CN101200747B - Physical fields aided metal-coordination chitosan positioning enzymolysis method - Google Patents
Physical fields aided metal-coordination chitosan positioning enzymolysis method Download PDFInfo
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Abstract
The invention discloses a physical field strengthening metal coordination chitosan location enzymolysis method, including the following steps: dissolving chitosan in acetate solution which is added with metal salt to obtain highly coordinated chitosan-metal complex after the reaction; adding acetate-acetate sodium solution and papain in the mixed solution for the stirring reaction in the magnetic field and out of the magnetic field; removing enzyme and incompletely reacted macromolecule chitosan-metal complex in the obtained enzymatic hydrolyzed reaction product; removing metal ions to obtain oligomeric chitosan solution; spray-drying the oligomeric chitosan solution to obtain the finished product of oligomeric chitosan. The invention introduces the degradation effect of protease on the chitosan, improves the degradation efficiency, realizes the efficient location degradation and prepares oligomeric chitosan- metal complex. The complex has the advantages of good water-soluble property and application characteristics of both the chitosan and the metal ions and is widely applied in various fields of medicine, health, foods, etc.
Description
Technical field
The present invention relates to a kind of chitosan enzymolysis method, particularly a kind of physical fields aided metal-coordination chitosan positioning enzymolysis method.
Background technology
Chitosan (Chitosan) is the product behind the natural polymer de-acetyl chitin, is the interpolymer of being made up of β-(1,4)-2-amino-2-deoxy-D-glucose unit and β-(1,4)-2-acetamido 2-deoxy-D-glucose unit.Compare with other macromolecular materials, that chitosan has is biodegradable, nontoxicity, have characteristics such as good biocompatibility, obtains application more and more widely in recent years at aspects such as food, medicine, makeup, chemical industry, environmental protection.But chitosan is a macromolecular compound, and molecular weight is very big usually, and molecular structure is tight, and is poorly soluble, is difficult to be absorbed and used, and limited its application greatly.With the chitosan oligomer that obtains after the degradation of chitosan, water-soluble, physiologically active is all more superior than chitosan.Chitosan oligomer energy activating human body lymphocyte promotes spleen antibody to generate the breeding of anticancer and diffusion; Strengthen liver function, prevent stomach ulcer; Bring high blood pressure down, blood sugar, cholesterol etc.; Have stronger anti-microbial effect and significant moisture-retaining capacity; Help the growth of human body probiotic bacterium and suppress pathogenic bacterium simultaneously.When chitosan oligomer was 6～8 sugar, bioavailability was best, and the effect aspect the inhibition tumour is encouraging, was the current focus of research and development both at home and abroad.Therefore, the chitosan oligomer that how to obtain narrow molecular weight distributions is the key that improves the chitosan using value.
The method for preparing chitosan oligomer is broadly divided into acid degradation method, oxidation degradation method, enzyme liberating method and mechanical degradation method.Mechanical degradation fado wherein is to be used to cooperate other method, the enhancing degradation effect.Acid-hydrolysis method is cheap, and is simple to operate, and shortcoming is that the molecular weight distribution of reaction process and the chitosan oligomer that obtains is difficult to control, and environmental pollution is serious.Oxidation degradation method has advantages such as speed of response is fast, productive rate is high, reactant is nontoxic, but the effect of controlling the molecular weight of product distribution is undesirable.The enzyme liberating method can be carried out under relatively mild condition, can optionally cut off β-(1,4) glycosidic link of chitosan, and the molecular weight distribution of degraded product is had certain control action kou, and reaction process is controlled easily.Wherein used enzyme can be divided into specificity enzyme and non-specificity enzyme.The specificity enzyme is difficult for obtaining, and costs an arm and a leg, and is difficult to commercialization.In the non-specificity enzyme of degrade chitosan, the proteolytic enzyme effect is obvious, its degradation capability even surpassed chitoanase and N,O-Diacetylmuramidase.And protease price is cheap, and good stability has excellent industrial application foreground, so come into one's own day by day.Though comparing with other method, the enzyme liberating method has greater advantage, still not ideal enough to the molecular weight control effect of product, can not satisfy the high request in market.Therefore, how more effective control chitosan oligomer range of molecular weight distributions is to improve its key in application.
2 of the chitosan glycogen are-NHCOCH
3Or free-NH
2, 3 is primary-OH for the second month in a season-OH and 6, can form similar cancellated cage type molecule by hydrogen bond and sat linkage, thereby chitosan has fabulous mating capability to transition metal and rare earth ion, can prepare chitosan-metal complexes.In recent years, people cooperated with chitosan different metal ions and had carried out comparatively concrete research, had disclosed chitosan to the kinetics of adsorption rule of metal ion and the structure of chitosan-metal complexes.After metal ion and chitosan played complex reaction, the chitosan chain took place folding, made the fracture easier generation of polymer chitosan chain at privileged site.The existence of coordinate bond may make the Intermolecular Forces of chitosan and some key of intramolecularly change in the chitosan metal complexes, is formed with the weak tendency structure that is beneficial to the chitosan molecule splitting of chain.More than research, the feasibility of degrading for metal-complexing chitosan location provides evidence.
On the other hand, introduce physical actions such as ultrasonic, magnetic field, microwave field, can produce very important influence the correlated response process of chitosan.Existing microwave promotes Cu (II), Ca (II), Zn (II) ion to cooperate with chitosan, act on chitosan with ultrasonic wave, can improve the adsorptive capacity of chitosan to Fe (II), there are research reports such as promoter action in the magnetic field of certain intensity to the reaction of degrading chitosan by using papain.
Summary of the invention
The present invention is directed to above-mentioned technical disadvantages, propose a kind of physical fields aided metal-coordination chitosan positioning enzymolysis method, prepare the chitosan oligomer-metal complexes and the chitosan oligomer of even molecular weight distribution.
The present invention adopts following technical scheme to realize this purpose:
Physical fields aided metal-coordination chitosan positioning enzymolysis method of the present invention comprises the steps:
(1) chitosan being dissolved in 3%～5%pH is that the final concentration that makes chitosan is 2～5mg/ml, adds metal-salt again in 1.0～3.5 the acetum, and wherein, chitosan: the mol ratio of metal-salt is 1: 4～10, be stirred to dissolving fully after, place 90～150W/cm
2Ultrasonic field or the microwave field of the magnetic field of 50～100mT or 100～600W under, effect 10min～2h leaves standstill 9～18h at last; Filter,, to elutant, detect, obtain high coordinate chitosan-metal complexes less than metal ion with 0.5-2% formic acid solution, acetone-alcoholic acid mixing solutions and the washing precipitation successively of 60%～75% ethanolic soln;
(2) in pH3.0～5.0, concentration is in acetate-sodium acetate solution of 0.1～0.3mol/L, add chitosan-metal complexes that step (1) obtains, the final concentration that makes chitosan-metal complexes is 10～20g/L, add papoid again, make the quality of enzyme account for 1～8% of solution total mass, in temperature is 35～50 ℃, stirring reaction 0.5～1.5h in 50～100mT magnetic field, stirring reaction 6～12h outside magnetic field again;
(3) the enzyme digestion reaction product that step (2) is obtained removes enzyme and unreacted macromole chitosan-metal complexes completely with ultra-filtration equipment, obtain the chitosan oligomer-metal complexes of narrow molecular weight distributions, remove metal ion by ion exchange column again, obtain the oligopolymerization chitosan sugar soln, with its spraying drying, obtain the finished product chitosan oligomer.
The metal-salt of described step (1) comprises mantoquita, zinc salt, divalent iron salt, trivalent iron salt, manganese salt or nickel salt.
Acetone in the acetone of described step (1)-alcoholic acid mixing solutions: the ethanol volume ratio is 1: 1.
The narrow molecular weight distributions of described step (3) is 4～16 sugar.
The present invention has following beneficial effect:
By preparing chitosan oligomer with proteasome degradation chitosan-metal complexes, introduce the Degradation of magnetic field-intensification proteolytic enzyme to chitosan, improve degradation efficiency, realize the degraded of efficient location, prepare chitosan oligomer-metal complexes, this title complex has good water-solubility, and possesses the application characteristic of chitosan and metal ion simultaneously, is widely used in a plurality of fields such as medicine, health care, food.
Embodiment
Provide specific embodiment further to set forth technical scheme of the present invention below, but technology of the present invention application is not limited only to embodiment.
Embodiment 1:
Step 1: under weakly acidic condition, preparation chitosan-metal ion match and with this process of magnetic field-intensification.
1. chitosan is dissolved in 5% acetum (pH2.5), be made into the 2mg/ml chitosan solution, add a certain amount of oxammonium hydrochloride and ferrous sulfate ammonium salt, be stirred to dissolving fully, making the oxammonium hydrochloride final concentration is 5%, and the ferrous ammonium sulphate final concentration is 15mg/ml (chitosan: the mol ratio of ferrous sulfate ammonium salt is 1: 6.4).
2. above reaction system is placed under the magnetic field of 90mT, effect 30min leaves standstill 15h again.
3. filter,, to elutant, detect less than ferrous ion with 1% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 75% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-ferrous title complex.
1. be in acetate-sodium acetate buffer of 0.2mol/L in pH=4.5, concentration, add certain amount of chitosan-ferrous title complex, be mixed with chitosan-ferrous complex solution of 20mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 2%, and 45 ℃ of temperature place 80mT magnetic field stirring reaction 30min, again stirring reaction 6h outside magnetic field.Make chitosan-ferrous title complex degraded.
Step 3: separation, purifying chitosan oligomer-ferrous title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-ferrous title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-ferrous title complex of narrow molecular weight distributions.
2. chitosan oligomer-ferrous complex solution is removed ferrous ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 2:
Step 1: under weakly acidic condition, preparation chitosan-metal ion match and with this process of magnetic field-intensification.
1. chitosan is dissolved in 5% acetum (pH2.5), is made into the 2mg/ml chitosan solution, add a certain amount of mantoquita, be stirred to dissolving fully,, chitosan: the mol ratio of mantoquita is 1: 4.
2. above reaction system is placed under the magnetic field of 50mT, effect 30min leaves standstill 15h again.
3. filter,, to elutant, detect less than ferrous ion with 1% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 75% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-mantoquita title complex.
1. be in acetate-sodium acetate buffer of 0.2mol/L in pH=4.5, concentration, add certain amount of chitosan-mantoquita title complex, be mixed with chitosan-mantoquita complex solution of 20mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 2%, and 45 ℃ of temperature place 80mT magnetic field stirring reaction 30min, again stirring reaction 6h outside magnetic field.Make chitosan-mantoquita title complex degraded.
Step 3: separation, purifying chitosan oligomer-mantoquita title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-mantoquita title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-mantoquita title complex of narrow molecular weight distributions.
2. chitosan oligomer-mantoquita complex solution is removed cupric ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 3:
Step 1: under weakly acidic condition, preparation chitosan-metal ion match and with this process of magnetic field-intensification.
1. chitosan is dissolved in 5% acetum (pH2.5), is made into the 2mg/ml chitosan solution, add a certain amount of zinc salt, be stirred to dissolving fully, chitosan: the mol ratio of ferrous sulfate ammonium salt is 1: 8.
2. above reaction system is placed under the magnetic field of 100mT, effect 30min leaves standstill 15h again.
3. filter,, to elutant, detect less than ferrous ion with 1% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 75% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-zinc salt title complex.
1. be in acetate-sodium acetate buffer of 0.2mol/L in pH=4.5, concentration, add certain amount of chitosan-zinc salt title complex, be mixed with chitosan-zinc salt complex solution of 20mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 2%, and 45 ℃ of temperature place 80mT magnetic field stirring reaction 30min, again stirring reaction 6h outside magnetic field.Make chitosan-zinc salt title complex degraded.
Step 3: separation, purifying chitosan oligomer-zinc salt title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-zinc salt title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-zinc salt title complex of narrow molecular weight distributions.
2. chitosan oligomer-zinc salt complex solution is removed zine ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 4:
Step 1: under weakly acidic condition, prepare chitosan-metal ion match and strengthen this process with ultrasonic field.
1. chitosan is dissolved in 4% acetum (pH3.5), is made into the 4mg/ml chitosan solution, add a certain amount of trivalent iron salt, be stirred to dissolving fully, make chitosan: the mol ratio of trivalent iron salt is 1: 7.
2. above reaction system is placed 90W/cm
2Ultrasonic field in, the effect 2h, restir 5h leaves standstill 18h.
3. filter,, to elutant, detect less than ferrous ion with 2% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 70% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-trivalent iron salt title complex.
1. be in acetate-sodium acetate buffer of 0.3mol/L in pH=5-0, concentration, add certain amount of chitosan-trivalent iron salt title complex, be mixed with chitosan-trivalent iron salt complex solution of 15mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 8%, and 50 ℃ of temperature place 100mT magnetic field stirring reaction 1.5h, again stirring reaction 10h outside magnetic field.Make chitosan-trivalent iron salt title complex degraded.
Step 3: separation, purifying chitosan oligomer-trivalent iron salt title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-trivalent iron salt title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-trivalent iron salt title complex of narrow molecular weight distributions.
2. chitosan oligomer-trivalent iron salt complex solution is removed the molysite ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, just can obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 5:
Step 1: under weakly acidic condition, prepare chitosan-metal ion match and strengthen this process with ultrasonic field.
1. chitosan is dissolved in 3% acetum (pH1.0), is made into the 5mg/ml chitosan solution, add a certain amount of manganese salt, be stirred to dissolving fully, make chitosan: the mol ratio of manganese salt is 1: 4.3.
2. above reaction system is placed 150W/cm
2Ultrasonic field in, the effect 10min, restir 5h leaves standstill 9h.
3. filter,, to elutant, detect less than ferrous ion with 0.5% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 60% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-manganese salt complex.
1. be in acetate-sodium acetate buffer of 0.1mol/L in pH=3.0, concentration, add certain amount of chitosan-manganese salt complex, be mixed with chitosan-manganese salt complex solution of 10mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 1%, and 35 ℃ of temperature place 50mT magnetic field stirring reaction 50min, again stirring reaction 12h outside magnetic field.Make chitosan-manganese salt complex degraded.
Step 3: separation, purifying chitosan oligomer-manganese salt complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-manganese salt complex completely with ultra-filtration equipment, obtains the chitosan oligomer-manganese salt complex of narrow molecular weight distributions.
2. chitosan oligomer-manganese salt complex solution is removed mn ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, just can obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 6:
Step 1: under weakly acidic condition, prepare chitosan-metal ion match and strengthen this process with ultrasonic field.
1. chitosan is dissolved in 3% acetum (pH1.0), is made into the 5mg/ml chitosan solution, add a certain amount of nickel salt, be stirred to dissolving fully, make chitosan: the mol ratio of nickel salt is 1: 4.
2. above reaction system is placed 100W/cm
2Ultrasonic field in, the effect 10min, restir 5h leaves standstill 9h.
3. filter,, to elutant, detect less than ferrous ion with 0.5% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 60% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-nickel salt title complex.
1. be in acetate-sodium acetate buffer of 0.1mol/L in pH=3.0, concentration, add certain amount of chitosan-nickel salt title complex, be mixed with chitosan-nickel salt complex solution of 10mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 1%, and 35 ℃ of temperature place 50mT magnetic field stirring reaction 50min, again stirring reaction 12h outside magnetic field.Make chitosan-nickel salt title complex degraded.
Step 3: separation, purifying chitosan oligomer-nickel salt title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-nickel salt title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-nickel salt title complex of narrow molecular weight distributions.
2. chitosan oligomer-nickel salt complex solution is removed nickel ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, just can obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 7:
Step 1: under weakly acidic condition, preparation chitosan-metal ion match and with microwave field reinforced this process.
1. chitosan is dissolved in 3% acetum (pH1.0), be made into the 5mg/ml chitosan solution, add a certain amount of oxammonium hydrochloride and ferrous sulfate ammonium salt, be stirred to dissolving fully, making the oxammonium hydrochloride final concentration is 5%, and the ferrous ammonium sulphate final concentration is that (chitosan: the mol ratio of ferrous sulfate ammonium salt is 1: 4.3 to 10mg/ml.
2. above reaction system is placed the ultrasonic field of 100W, effect 10min, restir 5h leaves standstill 9h.
3. filter,, to elutant, detect less than ferrous ion with 0.5% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 60% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-ferrous title complex.
1. be in acetate-sodium acetate buffer of 0.1mol/L in pH=3.0, concentration, add certain amount of chitosan-ferrous title complex, be mixed with chitosan-ferrous complex solution of 10mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 1%, and 35 ℃ of temperature place 50mT magnetic field stirring reaction 50min, again stirring reaction 12h outside magnetic field.Make chitosan-ferrous title complex degraded.
Step 3: separation, purifying chitosan oligomer-ferrous title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-ferrous title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-ferrous title complex of narrow molecular weight distributions.
2. chitosan oligomer-ferrous complex solution is removed ferrous ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, just can obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 8:
Step 1: under weakly acidic condition, preparation chitosan-metal ion match and with microwave field reinforced this process.
1. chitosan is dissolved in 3% acetum (pH1.0), be made into the 5mg/ml chitosan solution, add a certain amount of oxammonium hydrochloride and ferrous sulfate ammonium salt, be stirred to dissolving fully, making the oxammonium hydrochloride final concentration is 5%, and the ferrous ammonium sulphate final concentration is 10mg/ml (chitosan: the mol ratio of ferrous sulfate ammonium salt is 1: 4.3).
2. above reaction system is placed the ultrasonic field of 600W, effect 10min, restir 5h leaves standstill 9h.
3. filter,, to elutant, detect less than ferrous ion with 0.5% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 60% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-ferrous title complex.
1. be in acetate-sodium acetate buffer of 0.1mol/L in pH=3.0, concentration, add certain amount of chitosan-ferrous title complex, be mixed with chitosan-ferrous complex solution of 10mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 1%, and 35 ℃ of temperature place 50mT magnetic field to feel and mix reaction 50min, stirring reaction 12h outside magnetic field again.Make chitosan-ferrous title complex degraded.
Step 3: separation, purifying chitosan oligomer-ferrous title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-ferrous title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-ferrous title complex of narrow molecular weight distributions.
2. chitosan oligomer-ferrous complex solution is removed ferrous ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, just can obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Embodiment 9:
Step 1: under weakly acidic condition, preparation chitosan-metal ion match and with microwave field reinforced this process.
1. chitosan is dissolved in 3% acetum (pH1.0), be made into the 5mg/ml chitosan solution, add a certain amount of oxammonium hydrochloride and ferrous sulfate ammonium salt, be stirred to dissolving fully, making the oxammonium hydrochloride final concentration is 5%, and the ferrous ammonium sulphate final concentration is 10mg/ml (chitosan: the mol ratio of ferrous sulfate ammonium salt is 1: 4.3).
2. above reaction system is placed the ultrasonic field of 150W, effect 10min, restir 5h leaves standstill 9h.
3. filter,, to elutant, detect less than ferrous ion with 0.5% formic acid solution, 1: 1 (V/V) acetone ethanol solution, the washing precipitation successively of 60% ethanolic soln.Promptly obtain high coordinate chitosan-ferrous title complex.
Step 2: under the action of a magnetic field, degrade chitosan-ferrous title complex.
1. be in acetate-sodium acetate buffer of 0.1mol/L in pH=3.0, concentration, add certain amount of chitosan-ferrous title complex, be mixed with chitosan-ferrous complex solution of 10mg/ml.
2. add a certain amount of papoid in solution, the mass concentration that makes enzyme is 1%, and 35 ℃ of temperature place 50mT magnetic field stirring reaction 50min, again stirring reaction 12h outside magnetic field.Make chitosan-ferrous title complex degraded.
Step 3: separation, purifying chitosan oligomer-ferrous title complex and chitosan oligomer.
1. the enzyme digestion reaction product removes enzyme and unreacted macromole chitosan-ferrous title complex completely with ultra-filtration equipment, obtains the chitosan oligomer-ferrous title complex of narrow molecular weight distributions.
2. chitosan oligomer-ferrous complex solution is removed ferrous ion by ion exchange column, promptly obtain the oligopolymerization chitosan sugar soln,, just can obtain the finished product chitosan oligomer, be distributed as 4-16 sugar its spraying drying.
Claims (3)
1. a physical fields aided metal-coordination chitosan positioning enzymolysis method is characterized in that, comprises the steps:
(1) chitosan being dissolved in 3%～5%pH is that the final concentration that makes chitosan is 2～5mg/ml, adds metal-salt again in 1.0～3.5 the acetum, and wherein, chitosan: the mol ratio of metal-salt is 1: 4～10, be stirred to dissolving fully after, place 90～150W/cm
2Ultrasonic field or the microwave field of the magnetic field of 50～100mT or 100～600W under, effect 10min～2h leaves standstill 9～18h at last; Filter,, to elutant, detect, obtain chitosan-metal complexes less than metal ion with 0.5～2% formic acid solution, acetone-alcoholic acid mixing solutions and the washing precipitation successively of 60%～75% ethanolic soln;
(2) in pH3.0～5.0, concentration is in acetate-sodium acetate solution of 0.1～0.3mol/L, add chitosan-metal complexes that step (1) obtains, the final concentration that makes chitosan-metal complexes is 10～20g/L, add papoid again, what make that the quality of enzyme accounts for the solution total mass is 1～8%, at 35～50 ℃, stirring reaction 0.5～1.5h in 50～100mT magnetic field, stirring reaction 6～12h outside magnetic field again;
(3) the enzyme digestion reaction product that step (2) is obtained removes enzyme and unreacted macromole chitosan-metal complexes completely with ultra-filtration equipment, obtain the chitosan oligomer-metal complexes of narrow molecular weight distributions, remove metal ion by ion exchange column again, obtain the oligopolymerization chitosan sugar soln, with its spraying drying, obtain the finished product chitosan oligomer;
The metal-salt of described step (1) is mantoquita, zinc salt, divalent iron salt, trivalent iron salt, manganese salt or nickel salt.
2. enzyme solution according to claim 1, it is characterized in that the acetone in the acetone-alcoholic acid mixing solutions of described step (1): the ethanol volume ratio is 1: 1.
3. enzyme solution according to claim 1, the narrow molecular weight distributions that it is characterized in that described step (3) is 4～16 sugar.
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