CN104046670A - Method for preparing chitosan through enzymatic hydrolysis acetyl removal - Google Patents
Method for preparing chitosan through enzymatic hydrolysis acetyl removal Download PDFInfo
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- CN104046670A CN104046670A CN201310450777.3A CN201310450777A CN104046670A CN 104046670 A CN104046670 A CN 104046670A CN 201310450777 A CN201310450777 A CN 201310450777A CN 104046670 A CN104046670 A CN 104046670A
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Abstract
The invention discloses a method for preparing chitosan through enzymatic hydrolysis acetyl removal. The method comprises the following steps: drying crab shell or shrimp shell, and crushing; removing calcium by using hydrochloric acid; carrying out enzymatic hydrolysis calcium removal and protein removal; and carrying out enzymatic hydrolysis acetyl removal, washing with water, drying, and crushing. Compared with the prior art, the above enzymatic hydrolysis method has the advantages of realization of calcium removal and protein removal in a same step, substantial improvement of the purity of the obtained chitosan, strong aid consumption reduction, low energy consumption, no pollution and environmental protection.
Description
Technical field
The present invention relates to chitosan preparation method, belong to manufacture field.
Background technology
Chitin is a kind of natural nitrogenous polysaccharide material, extensively in the shell or stratum corneum of existence and invertebrates, especially in the shell of shrimp crab class.Chitin is easy to obtain, renewable, is to be only second to cellulosic natural homopolymer, but because intramolecule has very strong hydrogen bond action, solvability is low, and range of application is restricted.
Generating chitosan after chitin deacetylase.The preparation of chitosan at present adopts chemical method and the de-acetyl of enzymolysis process more.In chemical method deacetylation, the factor of the de-acetyl of impact is a lot, but major influence factors comprises: concentration of lye, temperature of reaction and time.In general, improve temperature of reaction and prolongation reaction times and all can improve deacetylation, but can cause the degraded of chitin main chain to increase, thereby affect viscosity and the molecular weight of product.Pass into the degraded that nitrogen can slow down main chain to a certain extent.The concentration of lye using due to chemical method is high, long reaction time, and unstable product quality, environmental pollution is serious.
The non-degradable main chain of enzymolysis process, and pollution-free, there are important research and development and be worth.The factor that affects enzymolysis is a lot, mainly comprises pH value and temperature.Document is recorded optimum pH value 4-8, optimal temperature 30-70 DEG C " research and development of natural products " 2003 vol 15 No.6 such as () " chitinase characteristic and applied research " Wan Yunyang.The fungi chitin deacetylase (CDA) of finding is up to now all glycoprotein, and has good thermostability.But the location of different sources CDA, optimum pH value (PH4.5-12), carbohydrate content, relative molecular mass and ion impact etc. have larger difference.
The CDA of hitherto reported substantially derives from fungi.The CDA Main Function of originated from fungus is the synthetic of self cell walls, and the suitableeest substrate is generally chitinous oligomers, lower to chitinous activity, is not suitable for producing chitosan with the de-acetyl of enzyme process.The CDA wide material sources of originated from fungus, obtain easily, and cost is relatively low.Therefore how the CDA of originated from fungus can be become to those skilled in the art's urgent problem for the preparation of chitosan.
Summary of the invention
The object of the present invention is to provide a kind of new chitosan preparation method, described method can be compared with more energy-conserving and environment-protective of prior art.
Another object of the present invention is to provide a kind of method that CDA with originated from fungus prepares chitosan, and described method can solve prior art CDA and be unsuitable for producing the technical barrier of chitosan.
Another object of the present invention is to provide a kind of safe chitosan preparation method, and the chitosan that described method makes is residual without strong acid or highly basic, and product is safer.
Another object of the present invention is to provide a kind of chitosan with the market competitiveness, and described product is not because the improvement of technique increases production cost.
For achieving the above object, the present invention adopts following technical proposal to realize:
Dry, pulverizing, hydrochloric acid decalcification, enzymolysis decalcification and deproteinated, de-acetyl, washing and drying are pulverized and be get final product.
Crab shell or shrimp shell before the present invention adopts preparation method to decalcification carry out pre-treatment, and then carry out enzymolysis decalcification and deproteinated.
For strengthening decalcification and deproteinated effect, the present invention is preferably broken to 0.5～1.0mm particle by crab shell or shrimp shell meal, the too small production cost that increases of particle, and the excessive effect of particle is poor.Contriver, through test of many times and experience, finally determines that 0.5～1.0mm granulometric range reaches the best cost performance of decalcification and cost.
Raw material after crushed, adds 8～15% hydrochloric acid decalcifications, fully, after reaction, filters, and is washed to neutrality, leaves and takes residue.
Enzymolysis of the present invention adopts complex enzyme hydrolysis method to carry out deproteinated, also has the effect of further decalcification simultaneously.After screening, final discovery is carried out enzymolysis with bromeline and neutral protease and is had better effect compared with other proteolytic enzyme.Certainly, select other proteolytic enzyme to carry out enzymolysis and also can realize object of the present invention.In technique of the present invention, best enzymolysis scheme is in the residue after crab shell or the decalcification of shrimp shell hydrochloric acid, to add the water of 4-8 times of weightmeasurement ratio to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:2～5, adding citric acid is adjusted pH value to 5.5～6.5, and 40～60 DEG C are stirred 1～2h; Separating liquid, residue repeats to extract again.
It is 0.05～0.2% for good that enzymolysis shell material compound protease used accounts for shell material weight ratio.
After enzymolysis decalcification and deproteinated, filter, abandon filtrate, leave and take residue, residue is chitin.Chitin is added to the de-acetyl of the fermented liquid that contains chitin deacetylase, weightmeasurement ratio (g/l) 1～4:2～5 of chitin and fermented liquid, 30～32 DEG C, 48～72h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
Described can buy from open market, after also can fermenting by aspergillus nidulans, makes.
Described chitin deacetylase preparation method is as follows:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, inoculation aspergillus nidulans, 30～35 DEG C, PH6.0～7.0,72～96h.
After enzymolysis decalcification and deproteinated, the calcium ion in raw material, other organic substance of protein are removed.
Chitosan deacetylation of the present invention is up to more than 95%.
The various formulations that in product of the present invention and any or more than one pharmaceuticies, auxiliary material is mixed as starch, dextrin, lactose, Microcrystalline Cellulose, HPMC, polyoxyethylene glycol, Magnesium Stearate, micropowder silica gel, Xylitol, Saccharum lactis, glucose, glycine, N.F,USP MANNITOL, glycine etc., for example, can be made into tablet, slow releasing tablet, dripping pill, granule, capsule, microgranules.Preferred dosage form is capsule or granule.
Enzymolysis process of the present invention is compared with prior art system, and decalcification and deproteinated are realized in same step, greatly improves the purity of product, reduces the usage quantity of strong acid, consumes energy low, pollution-free, environmental protection more.
Embodiment
embodiment 1
Prepare chitin deacetylase:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation: fermention medium sterilizing, inoculation aspergillus nidulans, 35 DEG C, PH6.0, fermentation 72h.
Crab shell or shrimp shell drying and crushing, to 0.5mm particle, are added to 8% hydrochloric acid decalcification, fully, after reaction, filter, be washed to neutrality, leave and take residue; In residue, add the water of 4 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:2, and it is 0.05% that proteolytic enzyme accounts for shell material weight ratio, and adding citric acid is adjusted pH value to 5.5, and 40 DEG C are stirred 1h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leave and take residue, add the de-acetyl of the fermented liquid that contains chitin deacetylase, the weightmeasurement ratio 1:2 of chitin and fermented liquid, 30 DEG C, 48h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
embodiment 2
Prepare chitin deacetylase:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, inoculation aspergillus nidulans, 30 DEG C, PH7.0,72h.
Crab shell or shrimp shell drying and crushing, to 1.0mm particle, are added to 15% hydrochloric acid decalcification, fully, after reaction, filter, be washed to neutrality, leave and take residue; In residue, add the water of 8 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:5, and it is 0.2% that proteolytic enzyme accounts for shell material weight ratio, and adding citric acid is adjusted pH value to 6.5, and 60 DEG C are stirred 2h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leave and take residue, add the de-acetyl of the fermented liquid that contains chitin deacetylase, the weightmeasurement ratio 1:5 of chitin and fermented liquid, 32 DEG C, 72h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
embodiment 3
Prepare chitin deacetylase:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, inoculation aspergillus nidulans, 33 DEG C, PH6.5,96h.
Crab shell or shrimp shell drying and crushing, to 0.5mm particle, are added to 10% hydrochloric acid decalcification, fully, after reaction, filter, be washed to neutrality, leave and take residue; In residue, add the water of 8 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:5, and it is 0.1% that proteolytic enzyme accounts for shell material weight ratio, and adding citric acid is adjusted pH value to 6.5, and 60 DEG C are stirred 2h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leave and take residue, add the de-acetyl of the fermented liquid that contains chitin deacetylase, the weightmeasurement ratio 1:5 of chitin and fermented liquid, 31.5 DEG C, 64h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
embodiment 4
Prepare chitin deacetylase:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, inoculation aspergillus nidulans, 32 DEG C, PH6.6,72h.
Crab shell or shrimp shell drying and crushing, to 1.0mm particle, are added to 12% hydrochloric acid decalcification, fully, after reaction, filter, be washed to neutrality, leave and take residue; In residue, add the water of 4 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:2, and it is 0.08% that proteolytic enzyme accounts for shell material weight ratio, and adding citric acid is adjusted pH value to 6, and 40 DEG C are stirred 2h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leave and take residue, add the de-acetyl of the fermented liquid that contains chitin deacetylase, the weightmeasurement ratio 4:5 of chitin and fermented liquid, 31 DEG C, 72h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
embodiment 5
Prepare chitin deacetylase:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, inoculation aspergillus nidulans, 34 DEG C, PH6.1,72h.
Crab shell or shrimp shell drying and crushing, to 0.8mm particle, are added to 13% hydrochloric acid decalcification, fully, after reaction, filter, be washed to neutrality, leave and take residue; In residue, add the water of 6 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:3, and it is 0.15% that proteolytic enzyme accounts for shell material weight ratio, and adding citric acid is adjusted pH value to 5.5, and 50 DEG C are stirred 2h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leave and take residue, add the de-acetyl of the fermented liquid that contains chitin deacetylase, the weightmeasurement ratio 1:3 of chitin and fermented liquid, 32 DEG C, 48～72h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
embodiment 6
Prepare chitin deacetylase:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, access aspergillus nidulans, 32 DEG C, PH6.0,80h.
Crab shell or shrimp shell drying and crushing, to 0.6mm particle, are added to 11% hydrochloric acid decalcification, fully, after reaction, filter, be washed to neutrality, leave and take residue; In residue, add the water of 5 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:2.5, and it is 0.18% that proteolytic enzyme accounts for shell material weight ratio, and adding citric acid is adjusted pH value to 6.5, and 55 DEG C are stirred 1h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leave and take residue, enter to contain the de-acetyl of fermented liquid of chitin deacetylase, the weightmeasurement ratio 1:3 of chitin and fermented liquid, 30 DEG C, 72h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
embodiment 7
Prepare chitin deacetylase:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, cobalt dichloride (0.01mol/l) 10ml, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, access aspergillus nidulans, 35 DEG C, PH6.0,96h.
Crab shell or shrimp shell drying and crushing, to 0.7mm particle, are added to 9% hydrochloric acid decalcification, fully, after reaction, filter, be washed to neutrality, leave and take residue; In residue, add the water of 7 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and bromeline weight ratio 1:4, and it is 0.12% that proteolytic enzyme accounts for shell material weight ratio, and adding citric acid is adjusted pH value to 5.5, and 45 DEG C are stirred 1.5h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leave and take residue, add the de-acetyl of the fermented liquid that contains chitin deacetylase, the weightmeasurement ratio 2:1 of chitin and fermented liquid, 32 DEG C, 56h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
Claims (8)
1. the de-acetyl of enzymolysis is prepared a method for chitosan, comprises the steps:
(1) by dry to crab shell or shrimp shell, pulverizing;
(2) in raw material, add 8～15% hydrochloric acid decalcifications;
(3) protease hydrolyzed decalcification and deproteinated;
(4) the de-acetyl of enzymolysis, washing and drying are pulverized and be get final product.
2. preparation method according to claim 1, is characterized in that, crab shell or shrimp shell meal are broken to 0.5～1.0mm size.
3. preparation method according to claim 1 and 2, is characterized in that, adds the water of 4-8 times of weightmeasurement ratio to make feed liquid on crab shell or shrimp shell meal in the raw material after broken, and feed liquid adds neutral protease and bromeline, adjusts pH value, and 40～60 DEG C are stirred 1～2h.
4. preparation method according to claim 3, is characterized in that, described neutral protease and bromeline weight ratio 1:2～5.
5. according to the preparation method described in claim 3 or 4, it is characterized in that, adding citric acid is adjusted pH value to 5.5～6.5.
6. according to the preparation method described in the arbitrary claim of claim 1-5, it is characterized in that, it is 0.05～0.2% that enzymolysis shell material compound protease used accounts for shell material weight ratio.
7. according to the preparation method described in the arbitrary claim of claim 1-6, it is characterized in that, chitin is added to the de-acetyl of the fermented liquid that contains chitin deacetylase, weightmeasurement ratio 1～4:2～5 of chitin and fermented liquid, 30～32 DEG C, 48～72h, 0.2MPa, then 120 DEG C of sterilizing 20min, are drying to obtain chitosan.
8. preparation method according to claim 7, is characterized in that, described chitin deacetylase preparation method is as follows:
(1) prepare fermention medium: yeast powder 8g, chitosan 15g, glucose 30g, potassium primary phosphate 2g, anhydrous magnesium sulfate 1g, the cobalt dichloride 10ml of 0.01mol/l, water 1000ml;
(2) fermentation condition: fermention medium sterilizing, inoculation aspergillus nidulans, 30～35 DEG C, PH6.0～7.0,72～96h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262507A (en) * | 2014-10-31 | 2015-01-07 | 江苏财经职业技术学院 | Method for preparing lobster shell chitosan through cooperation of ultrasonic wave and CDA (Chitin Deacetylase) |
| CN108308196A (en) * | 2018-02-06 | 2018-07-24 | 厦门昶青生物科技有限公司 | A kind of chitosan composite bacteria agent and its preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900126A (en) * | 2006-05-19 | 2007-01-24 | 华南理工大学 | Process for preparing chitin and chitosan |
| CN101144097A (en) * | 2007-09-18 | 2008-03-19 | 重庆百奥帝克微生态科技有限公司 | Method for preparing chitin and its chitosan and chitosan oligosaccharide |
| CN101736583A (en) * | 2009-12-18 | 2010-06-16 | 东华大学 | Method for preparing chitin size modified by biological enzyme method |
-
2013
- 2013-09-29 CN CN201310450777.3A patent/CN104046670A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900126A (en) * | 2006-05-19 | 2007-01-24 | 华南理工大学 | Process for preparing chitin and chitosan |
| CN101144097A (en) * | 2007-09-18 | 2008-03-19 | 重庆百奥帝克微生态科技有限公司 | Method for preparing chitin and its chitosan and chitosan oligosaccharide |
| CN101736583A (en) * | 2009-12-18 | 2010-06-16 | 东华大学 | Method for preparing chitin size modified by biological enzyme method |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262507A (en) * | 2014-10-31 | 2015-01-07 | 江苏财经职业技术学院 | Method for preparing lobster shell chitosan through cooperation of ultrasonic wave and CDA (Chitin Deacetylase) |
| CN104262507B (en) * | 2014-10-31 | 2016-05-18 | 江苏财经职业技术学院 | A kind of ultrasonic synergistic CDA enzyme is prepared the method for lobster shell shitosan |
| CN108308196A (en) * | 2018-02-06 | 2018-07-24 | 厦门昶青生物科技有限公司 | A kind of chitosan composite bacteria agent and its preparation method and application |
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Application publication date: 20140917 |