CN106883352A - A kind of method of chitobiose/chitotriose monomer prepare with scale - Google Patents
A kind of method of chitobiose/chitotriose monomer prepare with scale Download PDFInfo
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- CN106883352A CN106883352A CN201710160151.7A CN201710160151A CN106883352A CN 106883352 A CN106883352 A CN 106883352A CN 201710160151 A CN201710160151 A CN 201710160151A CN 106883352 A CN106883352 A CN 106883352A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F257/00—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00
- C08F257/02—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00 on to polymers of styrene or alkyl-substituted styrenes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/16—Organic material
- B01J39/18—Macromolecular compounds
- B01J39/20—Macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/12—Polymerisation in non-solvents
- C08F2/16—Aqueous medium
- C08F2/20—Aqueous medium with the aid of macromolecular dispersing agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F212/02—Monomers containing only one unsaturated aliphatic radical
- C08F212/04—Monomers containing only one unsaturated aliphatic radical containing one ring
- C08F212/06—Hydrocarbons
- C08F212/08—Styrene
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F212/34—Monomers containing two or more unsaturated aliphatic radicals
- C08F212/36—Divinylbenzene
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/34—Introducing sulfur atoms or sulfur-containing groups
- C08F8/36—Sulfonation; Sulfation
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Abstract
The present invention relates to a kind of method of chitobiose/chitotriose monomer prepare with scale, comprise the following steps：(1) take chitosan oligosaccharide component and be dissolved in water wiring solution-forming, decolourize, filtering pumps into highly acidic cation exchange column and starts loading, is eluted with hydrochloric acid solution, collect one every same time and manage；(2) saccharic composition in each pipe sampling detection collection liquid, merges identical saccharic composition；(3) chitobiose of merging and the component of chitotriose two are rotated to dry, sloughs hydrochloric acid, that is, respectively obtain chitobiose elemental solid and chitotriose elemental solid.Compared with prior art, technique of the invention is easy, and yield is high, it is possible to achieve high purity of shell disaccharides, chitotriose monomer of feather weight preparative-scale etc..
Description
Technical field
Field is isolated and purified the present invention relates to a kind of function ocean amino-oligosacchride, more particularly, to a kind of chitobiose/shell
The method of three sugar monomer prepare with scale.
Background technology
Chitosan oligosaccharide is that to birds of the same feather flock together right be 2~10, and molecular weight is less than 3000Da, the alkalescence widow of unique positively charged in nature
Sugar.Its structure is formed by connecting by β -1,4- glycosidic bonds by D- Glucosamines and 2-acetylamino-2-deoxy-D-glucose.Chitosan oligosaccharide
Possess various physiologically actives containing hydroxyl and amino active group in self structure.Such as antibacterial, anti-oxidant, antitumor, anticancer
Activity, reduces cholesterol and enhance immunity etc..Because of itself small molecule, good aqueous solubility, good organization's compatibility, natural nothing
Malicious the features such as, is usually used in the fields such as biological medicine, health food, agriculture herding, sewage disposal.
The preparation method of chitosan oligosaccharide can be divided into two classes, one is, be formed by connecting for monose by specific enzyme by synthetic method.
The second is edman degradation Edman, edman degradation Edman has Physical, chemical method and enzymatic isolation method.Physical inefficiency, meeting in chemical method preparation process
Consume substantial amounts of chemical reagent and produce substantial amounts of acidic and alkaline waste water pollution environment.It is a kind of environmental protection with respect to enzymatic isolation method, can be high
Energy-conservation and can effectively prepare target oligosaccharides.At present, chitosan oligosaccharide activity research uses blending ingredients mostly, seldom uses chitooligose monomer
Tested, cause to be difficult clearly specific which kind of chitooligose monomer or which chitooligose monomer plays a role.Because of chitosan oligosaccharide list
Difference between body is small, and separating difficulty is big, and production scale is small, expensive, using limited.Have been reported chitobiose, chitotriose
Possess stronger antioxidation activity, therefore, chitobiose, chitotriose monomer prepare with scale can be the activity research and medicine of chitosan oligosaccharide
Provided safeguard with value popularization.
Chinese patent ZL201110068951.9 discloses a kind of method of preparing chitosan oligosaccharide monomers by gel chromatography, specific step
Suddenly it is：Chitosan oligosaccharide is dissolved in dual distilled water with the mass concentration of 20-70%, until completely dissolved, suction filtration, filtrate is shell
Oligosaccharide solution；Chitosan oligosaccharide solution is splined on gel chromatography column, with dual distillation water elution, every 30 points with automatic fraction collector
Clock is collected one and is managed, and often pipe takes 0.1ml, after acetone precipitation, by precipitation distillation water dissolves, TLC quick detections is then used, it is determined that often
The species of the contained chitooligose monomer of pipe；Often manage and separately take a small amount of liquid freezing drying, claim solid content, to determine contained by often pipe
The concentration of chitosan oligosaccharide, draws elution curve；The isolated solution containing chitooligose monomer；Concentrated under reduced pressure, freeze-drying is obtained respectively
Chitooligose monomer.But, the filler in above-mentioned patent is relatively costly, and it is difficult regeneration, and applied sample amount is also smaller, is not suitable for rule
Modelling production prepares chitooligose monomer.
The content of the invention
The purpose of the present invention is exactly to provide a kind of chitobiose/chitotriose for the defect for overcoming above-mentioned prior art to exist
The method of monomer prepare with scale.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of chitobiose/chitotriose monomer prepare with scale, comprises the following steps：
(1) take chitosan oligosaccharide component and be dissolved in water wiring solution-forming, decolourize, filtering is pumped on highly acidic cation exchange column starts
Sample, is eluted with hydrochloric acid solution, is collected one every same time and is managed；
(2) each pipe sampling revolving detects the saccharic composition of collection liquid to dry, merges identical saccharic composition；
(3) chitobiose of merging and the component of chitotriose two are rotated to dry, sloughs hydrochloric acid, that is, respectively obtain chitobiose monomer
Solid and chitotriose elemental solid.Obtained chitobiose, chitotriose structural formula be respectively I (a), shown in (b),
Preferably, in the chitosan oligosaccharide component described in step (1), the total content ＞ 95% of chitobiose and chitotriose.
It is furthermore preferred that described chitosan oligosaccharide component is made by the following method：
Take shitosan add water fully it is swelling, add acetic acid, stir, adjust pH to 5.0~5.6, add shitosan
Enzyme, stirring carries out enzyme digestion reaction, and after reaction terminates, centrifugation, filtering is dried, and obtains final product the chitosan oligosaccharide component.
Even more preferably, the deacetylation of described shitosan>95%, viscosity<60mPa·S.
Preferably, described highly acidic cation exchange column is made by the following method：
The preparation of (a) Aqueous Phase Raw Material
Deionized water, ethylenediamine, sodium chloride, sodium peroxydisulfate and polyvinyl alcohol are weighed, regulation system PH is neutrality, instead
Should, obtain Aqueous Phase Raw Material；
The preparation of (b) oil phase raw material
Benzoyl peroxide, azodiisobutyronitrile, styrene, ferrocene and divinylbenzene are weighed, is mixed, follow procedure A liters
Temperature, obtains intermediate product A, is subsequently adding the Aqueous Phase Raw Material mixing that step (a) is obtained, and suspension polymerization obtains bead friendship
Ally the communists polymer backbone；
C () after polymerization prepares intermediate product B
Copolymer skeleton, styrene, deionized water, divinylbenzene and azodiisobutyronitrile are weighed, is mixed, follow procedure A liters
Temperature, obtains intermediate product B；
(d) sulfonating reaction
Intermediate product B, dichloroethanes, ethyl acetate, solvent naphtha and the concentrated sulfuric acid are weighed, follow procedure B heats up, vacuum distillation,
Cooled down after reaction, cleaned, dried, multistage is sieved, that is, obtain purpose product storng-acid cation exchange resin.
It is furthermore preferred that deionized water in step (a):Ethylenediamine:Sodium chloride:Sodium peroxydisulfate:The addition quality of polyvinyl alcohol
Than being (1000~1200):(30~45):(85~95):(1.5~4):(75~120)；
Benzoyl peroxide in step (b):Azodiisobutyronitrile:Styrene:Ferrocene:The mass ratio of divinylbenzene is
(1.5~4.0):(3~6):(900~1000):(25~40):(50~65)；
Copolymer skeleton in step (c):Styrene:Deionized water:Divinylbenzene:The mass ratio of azodiisobutyronitrile is
(240~350):(250~360):(200~300):(25~32):(2~6)；
Step (d) intermediate product B:Dichloroethanes:Ethyl acetate:Solvent naphtha:The mass ratio of the concentrated sulfuric acid for (1000~
1300):(400~500):(50~65):(80~105):(2500~3000).Heated up in resins synthesis step (b) (c) (d)
Mode influences formation and the cross-linkage of resin of resin particle, controls what is prepared using the screen cloth multi-stage screening of 150~300 mesh
The granularity of storng-acid cation exchange resin, it is abundant to realize reaction in step (d) sulfonating reaction, use excessive dense sulphur
Acid so that more multi-functional group i.e. sulfonic acid group is bonded in repeat unit skeleton.Have compared with other cationic ion-exchange resins
Bigger exchange capacity.It is furthermore preferred that the process conditions of reaction are in step (a)：Temperature control is 45 DEG C~55 DEG C, stirring speed
Degree 120rmp/min, reacts 24h；
Step (b) Program A heats up and is specially：Temperature is risen to by 78~80 DEG C, perseverance with the programming rate of 0.5 DEG C/min
5~6h of temperature, is further continued for progressively being warming up to 93~95 DEG C, 5~6h of constant temperature；The process conditions of suspension polymerization are：Temperature control
To 65~70 DEG C, 45~8h is reacted；
Step (d) Program B heat up technique be：78~80 DEG C, perseverance are warming up to 0.5~0.7 DEG C/min programming rates
Temperature 8~10h of reaction, then 805~85 DEG C, 4~6h of isothermal reaction, finally with 0.1~0.2 are warming up to 0.02~0.05 DEG C/min
DEG C/speed of min is warming up to 115 DEG C；The process conditions of vacuum distillation are：Pressure≤0.01MPa.
Above-mentioned storng-acid cation exchange resin uses preceding its pre- hydrochloric acid and 1mol/ for first passing through 1mol/L~3mol/L
The alkali lye of L~2mol/L is alternately pre-processed, pretreatment time 12h~24h.
Even more preferably, the ratio between addition of shitosan and acetic acid is (100~200) g:(40~80) mL, shell gathers
The addition of carbohydrase is 30~60U/g；
The temperature of enzyme digestion reaction is 30~40 DEG C.
Preferably, the strongly acidic cation exchange tree in step (1) in chitosan oligosaccharide component and highly acidic cation exchange column
Fat amount ratio is (80~200) g:(1200~2000) mL.
Preferably, decolourize to use activated carbon decolorizing in step (1), the mass ratio of activated carbon and chitosan oligosaccharide component for (0.1~
0.6)g:(80~200) g.
Preferably, the concentration of hydrochloric acid solution is 1.0~4mol/L in step (1), and its flow velocity is 0.3~0.6BV/h.
It is furthermore preferred that collection procedure is in step (1)：One is collected every 2~3h to manage, and label successively, 30 are collected altogether manages；
Saccharic composition is detected using TLC methods in step (2), wherein, 7~No. 13 pipes are chitobiose, and 16~No. 28 pipes are shell three
Sugar.
Preferably, it is filtered into using sand core filter filtering, the mesh of its aperture ＞ 200.
Preferably, the judgement of reaction end is：Sampled every 8h and use TLC methods to detect, if detecting enzymolysis product only
For chitobiose, chitotriose are that can be considered reaction end；
The measure of enzymolysis reaction is：It is heated to 100 DEG C and keeps 10min.
Preferably, adjustment pH used by solution be NaAc_HAc buffer solution, its concentration be 0.1mol/L~
0.3mol/L。
The present invention with contrast patent than relatively similar, main distinction point is：The filler that both use is different, and the present invention is developed
Go out epigranular, the storng-acid cation exchange resin higher to chitosan oligosaccharide adsorption capacity, with hydrochloric acid as eluent, using gradient
Type of elution, eluent flow rate is not equal.Chitobiose chitotriose is separated using storng-acid cation exchange resin in the present invention.Its
Principle be according to chitobiose, chitotriose on strong-acid ion exchange resin absorption affinity difference in size and by various concentrations salt pickling
Take off.Separating effect is influenceed by concentration of hydrochloric acid, flow velocity etc..Concentration of hydrochloric acid is too low, and the chitosan oligosaccharide adsorbed on resin can not be solved
Analysis.If concentration of hydrochloric acid is too high, eluting power is strong, causes chitobiose chitotriose separating degree low.
Additionally, the invention further particularly discloses the preparation method of chitosan oligosaccharide component, it is made using preparation method of the invention
Standby chitosan oligosaccharide component, for general chitosan oligosaccharide product, possesses that molecular weight is low and chitobiose chitotriose composition accounts for 90%
More than, the component of preparation is influenceed by the factor such as addition, temperature, rotating speed, acid-base value of enzyme.
Compared with prior art, the present invention has advantages below：
1) chitobiose, the chitotriose monomer of high-purity, and scale level are may separate out using storng-acid cation exchange resin
It is capable of achieving feather weight.
2) chitobiose, the yield of chitotriose monomer that prepared by the present invention detect it higher than 90% through high performance liquid chromatography
Purity chitobiose is higher than 98%, and chitotriose is higher than 96%.Storng-acid cation exchange resin filler used is renewable, produces work
Skill stabilization, operating cost is low.
Brief description of the drawings
Fig. 1 is the sugared concentration wash-out stream that storng-acid cation exchange resin of the invention prepares chitobiose, chitotriose monomer
Go out curve map；
Fig. 2 is the TLC detection collection of illustrative plates for separating and collecting liquid of the invention；
Fig. 3 is the TLC analysis collection of illustrative plates that the present invention merges the monomer after collecting component；
Fig. 4 is the HPLC collection of illustrative plates of the hybrid standard product of the chitosan oligosaccharide degree of polymerization 1~7；
Fig. 5 is the HPLC collection of illustrative plates of the obtained enzymolysis component of the present invention；
Fig. 6 is the HPLC collection of illustrative plates of chitobiose monomer obtained in the present invention；
Fig. 7 is the HPLC collection of illustrative plates of chitotriose monomer obtained in the present invention
Fig. 8 is the structure infrared spectrogram that the present invention characterizes its obtained chitobiose, chitotriose monomer using FT-IR methods
Spectrum；
Fig. 9 is used for the present invention1The collection of illustrative plates of the obtained chitobiose monomer structure of HNMR methods analysis；
Figure 10 is used for the present invention1The collection of illustrative plates of the obtained chitotriose monomer structure of HNMR methods analysis.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
(1) synthesis of storng-acid cation exchange resin is to combine to form copolymer pearl with styrene and divinylbenzene crosslink
Grain is skeleton, and introducing sulfonic group by the sulfonation of concentrated sulfuric acid strong oxidizer afterwards is made.Specifically there is procedure below：
The preparation of (a) Aqueous Phase Raw Material
By ratio of weight and the number of copies 1000:30:85:1.5:75 weigh deionized water:Ethylenediamine:Sodium chloride:Sodium peroxydisulfate:Poly- second
Enol, regulation system PH is neutrality, and temperature control is 45 DEG C~55 DEG C, and mixing speed 120rmp/min reacts 24h, obtains water
Phase raw material；
The preparation of (b) oil phase raw material
Benzoyl peroxide is weighed by weight:Azodiisobutyronitrile:Styrene:Ferrocene:Divinylbenzene=1.5:
3:900:25:50, temperature is risen to 78~80 DEG C by mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature, be further continued for by
Step is warming up to 93~95 DEG C, and 5~6h of constant temperature obtains intermediate product A, is subsequently adding the Aqueous Phase Raw Material mixing that step (a) is obtained, and rises
To 65~67 DEG C, suspension polymerization 7h obtains bead cross-linked copolymer skeleton to temperature；
C () after polymerization prepares intermediate product B
Copolymer skeleton is weighed by weight:Styrene:Deionized water:Divinylbenzene:Azodiisobutyronitrile=240:
250:200:25:2, temperature is risen to 78~80 DEG C by mixing with the programming rate of 0.5 DEG C/min, and 5~6h of constant temperature is further continued for
93~95 DEG C are progressively warming up to, 5~6h of constant temperature obtains intermediate product B；
(d) sulfonating reaction
Intermediate product B is weighed by weight:Dichloroethanes:Ethyl acetate:Solvent naphtha:The concentrated sulfuric acid=1000:400:50:
80:2500, it is warming up to 78~80 DEG C with 0.5~0.7 DEG C/min programming rates, 8~10h of isothermal reaction, then with 0.02~0.05
DEG C/min is warming up to 83~85 DEG C, isothermal reaction 4h is finally warming up to 113~115 DEG C with the speed of 0.1~0.2 DEG C/min,
Vacuum distillation under the conditions of 0.005MPa, cooled down after reaction, cleaning, dry, multistage sieving, that is, obtain purpose product highly acid sun from
Sub-exchange resin.The exchange capacity that obtained resin chitosan oligosaccharide is determined is 87.5mg/mL, takes 1.0L dress posts.
Embodiment 2
The synthesis of storng-acid cation exchange resin is to combine to form copolymer bead with styrene and divinylbenzene crosslink
It is skeleton, introducing sulfonic group by the sulfonation of concentrated sulfuric acid strong oxidizer afterwards is made.Specifically there is procedure below：
The preparation of (a) Aqueous Phase Raw Material
1040g by ratio of weight and the number of copies:33g:87g:2g:80g weighs deionized water:Ethylenediamine:Sodium chloride:Sodium peroxydisulfate:
Polyvinyl alcohol, regulation system PH is neutrality, and temperature control is 45 DEG C~55 DEG C, and mixing speed 120rmp/min reacts 24h, obtains
To Aqueous Phase Raw Material；
The preparation of (b) oil phase raw material
Benzoyl peroxide is weighed by weight:Azodiisobutyronitrile:Styrene:Ferrocene:Divinylbenzene=2.0g:
3.5g:920g:30g:Temperature is risen to 78~80 DEG C by 53g, mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature, then
Continuation is progressively warming up to 93~95 DEG C, and 5~6h of constant temperature obtains intermediate product A, is subsequently adding the Aqueous Phase Raw Material that step (a) is obtained
Mixing, by temperature control to 67-69 DEG C, suspension polymerization 6h obtains bead cross-linked copolymer skeleton；
C () after polymerization prepares intermediate product B
Copolymer skeleton is weighed by weight:Styrene:Deionized water:Divinylbenzene:Azodiisobutyronitrile=261g:
270g:220g:26.5g:Temperature is risen to 78~80 DEG C by 3.0g, mixing with the programming rate of 0.5 DEG C/min, and constant temperature 5~
6h, is further continued for progressively being warming up to 93~95 DEG C, and 5~6h of constant temperature obtains intermediate product B；
(d) sulfonating reaction
Intermediate product B is weighed by weight:Dichloroethanes:Ethyl acetate:Solvent naphtha:The concentrated sulfuric acid=1050g:420g:
53g:85g:2600g, 78~80 DEG C are warming up to 0.5~0.7 DEG C/min programming rates, 8~10h of isothermal reaction, then with 0.02
~0.05 DEG C/min is warming up to 83~85 DEG C, isothermal reaction 5h, finally 113 are warming up to the speed of 0.1~0.2 DEG C/min~
115 DEG C, vacuum distillation under the conditions of 0.008MPa, cooling, cleaning, dry, multistage sieving, that is, obtain purpose product strong acid after reaction
Property cationic ion-exchange resin.The exchange capacity that obtained resin chitosan oligosaccharide is determined is 87.5mg/mL, takes 1.0L dress posts.
Embodiment 3
The preparation of (a) Aqueous Phase Raw Material
1080g by ratio of weight and the number of copies:36g:90g:2.5g:90g weighs deionized water:Ethylenediamine:Sodium chloride:Persulfuric acid
Sodium:Polyvinyl alcohol, regulation system PH is neutrality, and temperature control is 45 DEG C~55 DEG C, and mixing speed 120rmp/min reacts 24h,
Obtain Aqueous Phase Raw Material；
The preparation of (b) oil phase raw material
Benzoyl peroxide is weighed by weight:Azodiisobutyronitrile:Styrene:Ferrocene:Divinylbenzene=2.5g:
4.5g:940g:35g:Temperature is risen to 78~80 DEG C by 60g, mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature, then
Continuation is progressively warming up to 93~95 DEG C, and 5~6h of constant temperature obtains intermediate product A, is subsequently adding the Aqueous Phase Raw Material that step (a) is obtained
Mixing, by temperature control to 65~67 DEG C, suspension polymerization 8h obtains bead cross-linked copolymer skeleton；
C () after polymerization prepares intermediate product B
Copolymer skeleton is weighed by weight:Styrene:Deionized water:Divinylbenzene:Azodiisobutyronitrile=285g:
300g:240g:28g:Temperature is risen to 78~80 DEG C by 3.8g, mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature,
It is further continued for progressively being warming up to 93~95 DEG C, 5~6h of constant temperature obtains intermediate product B；
(d) sulfonating reaction
Intermediate product B is weighed by weight:Dichloroethanes:Ethyl acetate:Solvent naphtha:The concentrated sulfuric acid=1200g:480g:
62g:92g:2850g, 78~80 DEG C are warming up to 0.5~0.7 DEG C/min programming rates, 8~10h of isothermal reaction, then with 0.02
~0.05 DEG C/min is warming up to 80-82 DEG C, and isothermal reaction 6h is finally warming up to 113~115 with the speed of 0.1~0.2 DEG C/min
DEG C, vacuum distillation under the conditions of 0.003MPa, cooling, cleaning, dry, multistage sieving, that is, obtain purpose product highly acid after reaction
Cationic ion-exchange resin.The exchange capacity that obtained resin chitosan oligosaccharide is determined is 92.54mg/mL, takes 1.8L dress posts.
Embodiment 4
The synthesis of storng-acid cation exchange resin is to combine to form copolymer bead with styrene and divinylbenzene crosslink
It is skeleton, introducing sulfonic group by the sulfonation of concentrated sulfuric acid strong oxidizer afterwards is made.Specifically there is procedure below：
The preparation of (a) Aqueous Phase Raw Material
1150g by ratio of weight and the number of copies:42g:90g:3.4g:105g weighs deionized water:Ethylenediamine:Sodium chloride:Persulfuric acid
Sodium:Polyvinyl alcohol, regulation system PH is neutrality, and temperature control is 45 DEG C~55 DEG C, and mixing speed 120rmp/min reacts 24h,
Obtain Aqueous Phase Raw Material；
The preparation of (b) oil phase raw material
Benzoyl peroxide is weighed by weight:Azodiisobutyronitrile:Styrene:Ferrocene:Divinylbenzene=3.2g:
5.0g:970g:34g:Temperature is risen to 78~80 DEG C by 62g, mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature, then
Continuation is progressively warming up to 93~95 DEG C, and 5~6h of constant temperature obtains intermediate product A, is subsequently adding the Aqueous Phase Raw Material that step (a) is obtained
Mixing, by temperature control to 67-69 DEG C, suspension polymerization 5h obtains bead cross-linked copolymer skeleton；
C () after polymerization prepares intermediate product B
Copolymer skeleton is weighed by weight:Styrene:Deionized water:Divinylbenzene:Azodiisobutyronitrile=318g:
330g:280g:30g:Temperature is risen to 78~80 DEG C by 5.0g, mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature,
It is further continued for progressively being warming up to 93~95 DEG C, 5~6h of constant temperature obtains intermediate product B；
(d) sulfonating reaction
Intermediate product B is weighed by weight:Dichloroethanes:Ethyl acetate:Solvent naphtha:The concentrated sulfuric acid=1250g:480g:
62g:102g:2900g, 78~80 DEG C are warming up to 0.5~0.7 DEG C/min programming rates, 8~10h of isothermal reaction, then with 0.02
~0.05 DEG C/min is warming up to 82-84 DEG C, and isothermal reaction 6h is finally warming up to 113~115 with the speed of 0.1~0.2 DEG C/min
DEG C, vacuum distillation under the conditions of 0.01MPa, cooling, cleaning, dry, multistage sieving, that is, obtain purpose product highly acid positive after reaction
Ion exchange resin.It is 89.43mg/mL that obtained resin chitosan oligosaccharide determines exchange capacity, takes 2.0L dress posts.
Embodiment 5
The synthesis of storng-acid cation exchange resin is to combine to form copolymer bead with styrene and divinylbenzene crosslink
It is skeleton, introducing sulfonic group by the sulfonation of concentrated sulfuric acid strong oxidizer afterwards is made.Specifically there is procedure below：
The preparation of (a) Aqueous Phase Raw Material
1200g by ratio of weight and the number of copies:45g:95g:4.0g:120g weighs deionized water:Ethylenediamine:Sodium chloride:Persulfuric acid
Sodium:Polyvinyl alcohol, regulation system PH is neutrality, and temperature control is 45 DEG C~55 DEG C, and mixing speed 120rmp/min reacts 24h,
Obtain Aqueous Phase Raw Material；
The preparation of (b) oil phase raw material
Benzoyl peroxide is weighed by weight:Azodiisobutyronitrile:Styrene:Ferrocene:Divinylbenzene=4.0g:
6g:1000g:40g:Temperature is risen to 78~80 DEG C by 65g, mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature, then
Continuation is progressively warming up to 93~95 DEG C, and 5~6h of constant temperature obtains intermediate product A, is subsequently adding the Aqueous Phase Raw Material that step (a) is obtained
Mixing, by temperature control to 66-68 DEG C, suspension polymerization 6h obtains bead cross-linked copolymer skeleton；
C () after polymerization prepares intermediate product B
Copolymer skeleton is weighed by weight:Styrene:Deionized water:Divinylbenzene:Azodiisobutyronitrile=350g:
360g:300g:32g:Temperature is risen to 78~80 DEG C by 6.0g, mixing with the programming rate of 0.5 DEG C/min, 5~6h of constant temperature,
It is further continued for progressively being warming up to 93~95 DEG C, 5~6h of constant temperature obtains intermediate product B；
(d) sulfonating reaction
Intermediate product B is weighed by weight:Dichloroethanes:Ethyl acetate:Solvent naphtha:The concentrated sulfuric acid=1300g:500g:
65g:105g:3000g, 78~80 DEG C are warming up to 0.5~0.7 DEG C/min programming rates, 8~10h of isothermal reaction, then with 0.02
~0.05 DEG C/min is warming up to 83~85 DEG C, and 4~6h of isothermal reaction is finally warming up to 113 with the speed of 0.1~0.2 DEG C/min
~115 DEG C, vacuum distillation under the conditions of 0.008MPa, cooling, cleaning, dry, multistage sieving, that is, obtain purpose product strong after reaction
Acid cation exchange resin.It is 93.67mg/mL that obtained resin chitosan oligosaccharide determines exchange capacity, takes 1.9L dress posts.
Embodiment 6
By 100g shitosans (DDA>95%) it is placed in the beaker of 5L, is added thereto to 1.96L water and stirs.Treat shell
After the abundant water absorption and swelling of glycan, start to adding 40mL acetic acid in system, uniform stirring to system is into after uniform state, starting to use
NaAc_HAc buffer solution adjusts pH to 5.6.Plus ChitosanaseOU01 enzymes, enzyme addition is 40U/g with substrate ratios,
With rotating speed be 100r/min uniform stirrings, reaction temperature for 37 DEG C, every 8h sampling TLC methods detections, judge reaction end.Then
Under 4000rpm rotating speeds, 10min, filtering, revolving to dry shelling oligosaccharide compositions is centrifuged.
The chitosan oligosaccharide component 80g that preparation will be taken is soluble in water, stirs.Activated carbon carries out desolventing technology, core filtering
Filtrate is pumped into the highly acidic cation exchange column that embodiment 1 is made after device filtering and starts loading, with concentration be 1.0~
The hydrochloric acid solution of 4.0mol/L is eluted, and flow velocity is 0.3BV/h；One is collected per 2.5h to manage：Each pipe sampling is rotated to dry measure
Its sugared concentration simultaneously draws elution curve (such as Fig. 1), it can be seen that mainly flowing out three kinds of components, the corresponding material in peak is successively
Aminoglucose hydrochloride, chitobiose, chitotriose.Aminoglucose hydrochloride content is relatively low, predominantly chitobiose chitotriose group
Into, the saccharic composition (as shown in Figure 2) of collection liquid is detected with TLC methods, show that 7~No. 13 are managed as chitobiose, 16~No. 28 pipes are shell
Trisaccharide, merges same composition.Two components for merging are rotated to dry, chitobiose, chitotriose monomer (structure is as follows) solid is obtained,
TLC analysis collection of illustrative plates is as shown in Figure 3.
The chitobiose that to prepare, chitotriose elemental solid use HPLC method purity assays, take and a small amount of be dissolved in water and be configured to 5%
(m/V), sample size 5uL.The purity of chitobiose, chitotriose is calculated using area normalization method, can be drawn, chitobiose purity is higher than
98%, chitotriose purity is higher than 96%.In the present embodiment, the hybrid standard product of the chitosan oligosaccharide degree of polymerization 1~7, obtained enzymolysis component,
The HPLC collection of illustrative plates of chitobiose monomer and chitotriose monomer is distinguished as also shown in e.g. figs. 4-7, according to chitobiose shell in hybrid standard product collection of illustrative plates
Trisaccharide appearance time understands；(1) enzymolysis component is mainly chitobiose, chitotriose.(2) prepared by chitobiose monomer and chitotriose monomer
Purity it is higher, scientific research requirement can be met.
Will prepare chitobiose, chitotriose monomer using FT-IR methods,1HNMR methods are characterized to its structure, infrared spectrum
Figure as shown in figure 8, hydrogen spectrogram as shown in Figure 9 and Figure 10.Wherein, in infrared spectrogram, as a result show, chitobiose and chitotriose two
The infrared scan collection of illustrative plates of person is very much like, there is identical characteristic peak.3426.3cm-1Left and right is that the flexible of O-H and N-H shakes in sugared ring
Dynamic absworption peak, in 2937.7cm-1Left and right is the stretching vibration absworption peak of C-H in sugared ring.1627.1cm-1Left and right is acid amides I spectrums
Band.1523.1cm-1Left and right is acid amides II bands of a spectrum, 1400.5cm-1Right and left rings hydroxyl C-H deformation vibrations, 1090.9cm-1Left and right and
1069.3cm-1The peak of left and right is C-O stretching vibration peaks.892.9cm-1Left and right is beta configuration glycosidic bond, 630.4cm-1Position refers to
The vibration peak of O-H out-of-plane bendings.
Embodiment 7
By 150g shitosans (DDA>95%) it is placed in the beaker of 5L, is added thereto to 2.94L water and stirs.Treat shell
After the abundant water absorption and swelling of glycan, start to adding 60mL acetic acid in system, uniform stirring to system is into after uniform state, starting to use
NaAc_HAc buffer solution adjusts pH to 5.6.Plus ChitosanaseOU01 enzymes, enzyme addition is 40U/g with substrate ratios,
It is 100r/min uniform stirrings with rotating speed, reaction temperature is 37 DEG C, every 8h sampling TLC method detections, judges reaction end.
4000rpm rotating speeds, are centrifuged 10min, filtering, and revolving is to doing into chitosan oligosaccharide blending ingredients.The chitosan oligosaccharide blending ingredients of preparation will be taken
120g is soluble in water, stirs.Activated carbon carries out desolventing technology, and filtrate is pumped into the institute of embodiment 2 after sand core filter filtering
The highly acidic cation exchange column being made starts loading, is eluted with the hydrochloric acid solution that concentration is 1.0~4.0mol/L, flow velocity
It is 0.4BV/h；One is collected per 2.5h to manage：Each pipe sampling revolving is to dry its sugared concentration of measure and draws elution curve, is examined with TLC methods
The saccharic composition of collection liquid is surveyed, show that 7~No. 13 are managed as chitobiose, 16~No. 28 pipes are chitotriose, merge same composition.To merge
Two components rotate to dry, obtain chitobiose chitotriose monomer (structure sees below formula) solid.
The chitobiose that to prepare, chitotriose elemental solid use HPLC method purity assays, take and a small amount of be dissolved in water and be configured to 5%
(m/V), sample size 5uL.The purity of chitobiose, chitotriose is calculated using area normalization method, chitobiose purity is higher than 98%, shell three
Sugared purity is higher than 96%.
Embodiment 8
By 200g shitosans (DDA>95%) it is placed in the beaker of 5L, is added thereto to 3.92L water and stirs.Treat shell
After the abundant water absorption and swelling of glycan, start to adding 80mL acetic acid in system, uniform stirring to system is into after uniform state, starting to use
NaAc_HAc buffer solution adjusts pH to 5.6.Plus ChitosanaseOU01 enzymes, enzyme addition is 40U/g with substrate ratios,
It is 100r/min uniform stirrings with rotating speed, reaction temperature is 37 DEG C, every 8h sampling TLC method detections, judges reaction end.
4000rpm rotating speeds, are centrifuged 10min, filtering, and revolving is to doing into chitosan oligosaccharide blending ingredients.
The chitosan oligosaccharide blending ingredients 160g that preparation will be taken is soluble in water, stirs.Activated carbon carries out desolventing technology, core
The highly acidic cation exchange column that pumps into filtrate made by embodiment 3 starts loading after filter filtering, with concentration be 1.0~
The hydrochloric acid solution of 4.0mol/L is eluted, and flow velocity is 0.45BV/h；One is collected per 2.5h to manage：Each pipe sampling is rotated to dry measure
Its sugared concentration simultaneously draws elution curve, and the saccharic composition of collection liquid is detected with TLC methods, show that 7~No. 13 pipes are chitobiose, 16~28
Number pipe be chitotriose, merge same composition.Two components for merging are rotated to doing, (structure sees below to obtain chitobiose chitotriose monomer
Formula) solid.
The chitobiose that to prepare, chitotriose elemental solid use HPLC method purity assays, take and a small amount of be dissolved in water and be configured to 5%
(m/V), sample size 5uL.The purity of chitobiose, chitotriose is calculated using area normalization method, chitobiose purity is higher than 98%, shell three
Sugared purity is higher than 96%.
Embodiment 9
By 160g shitosans (DDA>95%) it is placed in the beaker of 5L, is added thereto to 2.94L water and stirs.Treat shell
After the abundant water absorption and swelling of glycan, start to adding 50mL acetic acid in system, uniform stirring to system is into after uniform state, starting to use
NaAc_HAc buffer solution adjusts pH to 5.Plus ChitosanaseOU01 enzymes, enzyme addition is 30U/g with substrate ratios, with
Rotating speed is 100r/min uniform stirrings, and reaction temperature is 40 DEG C, every 8h sampling TLC method detections, judges reaction end.
4000rpm rotating speeds, are centrifuged 10min, filtering, and revolving is to doing into chitosan oligosaccharide blending ingredients.
The chitosan oligosaccharide blending ingredients 200g that preparation will be taken is soluble in water, stirs.Activated carbon carries out desolventing technology, core
The highly acidic cation exchange column that pumps into filtrate made by embodiment 4 starts loading after filter filtering, with concentration be 1.0~
The hydrochloric acid solution of 4.0mol/L is eluted, and flow velocity is 0.5BV/h；One is collected per 2.5h to manage：Each pipe sampling is rotated to dry measure
Its sugared concentration simultaneously draws elution curve, and the saccharic composition of collection liquid is detected with TLC methods, show that 7~No. 13 pipes are chitobiose, 16~28
Number pipe be chitotriose, merge same composition.Two components for merging are rotated to doing, (structure sees below to obtain chitobiose chitotriose monomer
Formula) solid.
The chitobiose that to prepare, chitotriose elemental solid use HPLC method purity assays, take and a small amount of be dissolved in water and be configured to 5%
(m/V), sample size 5uL.The purity of chitobiose, chitotriose is calculated using area normalization method, chitobiose purity is higher than 98%, shell three
Sugared purity is higher than 96%.
Embodiment 10
By 140g shitosans (DDA>95%) it is placed in the beaker of 5L, is added thereto to 2.94L water and stirs.Treat shell
After the abundant water absorption and swelling of glycan, start in system plus 45mL acetic acid, uniform stirring to system starts to use vinegar into after uniform state
Acid-sodium acetate buffer regulation pH to 5.2.Plus ChitosanaseOU01 enzymes, enzyme addition is 60U/g with substrate ratios, with
Rotating speed is 100r/min uniform stirrings, and reaction temperature is 30 DEG C, every 8h sampling TLC method detections, judges reaction end.
4000rpm rotating speeds, are centrifuged 10min, filtering, and revolving is to doing into chitosan oligosaccharide blending ingredients.
The chitosan oligosaccharide blending ingredients 150g that preparation will be taken is soluble in water, stirs.Activated carbon carries out desolventing technology, core
Filter filtering after the highly acidic cation exchange column that filtrate pumps into embodiment 5 is started into loading, with concentration be 1.0~
The hydrochloric acid solution of 4.0mol/L is eluted, and flow velocity is 0.6BV/h；One is collected per 2.5h to manage：Each pipe sampling is rotated to dry measure
Its sugared concentration simultaneously draws elution curve, and the saccharic composition of collection liquid is detected with TLC methods, show that 7~No. 13 pipes are chitobiose, 16~28
Number pipe be chitotriose, merge same composition.Two components for merging are rotated to doing, (structure sees below to obtain chitobiose chitotriose monomer
Formula) solid.
The chitobiose that to prepare, chitotriose elemental solid use HPLC method purity assays, take and a small amount of be dissolved in water and be configured to 5%
(m/V), sample size 5uL.The purity of chitobiose, chitotriose is calculated using area normalization method, chitobiose purity is higher than 98%, shell three
Sugared purity is higher than 96%.
The above-mentioned description to embodiment is to be understood that and use invention for ease of those skilled in the art.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel announcement of the invention, does not depart from improvement that scope made and modification all should be of the invention
Within protection domain.
Claims (10)
1. the method for a kind of chitobiose/chitotriose monomer prepare with scale, it is characterised in that comprise the following steps：
(1) take chitosan oligosaccharide component and be dissolved in water wiring solution-forming, decolourize, filtering pumps into highly acidic cation exchange column and starts loading, uses
Hydrochloric acid solution is eluted, and is collected one every same time and is managed；
(2) saccharic composition of each pipe sampling detection collection liquid, merges identical saccharic composition；
(3) chitobiose of merging and the component of chitotriose two are rotated to dry, sloughs hydrochloric acid, that is, respectively obtain the chitobiose of high-purity
Elemental solid and chitotriose elemental solid.
2. a kind of method of chitobiose according to claim 1/chitotriose monomer prepare with scale, it is characterised in that institute
The chitosan oligosaccharide component stated is made by the following method：
Take appropriate shitosan add water fully it is swelling, add acetic acid, stir, adjust pH to 5.0~5.6, add shitosan
Enzyme, stirring carries out enzyme digestion reaction, and after reaction is reached home, enzymolysis reaction, centrifugation, filtering is dried, and obtains final product the chitosan oligosaccharide
Component.
3. a kind of method of chitobiose according to claim 2/chitotriose monomer prepare with scale, it is characterised in that institute
The deacetylation of the shitosan stated>95%, viscosity<60mPa·S；
The ratio between addition of shitosan and acetic acid is (100~200) g:(40~80) mL, chitosan enzyme is selectivity chitosan enzyme
ChitosanaseOU01, its addition is 30~60U/g substrates；The temperature of enzyme digestion reaction is 30~40 DEG C.
4. the method for a kind of chitobiose according to claim 2/chitotriose monomer prepare with scale, it is characterised in that anti-
The judgement for answering terminal is：Sampled every 8h and use TLC methods to detect, if detecting enzymolysis product only chitobiose, chitotriose i.e.
Can be considered reaction end；
The measure of enzymolysis reaction is：It is heated to 100 DEG C and keeps 10min.
5. a kind of method of chitobiose according to claim 1/chitotriose monomer prepare with scale, it is characterised in that institute
The highly acidic cation exchange column stated is made by the following method：
The preparation of (a) Aqueous Phase Raw Material
Deionized water, ethylenediamine, sodium chloride, sodium peroxydisulfate and polyvinyl alcohol are weighed, regulation system PH is neutrality, is reacted,
Obtain Aqueous Phase Raw Material；
The preparation of (b) oil phase raw material
Benzoyl peroxide, azodiisobutyronitrile, styrene, ferrocene and divinylbenzene are weighed, is mixed, follow procedure A heats up, and obtains
To intermediate product A, the Aqueous Phase Raw Material mixing that step (a) is obtained is subsequently adding, suspension polymerization obtains bead copolymerzation with cross-linking
Thing skeleton；
C () after polymerization prepares intermediate product B
Copolymer skeleton, styrene, deionized water, divinylbenzene and azodiisobutyronitrile are weighed, is mixed, follow procedure A heats up, and obtains
To intermediate product B；
(d) sulfonating reaction
Intermediate product B, dichloroethanes, ethyl acetate, solvent naphtha and the concentrated sulfuric acid are weighed, follow procedure B heats up, vacuum distillation, reaction
Cool down afterwards, clean, drying, multistage is sieved, that is, obtain purpose product storng-acid cation exchange resin.
6. a kind of method of chitobiose according to claim 5/chitotriose monomer prepare with scale, it is characterised in that step
Suddenly deionized water in (a):Ethylenediamine:Sodium chloride:Sodium peroxydisulfate:The addition mass ratio of polyvinyl alcohol is (1000~1200):(30
~45):(85~95):(1.5~4):(75~120)；
Benzoyl peroxide in step (b):Azodiisobutyronitrile:Styrene:Ferrocene:The mass ratio of divinylbenzene for (1.5~
4.0):(3~6):(900~1000):(25~40):(50~65)；
Copolymer skeleton in step (c):Styrene:Deionized water:Divinylbenzene:The mass ratio of azodiisobutyronitrile for (240~
350):(250~360):(200~300):(25~32):(2~6)；
Step (d) intermediate product B:Dichloroethanes:Ethyl acetate:Solvent naphtha:The mass ratio of the concentrated sulfuric acid is (1000~1300):
(400~500):(50~65):(80~105):(2500~3000).
7. a kind of method of chitobiose according to claim 5/chitotriose monomer prepare with scale, it is characterised in that step
Suddenly the process conditions of reaction are in (a)：Temperature control is 45 DEG C~55 DEG C, and mixing speed 120rmp/min reacts 24h；
Step (b) Program A heats up and is specially：Temperature is risen to by 78~80 DEG C, constant temperature 5 with the programming rate of 0.5 DEG C/min
~6h, is further continued for progressively being warming up to 93~95 DEG C, 5~6h of constant temperature；The process conditions of suspension polymerization are：Temperature control is to 65
~70 DEG C, react 45~8h；
Step (d) Program B heat up technique be：78~80 DEG C are warming up to 0.5~0.7 DEG C/min programming rates, constant temperature is anti-
Answer 8~10h, then 80~85 DEG C be warming up to 0.02~0.05 DEG C/min, 4~6h of isothermal reaction, finally with 0.1~0.2 DEG C/
The speed of min is warming up to 115 DEG C；The process conditions of vacuum distillation are：Pressure≤0.01MPa.
8. a kind of method of chitobiose according to claim 1/chitotriose monomer prepare with scale, it is characterised in that step
Suddenly the storng-acid cation exchange resin amount ratio in (1) in chitosan oligosaccharide component and highly acidic cation exchange column for (80~
200)g：(1200~2000) mL.
9. a kind of method of chitobiose according to claim 1/chitotriose monomer prepare with scale, it is characterised in that step
Suddenly decolourize to use activated carbon decolorizing in (1), activated carbon is (0.1~0.6) g with the mass ratio of chitosan oligosaccharide component:(80~200) g；
It is filtered into using sand core filter filtering, the mesh of its aperture ＞ 200.
10. a kind of method of chitobiose according to claim 1/chitotriose monomer prepare with scale, it is characterised in that step
Suddenly the concentration of hydrochloric acid solution is 1.0~4.0mol/L in (1), and its flow velocity is 0.3~0.6BV/h.
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Application publication date: 20170623 |