclodextrin synthesis technical field.
Background technology
If cyclodextrin is the glucosyltransferase caused by bacillus act on starch and generate it is a kind of by
Dry D- glucopyranose units are by cyclic oligosaccharide made of α-Isosorbide-5-Nitrae glucosides key connection, during structure is in wide at the top and narrow at the bottom
Empty cylinder shape, it is outer hydrophilic interior hydrophobic.There are three types of natural common cyclodextrin, i.e. α-CD, β-CD and γ-CD.Wherein again with β-
CD is most widely used, but the water solubility of β-CD is relatively low（25 DEG C, 1.8g/100mL）, while β-CD also have haemolysis effect and kidney poison
Property, this makes the application of β-CD be restricted.Therefore, it is exactly to improve it in water to a β-CD important purpose being modified
In solubility, reduce haemolysis effect and renal toxicity.
It is either enzymatically treated by chemical method on original cyclodextrin side chain and introduces monosaccharide or oligosaccharide, can obtained
Branched cyclodextrin, such as glucosyl group cyclodextrin, malt-base cyclodextrin, galactolipin cyclodextrin and mannose cyclodextrin.Chemistry
Method is current most common method, is widely used in the fields such as drug, agricultural, environmental protection and chromatographic isolation, but its safety limits
Its application in field of food is made；The branched cyclodextrin of enzyme modification has raw material green, production process safety and environmental protection, yield
The higher and segregative advantage of product, can be applied in food, but disadvantage of this law is that technique is cumbersome, cost is higher.
Glucosyl-ss-cyclodextrin had not only had the inclusion characteristic of parent P-cyclodextrin, but also improved solubility, reduces
Haemolysis effect and renal toxicity, therefore in industries such as food, medicine, pesticide, fine chemistry industry, analysis detection, environmental protection with huge
Application prospect, but at present it is less to the research of glucosyl-ss-cyclodextrin both at home and abroad, it is described in detail below.
1984, Kobayashi etc., which has studied a kind of limited reactions using BME and acts on waxy corn starch, to be divided
Then the method for branch oligosaccharides acts on branch's oligosaccharides, in glucoamylase and Taka- in the presence of SDS with BME
G has been obtained under the synergy of amylase A1- α-CD, yield are 84.2% relative to branched cyclodextrin.
1984, Abe etc. isolated G from the corn syrup of commercialization1-β-CD。
1997, Watanabe etc., which is devised, a kind of preparing high yield G1The new method of-α-CD.This method includes two steps
Suddenly：Maltose and α-CD are condensed into G by Pullulanase2- α-CD and G2- α-CD are hydrolyzed under glucoamylase enzyme effect.Due to
Glucoamylase is by G2- α-CD are hydrolyzed to G1- α-CD, the conjunction of the balanced deflection malt-base α-CD of Pullulanase condensation reaction
At result leads to the increase of branching ratio.This method branching ratio is up to 69%.
2006, Cui Bo etc. utilized Pullulanase inverse composition property, and malt is synthesized as substrate using maltose and cyclodextrin
Glycosyl-CDs, then a molecule glucose is fallen in hydrolysis under the action of diastase, generates glucosyl group-CDs.
The separation of branched cyclodextrin and cyclodextrin all the time is the difficult point of industrialized production, such as G1- β-CD and β-CD
Only poor 162MW, and the substance of industrial separation packing material size separation difference relatively difficult to achieve so small-molecular-weight at present, and text at present
Offer report synthesis branched cyclodextrin method be that first monosaccharide or oligosaccharide are grafted on CDs, then use amylorrhexis, then into
Row separation causes β-CDs and CDs to detach difficult, therefore, how to improve synthetic ratio and separative efficiency and technological process is environmentally protective
Have become the technical problem that nowadays branched cyclodextrin field is urgently to be resolved hurrily.
In view of this, special propose the present invention.
Invention content
It is an object of the present invention to overcome the above deficiencies, provides based on long chain branches cyclodextrin synthesis of glucose base-
The method of beta-cyclodextrin solves the difficulty of separation, and the method has in easy to operate, preparation process without using organic molten
Agent, it is environmentally protective the advantages that.
According to technical solution provided by the invention, one kind being based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin
Method, steps are as follows：
（1）The preparation of long chain branches cyclodextrin：It is that substrate acts on reverse reaction through Pullulanase by Fructus Hordei Germinatus oligose and beta-cyclodextrin
Long chain branches cyclodextrin, then remove unreacted beta-cyclodextrin through chromatographic column separation；
（2）The preparation of glucosyl-ss-cyclodextrin：To step（1）It prepares and glucose shallow lake is added in gained long chain branches cyclodextrin
Thoroughly hydrolysis obtains glucosyl-ss-cyclodextrin and glucose to powder enzyme, then detaches removal glucose, remaining grape through chromatographic column
Glycosyl-beta-cyclodexterin aqueous solution is freeze-dried to obtain product glucosyl-ss-cyclodextrin powder.
It is as follows：
（1）The preparation of long chain branches cyclodextrin：By Fructus Hordei Germinatus oligose and beta-cyclodextrin according to molar ratio 8 ~ 12:It is used as bottom after 1 mixing
Pullulanase is added according to the ratio of 100 ~ 500 μ L/mL substrates wherein in object, and reaction temperature is 50 ~ 60 DEG C, and reaction system is
The acetic acid-sodium acetate buffer solution of pH 4.5 ~ 5.5, reaction time are 60 ~ 72h；
Gained reaction solution is removed into unreacted beta-cyclodextrin using chromatographic column separation, the separation molecular weight ranges of chromatographic stuffing are
100-1800MW；
（2）The preparation of glucosyl-ss-cyclodextrin：According to the amount of 50 ~ 100 μ L/mL substrates to step（1）Add in gained reaction solution
Add glucoamylase, 12 ~ 20h is hydrolyzed at 50 ~ 55 DEG C；Reaction solution detaches removal grape in chromatographic column after gained is hydrolyzed
The separation molecular weight ranges of sugar, chromatographic stuffing are 100 ~ 1500MW；
Gained glucosyl-ss-cyclodextrin aqueous solution after chromatography post separation is freeze-dried 48h at -10 DEG C~-50 DEG C, is obtained
Product glucosyl-ss-cyclodextrin powder.
The glucosyl-ss-cyclodextrin is in preparation process, using Fructus Hordei Germinatus oligose as substrate, the Fructus Hordei Germinatus oligose be by
Maltodextrin is through gel-filled column chromatography for separation and is lyophilized and obtains, and has the document report Pullulanase reverse reaction of Japanese scholars can
To utilize G3~G6And α-CD synthesize branched cyclodextrin, so being feasible using Fructus Hordei Germinatus oligose as substrate.
The step（1）In, the Pullulanase of selection is passed through by bacillus licheniformis made of submerged fermentation, Hydrolysis kinetics
Food grade enzyme is suitable for industrialized production.Containing a large amount of small molecule carbohydrate etc. in enzyme solution, cause enzyme solution sticky.In order to obtain
Purer enzyme enhances the effect of follow-up pillar layer separation, it is necessary to first remove the small-molecule substances such as carbohydrate therein, therefore select
Rough segmentation is carried out to Pullulanase with adaptability extensive dialysis, or uses ultra-filtration centrifuge tube, centrifugal filtration is fallen in enzyme solution
Small molecular sugar.
The step（1）In, the Fructus Hordei Germinatus oligose of selection and the molar ratio of beta-cyclodextrin are 8 ~ 12:1, since beta-cyclodextrin is
The important skeletal substance of glucosyl-ss-cyclodextrin is ultimately formed, maltotetraose to seven sugar is then main grafting material, and process is general
Reverse reaction, that is, transglycosylation of Shandong orchid enzyme is grafted on 6 hydroxyls of beta-cyclodextrin, and Pullulanase is under high concentration of substrate
Reverse reaction vigor it is maximum, and beta-cyclodextrin has obvious inhibiting effect, therefore two to the reverse reaction ability of Pullulanase
Ratio between person is particularly important, if the excessive Pullulanase of ratio mainly plays hydrolysis, if ratio is too small
The inhibiting effect of beta-cyclodextrin can cause synthetic product amount considerably less, therefore suitable ratio is preferably 8 ~ 12:1 or so, at it
According to 100 ~ 500 μ L/mL substrates ratio be added Pullulanase.
The step（1）In, with reference to pertinent literature use DNS methods measure Pullulanase backward reaction temperature range for
50 ~ 60 DEG C, the acetic acid-sodium acetate buffer solution of pH ranging from 4.5 ~ 5.5.More products in order to obtain, set the reaction time as
60~72h.Enzyme reactor temperature is risen into 100 DEG C of enzyme deactivation 15min after reaction.
Step（1）The substrate Fructus Hordei Germinatus oligose is mixture of the maltotetraose to seven sugar of malt, and preparation method is as follows：
（1）It weighs DE13-17 maltodextrins to be dissolved in ultra-pure water, is configured to the maltodextrin water that mass/volume ratio is 18% ~ 22%
Solution；It takes the solution to be splined on gel chromatography column, is eluted with the aqueous solution containing 0.1M ammonium hydrogen carbonate, automatic collector collection is washed
De- liquid stops collecting after collecting 40-50 pipes；The sample liquid of collection is subjected to TLC Preliminary Determinations, on same TLC plates successively
1 μ L of point sample spray developing solution after sampling point parches completely, are positioned on thin-layer chromatography baking sheet machine and are to slowly warm up to 160 DEG C of heating
Colour developing；
（2）Select the sample cell of the colour developing point sample again on the TLC plates for finish line；Wherein standard specimen is the G of 100mM1~G7Mixing
Then liquid is put in chromatography cylinder, developping solution did not had point line-transect, behind the top edge to be deployed to plank taking-up dried up with hair-dryer,
Developing solution is sprayed, is positioned on thin-layer chromatography baking sheet machine and is to slowly warm up to 160 DEG C of heating colour developings, and containing G1~G7Standard specimen comparison after
It filters out and mainly contains maltotetraose to the sample cell of seven sugar of malt, be freeze-dried.
The step（1）In, void column 2.5 × 50cm of size of gel chromatography column used, bed volume about 500cm3, separation
A concentration of the 20% of maltodextrin, each applied sample amount 2.5mL, automatic collector is collected one per 10min and is managed, after about 8 ~ 10h, by institute
Point sample, the expansion on the tlc plate of some collecting pipes, can judge the pipe number mainly containing maltotetraose to seven sugar of malt after colour developing,
For the main component of this more determining several pipe, HPLC measurement can be carried out, chromatographic condition is：Mobile phase：70% acetonitrile；Column temperature：30
℃；Flow velocity：1mL/min；Sample size：20μL；Chromatographic column：Lichrospher NH2(5μm 4.6×250 mm)；Pump：Shimadzu
Then this several pipe is freeze-dried, often manages and about obtain 10mg dry matters by LC-20A.
The step（2）In, glucoamylase activity is 36 ~ 50U/mg, and additive amount is 50 ~ 100 μ L/mL substrates, instead
It is 12 ~ 20h to answer 50 ~ 55 DEG C of temperature, reaction time.After reaction, enzyme reactor temperature rises to 100 DEG C of enzyme deactivation 15min.It will be anti-
Liquid is answered to carry out HPLC and UPLC-MS detections.
Step（1）And step（2）Reaction carried out in enzyme reactor, rotating speed be 500 ~ 1000rpm；Step（1）With
Step（2）After reaction, enzyme reactor temperature is risen into 100 DEG C of 14 ~ 16min of enzyme deactivation.
The developing solution is absolute methanol and water with volume ratio 1:1 mixing, is slowly added to the concentrated sulfuric acid, keeps concentrated sulfuric acid quality whole
A concentration of 10%；The developping solution is isobutanol:Ethyl alcohol:Water volume ratio 5:5:3.
Beneficial effects of the present invention：The preparation process and separation process of the present invention all have it is simple for process, without using organic
Solvent and it is environmentally protective the advantages that, in the process without poisonous and harmful substance remain.
Description of the drawings
Fig. 1 is glucosyl-ss-cyclodextrin liquid phase figure.
Fig. 2 is 8.36min G1The mass spectrogram of-β-CD.
Fig. 3 reacting flow charts of the present invention.
Specific implementation mode
Illustrate technical scheme of the present invention below with two embodiments, but protection scope of the present invention is not limited to this.
Wherein embodiment 1 is the feasibility test of early period, to obtain the basic experimental conditions of synthesis of glucose group-beta-cyclodextrin.
1 feasibility Experiment of embodiment
The preparation of Fructus Hordei Germinatus oligose：It weighs 2g DE13-17 maltodextrins to be dissolved in 10mL ultra-pure waters, that is, is configured to a concentration of 20%
(W/V) maltodextrin aqueous solution.This solution loading of 2.5mL is drawn to chromatography top end injection port with suction pipe, and mobile phase is
The often pipe acquisition time interval of 0.1M ammonium bicarbonate aqueous solutions, collector is initially set 2h, waits often receiving pipe after collecting 3 pipes
Collection time interval is set as 10min, after collecting pipe number reaches 45 pipes, stops collecting.At the beginning of 45 pipe samples of collection are carried out TLC
Pacing is fixed, and 1 μ L of point sample spray developing solution after sampling point parches completely successively on one block of TLC plate（First alcohol and water is with volume ratio
1:1 mixing, is slowly added to 10% concentrated sulfuric acid）, it is positioned on baking sheet machine and is to slowly warm up to 160 DEG C of heating colour developings.Selection colour developing
Sample cell such as 8 ~ No. 30, the point sample again on the TLC plates for finish line（Each block of plate will put standard specimen：G1~G7Mixed liquor,
100mM）, then it is put in chromatography cylinder, developping solution（Isobutanol:Ethyl alcohol:Water volume ratio=5:5:3）Have some line-transects, and wait opening up
Taking-up is dried up with hair-dryer after opening to plank top edge, sprays developing solution, is positioned on baking sheet machine and is to slowly warm up to 160 DEG C of heating
Colour developing, with standard specimen（Containing G1~G7）It is filtered out after comparison and mainly contains maltotetraose to the sample cell of seven sugar of malt, it is dry to carry out freezing
It is dry, often it is in control about 10mg samples.
Feasibility is verified：The reaction substrate Fructus Hordei Germinatus oligose prepared in the 10mg above methods and 2mg or so β-CD are taken, 100 μ are added
L pH4.5 acetic acid buffer solutions and 10 μ L Pullulanases, react 60h by 60 DEG C.After completion of the reaction, 100 DEG C of enzyme deactivation 15min.It is down to
After room temperature, 5 μ L glucoamylases are respectively added in every test tube, 50 DEG C, react 12h, after completion of the reaction, 100 DEG C of enzyme deactivation 15min, mistake
0.45 μm of filter membrane carries out liquid chromatography mass spectrometric combination detection, measures that UPLC-MS results are as depicted in figs. 1 and 2, and Fig. 1 is liquid phase figure, Fig. 2
For 8.36min G1The mass spectrogram of-β-CD.
The corresponding component of each retention times of 1 Fig. 1 of table
| Number | Retention time（min） | Sample |
| 1 | 6.53 | β-CD |
| 2 | 8.36 | G1-β-CD |
Embodiment 2
Weigh 20g embodiments 1 and prepare gained reaction substrate Fructus Hordei Germinatus oligose and 2g β-CD, add 5.0 acetate buffers of 10mL pH and
400 μ L/mL Pullulanases, 60 DEG C of insulation reaction 60h.Boiling water enzyme deactivation 15min after reaction adds a small amount of water to be diluted to 25mL,
Zymoprotein is removed in centrifugation, takes supernatant loading to gel chromatography post separation, according to the principle macromolecule of exclusion chromatography
Substance is first eluted out, therefore collects the component flowed out at first（That is long chain branches cyclodextrin, MW ＞ 1600）, add pH5.0 acetic acid
Buffer solution, 100 μ L/mL substrates of glucoamylase, 55 DEG C of insulation reaction 12h of enzyme reactor, after reaction 100 DEG C of enzyme deactivations
15min, centrifugation remove zymoprotein, take supernatant loading to gel chromatography post separation, it is G that this step flows out component at first1-β-
CD, the sample liquid of collection is freeze-dried, just obtain pure glucosyl-ss-cyclodextrin powder.The flow chart entirely reacted
As shown in Figure 3.
Embodiment 3
It is as follows：
（1）The preparation of long chain branches cyclodextrin：By Fructus Hordei Germinatus oligose and beta-cyclodextrin according to molar ratio 8:It is used as substrate after 1 mixing,
Pullulanase is added according to the ratio of 100 μ L/mL substrates wherein, reaction temperature is 50 DEG C, and reaction system is the second of pH 4.5
Acid-sodium acetate buffer, reaction time 60h；
Gained reaction solution is removed into unreacted β-CD using chromatographic column separation, the separation molecular weight ranges of chromatographic stuffing are
1200MW；
（2）The preparation of glucosyl-ss-cyclodextrin：According to the amount of 50 μ L/mL substrates to step（1）Portugal is added in gained reaction solution
Grape saccharogenic amylase hydrolyzes 20h at 50 DEG C；Gained reaction solution is detached into removal glucose, the separation of chromatographic stuffing in chromatographic column
Molecular weight ranges are 1000MW；Gained glucosyl-ss-cyclodextrin aqueous solution is freeze-dried 48h at -10 DEG C, obtains product
Glucosyl-ss-cyclodextrin powder.
Embodiment 4
It is as follows：
（1）The preparation of long chain branches cyclodextrin：By Fructus Hordei Germinatus oligose and beta-cyclodextrin according to molar ratio 12:It is used as substrate after 1 mixing,
Pullulanase is added according to the ratio of 500 μ L/mL substrates wherein, reaction temperature is 60 DEG C, and reaction system is the second of pH 5.5
Acid-sodium acetate buffer, reaction time 72h；
Gained reaction solution is removed into unreacted β-CD using chromatographic column separation, the separation molecular weight ranges of chromatographic stuffing are
1800MW；
（2）The preparation of glucosyl-ss-cyclodextrin：According to the amount of 100 μ L/mL substrates to step（1）It is added in gained reaction solution
Glucoamylase hydrolyzes 20h at 55 DEG C；Gained reaction solution is detached into removal glucose, point of chromatographic stuffing in chromatographic column
It is 1500MW from molecular weight ranges；Gained glucosyl-ss-cyclodextrin aqueous solution is freeze-dried 48h at -50 DEG C, is produced
Product glucosyl-ss-cyclodextrin powder.
Claims (6)
1. a kind of method based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin, it is characterised in that：
（1）The preparation of long chain branches cyclodextrin：It is that substrate acts on reverse reaction through Pullulanase by Fructus Hordei Germinatus oligose and beta-cyclodextrin
Long chain branches cyclodextrin, then remove unreacted beta-cyclodextrin through chromatographic column separation；
（2）The preparation of glucosyl-ss-cyclodextrin：To step（1）It prepares and glucose shallow lake is added in gained long chain branches cyclodextrin
Thoroughly hydrolysis obtains glucosyl-ss-cyclodextrin and glucose to powder enzyme, then detaches removal glucose, remaining grape through chromatographic column
Glycosyl-beta-cyclodexterin aqueous solution is freeze-dried to obtain product glucosyl-ss-cyclodextrin powder.
2. the method as described in claim 1 based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin, it is characterised in that
It is as follows：
（1）The preparation of long chain branches cyclodextrin：By Fructus Hordei Germinatus oligose and beta-cyclodextrin according to molar ratio 8 ~ 12:It is used as bottom after 1 mixing
Pullulanase is added according to the ratio of 100 ~ 500 μ L/mL substrates wherein in object, and reaction temperature is 50 ~ 60 DEG C, and reaction system is
The acetic acid-sodium acetate buffer solution of pH 4.5 ~ 5.5, reaction time are 60 ~ 72h；
Gained reaction solution is removed into unreacted beta-cyclodextrin using chromatographic column separation, the separation molecular weight ranges of chromatographic stuffing are
100-1800MW；
（2）The preparation of glucosyl-ss-cyclodextrin：According to the amount of 50 ~ 100 μ L/mL substrates to step（1）Add in gained reaction solution
Add glucoamylase, 12 ~ 20h is hydrolyzed at 50 ~ 55 DEG C；Reaction solution detaches removal grape through chromatographic column after gained is hydrolyzed
The separation molecular weight ranges of sugar, chromatographic stuffing are 100 ~ 1500MW；
Gained glucosyl-ss-cyclodextrin aqueous solution after chromatography post separation is freeze-dried 48h at -10 DEG C~-50 DEG C, is obtained
Product glucosyl-ss-cyclodextrin powder.
3. the method according to claim 2 based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin, feature exist
In：Step（2）The enzyme activity of the glucoamylase is 36 ~ 50U/mg.
4. the method according to claim 2 based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin, feature exist
In：Step（1）And step（2）Reaction carried out in enzyme reactor, rotating speed be 500 ~ 1000rpm；Step（1）And step
（2）After reaction, enzyme reactor temperature is risen into 100 DEG C of 14 ~ 16min of enzyme deactivation.
5. the method according to claim 2 based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin, feature exist
In step（1）The Fructus Hordei Germinatus oligose is mixture of the maltotetraose to seven sugar of malt, and preparation method is as follows：
（1）It weighs DE13-17 maltodextrins to be dissolved in ultra-pure water, is configured to the maltodextrin water that mass/volume ratio is 18% ~ 22%
Solution；It takes the solution to be splined on gel chromatography column, is eluted with the aqueous solution containing 0.1M ammonium hydrogen carbonate, automatic collector collection is washed
De- liquid stops collecting after collecting 40-50 pipes；The sample liquid of collection is subjected to TLC Preliminary Determinations, on same TLC plates successively
1 μ L of point sample spray developing solution after sampling point parches completely, are positioned on thin-layer chromatography baking sheet machine and are to slowly warm up to 160 DEG C of heating
Colour developing；
（2）Select the sample cell of the colour developing point sample again on the TLC plates for finish line；Wherein standard specimen is the G of 100mM1~G7Mixed liquor,
Then be put in chromatography cylinder, developping solution did not had point line-transect, behind the top edge to be deployed to plank taking-up dried up with hair-dryer, spray
Developing solution is positioned on thin-layer chromatography baking sheet machine and is to slowly warm up to 160 DEG C of heating colour developings, and containing G1~G7Standard specimen comparison after screen
Go out to mainly contain maltotetraose G4To seven sugar G of malt7Sample cell, be freeze-dried.
6. the method according to claim 5 based on long chain branches cyclodextrin synthesis of glucose group-beta-cyclodextrin, feature exist
In：The developing solution is absolute methanol and water with volume ratio 1:1 mixing, is slowly added to the concentrated sulfuric acid, makes concentrated sulfuric acid quality final concentration
It is 10%；The developping solution is isobutanol:Ethyl alcohol:Water volume ratio 5:5:3.
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| CN110747245A (en) * | 2019-11-29 | 2020-02-04 | 江南大学 | Method for preparing malt oligosaccharide syrup by using complex enzyme |
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