CN104059953A - Chitosan enzymatic hydrolysis method - Google Patents
Chitosan enzymatic hydrolysis method Download PDFInfo
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- CN104059953A CN104059953A CN201310450586.7A CN201310450586A CN104059953A CN 104059953 A CN104059953 A CN 104059953A CN 201310450586 A CN201310450586 A CN 201310450586A CN 104059953 A CN104059953 A CN 104059953A
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Abstract
The invention discloses a chitosan enzymatic hydrolysis method, which comprises the following steps: drying and crushing crab or shrimp shell; performing enzymolysis for calcium and protein removal; performing enzymolysis for acetyl removal, washing with water, and drying. Compared with the prior art, according to the enzymolysis technology of the chitosan enzymatic hydrolysis method, the calcium and protein removal can be realized in one step, the production efficiency is greatly improved, the use of a strong acid and strong alkali is not needed, and the chitosan enzymatic hydrolysis method is low in energy consumption, free of pollution, and more environmentally friendly.
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 ℃ < < research and development of natural products > > 2003 vol 15 No.6 such as () < < chitinase characteristic and applied research > > ten thousand Yun Yang.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 the technical barrier that prior art CDA is unsuitable for producing 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, micronizing, enzymolysis decalcification and deproteinated, de-acetyl, washing and drying is pulverized and 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.
Enzymolysis of the present invention adopts complex enzyme hydrolysis method, and after screening, final discovery is carried out enzymolysis with papoid and neutral protease and 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 raw material after broken, to add the water of 4-8 times of weightmeasurement ratio to make feed liquid on crab shell or shrimp shell meal, feed liquid adds the compound protease of neutral protease and papoid weight ratio 1:2～5, adding citric acid is adjusted pH value to 5.5～6.5, and 40～60 ℃ 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, the weightmeasurement ratio of chitin and fermented liquid (g/l) 1～4:2～5,30～32 ℃, 48～72h, 0.2MPa, then 120 ℃ 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 ℃, PH6.0～7.0,72～96h.
After enzymolysis decalcification and deproteinated, the calcium ion in raw material, other organic substance of protein have been removed.
Chitosan deacetylation of the present invention is up to more than 94%.
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 enhances productivity, and need not use strong acid and highly basic, 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 ℃, PH6.0, fermentation 72h.
By crab shell or shrimp shell drying and crushing to 0.5mm particle, add the water of 4 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and papoid weight ratio 1:2, and it is 0.05% that proteolytic enzyme accounts for shell material weight ratio, adding citric acid is adjusted pH value to 5.5, and 40 ℃ are stirred 1h; Separating liquid, residue repeats to extract again, filters, and abandons filtrate, leaves and takes residue, add the de-acetyl of the fermented liquid that contains chitin deacetylase, the weightmeasurement ratio 1:2 of chitin and fermented liquid, 30 ℃, 48h, 0.2MPa, 120 ℃, 20min, is 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 ℃, PH7.0,72h.
By crab shell or shrimp shell drying and crushing to 1.0mm particle, add the water of 8 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and papoid weight ratio 1:5, and it is 0.2% that proteolytic enzyme accounts for shell material weight ratio, adding citric acid is adjusted pH value to 6.5, and 60 ℃ 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 ℃, 72h, 0.2MPa, then 120 ℃ 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 ℃, PH6.5,96h.
By crab shell or shrimp shell drying and crushing to 0.5mm particle, add the water of 8 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and papoid weight ratio 1:5, and it is 0.1% that proteolytic enzyme accounts for shell material weight ratio, adding citric acid is adjusted pH value to 6.5, and 60 ℃ 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 ℃, 72h, 0.2MPa, then 120 ℃ 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 ℃, PH6.6,72h.
By crab shell or shrimp shell drying and crushing to 1.0mm particle, add the water of 4 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and papoid weight ratio 1:2, and it is 0.15% that proteolytic enzyme accounts for shell material weight ratio, adding citric acid is adjusted pH value to 6, and 40 ℃ 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 ℃, 48～72h, 0.2MPa, then 120 ℃ 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 ℃, PH6.1,72h.
By crab shell or shrimp shell drying and crushing to 0.8mm particle, add the water of 6 times of weightmeasurement ratios to make feed liquid, feed liquid adds the compound protease of neutral protease and papoid weight ratio 1:3, and it is 0.18% that proteolytic enzyme accounts for shell material weight ratio, adding citric acid is adjusted pH value to 5.5, and 50 ℃ 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 ℃, 64h, 0.2MPa, then 120 ℃ 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, inoculation aspergillus nidulans, 32 ℃, PH6.0,80h.
By crab shell or shrimp shell drying and crushing to 0.6mm particle, add the water of 5 times of weightmeasurement ratios to make feed liquid, feed liquid adds the prozyme of neutral protease and papoid weight ratio 1:2.5, and it is 0.12% that proteolytic enzyme accounts for shell material weight ratio, adding citric acid is adjusted pH value to 6.5, and 55 ℃ 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 2:1 of chitin and fermented liquid, 32 ℃, 56h, 0.2MPa, then 120 ℃ 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, inoculation aspergillus nidulans, 35 ℃, PH6.0,96h.
By crab shell or shrimp shell drying and crushing to 0.7mm particle, add the water of 7 times of weightmeasurement ratios to make feed liquid, feed liquid adds the prozyme of neutral protease and papoid weight ratio 1:4, and it is 0.08% that proteolytic enzyme accounts for shell material weight ratio, adding citric acid is adjusted pH value to 5.5, and 45 ℃ 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 1:3 of chitin and fermented liquid, 30 ℃, 72h, 0.2MPa, then 120 ℃ of sterilizing 20min, are drying to obtain chitosan.
Claims (8)
1. a chitosan enzyme solution, comprises the steps:
(1) crab shell or shrimp shell is dry, pulverizing;
(2) protease hydrolyzed decalcification and deproteinated;
(3) the de-acetyl of enzymolysis, washing and drying is pulverized and get final product.
2. enzyme solution according to claim 1, is characterized in that, crab shell or shrimp shell meal are broken to 0.5～1.0mm size.
3. enzyme solution 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 papoid, adjusts pH value, and 40～60 ℃ are stirred 1～2h.
4. enzyme solution according to claim 3, is characterized in that, described neutral protease and papoid weight ratio 1:2～5.
5. according to the enzyme solution 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 enzyme solution 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 ℃, 48～72h, 0.2MPa, then 120 ℃ of sterilizing 20min, are drying to obtain chitosan.
8. enzyme solution 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 ℃, PH6.0～7.0,72～96h.
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| CN201310450586.7A CN104059953A (en) | 2013-09-29 | 2013-09-29 | Chitosan enzymatic hydrolysis method |
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| CN201310450586.7A CN104059953A (en) | 2013-09-29 | 2013-09-29 | Chitosan enzymatic hydrolysis method |
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| CN104059953A true CN104059953A (en) | 2014-09-24 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105803019A (en) * | 2015-12-31 | 2016-07-27 | 珠海市金隆生物科技有限公司 | Method for producing chitosan oligosaccharide from home-made enzyme solution |
| CN107278208A (en) * | 2014-12-31 | 2017-10-20 | 昆虫公司 | Chitin, hydrolysate and the mode by means of enzymatic hydrolysis are used for the method that at least one desired product of target is produced from insect, and it is included in the step of being performed before enzymatic hydrolysis and combined |
| CN107428849A (en) * | 2014-12-31 | 2017-12-01 | 昆虫公司 | Chitin, hydrolysate and the mode by means of enzymatic hydrolysis are used for the method that one or more desired products are produced from insect |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736583A (en) * | 2009-12-18 | 2010-06-16 | 东华大学 | Method for preparing chitin size modified by biological enzyme method |
| CN102816808A (en) * | 2012-07-17 | 2012-12-12 | 肖丽凤 | Method for preparing chitosan by using shrimp shells |
| WO2012168618A1 (en) * | 2011-05-26 | 2012-12-13 | Ifremer (Institut Français De Recherche Pour L'exploitation De La Mer) | Extraction of chitins in a single step by enzymatic hydrolysis in an acid medium |
| CN103113496A (en) * | 2013-02-21 | 2013-05-22 | 华东理工大学 | Clean chitosan production technology |
-
2013
- 2013-09-29 CN CN201310450586.7A patent/CN104059953A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736583A (en) * | 2009-12-18 | 2010-06-16 | 东华大学 | Method for preparing chitin size modified by biological enzyme method |
| WO2012168618A1 (en) * | 2011-05-26 | 2012-12-13 | Ifremer (Institut Français De Recherche Pour L'exploitation De La Mer) | Extraction of chitins in a single step by enzymatic hydrolysis in an acid medium |
| CN102816808A (en) * | 2012-07-17 | 2012-12-12 | 肖丽凤 | Method for preparing chitosan by using shrimp shells |
| CN103113496A (en) * | 2013-02-21 | 2013-05-22 | 华东理工大学 | Clean chitosan production technology |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107278208A (en) * | 2014-12-31 | 2017-10-20 | 昆虫公司 | Chitin, hydrolysate and the mode by means of enzymatic hydrolysis are used for the method that at least one desired product of target is produced from insect, and it is included in the step of being performed before enzymatic hydrolysis and combined |
| CN107428849A (en) * | 2014-12-31 | 2017-12-01 | 昆虫公司 | Chitin, hydrolysate and the mode by means of enzymatic hydrolysis are used for the method that one or more desired products are produced from insect |
| CN107278208B (en) * | 2014-12-31 | 2021-05-25 | 昆虫公司 | Chitin, hydrolysate and production of at least one target product from insects by means of enzymatic hydrolysis |
| US11891459B2 (en) | 2014-12-31 | 2024-02-06 | Ynsect | Chitin, hydrolysate and method for the production of one or more desired products from insects by means of enzymatic hydrolysis |
| CN107428849B (en) * | 2014-12-31 | 2024-03-26 | 昆虫公司 | Chitin, hydrolysate and method for producing one or more desired products from insects by means of enzymatic hydrolysis |
| CN105803019A (en) * | 2015-12-31 | 2016-07-27 | 珠海市金隆生物科技有限公司 | Method for producing chitosan oligosaccharide from home-made enzyme solution |
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