CN1228350C - Water-soluble Chitooligosaccharide preparation method - Google Patents
Water-soluble Chitooligosaccharide preparation method Download PDFInfo
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Abstract
The present invention relates to a method for preparing water-solubility chitosan oligosaccharide. In the present invention, water-solubility chitosan oligosaccharide is prepared by the difference of the recognition capability and the cutting-off capability of enzyme to different glycosidic bond and the inhomogeneous distribution in chitosan raw materials. Consequently, the technology is simple. When the chitosan oligosaccharide is prepared, water-solubility chitosan oligosaccharide with the polymerization from 8 to 200 is obtained by an ultrafiltration membrane separating method. Experiment indicates that the obtained products have the action of tumor prevention and immunological enhancement. An experiment of Sacroma 180 solid tumor inhibition shows that the chitosan oligosaccharide prepared by the present invention has preferable activity for preventing tumors; the tumor preventing rate of intraperitoneal injection is more than 40% and reaches 64%; tumor preventing rate of oral medication reaches 33.7%.
Description
Technical field
The present invention relates to a kind of preparation method of water soluble shells oligose, especially prepare the polymerization degree and be the method for 8 to 200 water soluble shells oligose.
Background technology
Chitosan is as a kind of natural polysaccharide, and especially An Ji a large amount of existence are a kind of alkaline polysaccharides, thereby has the functional of some uniquenesses.The research announcement polymerization degree has better antitumor action and immunologic enhancement greater than 6 water-soluble chitin oligosaccharide and chitooligosaccharide-than corresponding oligosaccharides.The usually said chitin (deacetylation is less than 20%) and chitosan (deacetylation is greater than the 70%) polymerization degree are greater than 7 o'clock, because of its water-soluble bad directly influence digestion and absorption in vivo; For this reason, many investigators are devoted to the research of high-polymerization degree water soluble shells oligose, find that the chitosan that the deacetylation stochastic distribution is about about 50% can be water-soluble.High-polymerization degree water soluble shells oligose is by earlier that the chitin height is deacetylated at present, again degradation of chitosan is arrived certain polymerization degree, comes the method for partial acetylation to prepare with acetic anhydride then, and technology is more loaded down with trivial details.
Summary of the invention
The present invention proposes the preparation method of the better simply water soluble shells oligose of a kind of technology.
Technical scheme provided by the invention is: a kind of preparation method of water soluble shells oligose, get deacetylation and be 50～85% chitosan, dissolve with dilute acid soln, transferring pH value of solution with alkali is 5-6, the control solution temperature is 40-55 ℃, enzyme-added liquid catalyzed degradation chitosan, filtering insolubles then, gained filtrate is used the ultra-filtration membrane ultrafiltration, obtains required water soluble shells oligose.
In aforesaid method, with the ultra-filtration membrane ultrafiltration of filtering insolubles gained filtrate with 10kDa, after unsanctioned solution boiled and dezymotizes, obtaining the polymerization degree was the water soluble shells oligose of 60-200; Solution by the 10kDa ultra-filtration membrane further obtains the water soluble shells oligose that the polymerization degree is 30-60 with the classification of 3kDa ultra-filtration membrane, solution by the 3kDa ultra-filtration membrane obtains the water soluble shells oligose that the polymerization degree is 8-30 with the classification of 1kDa ultra-filtration membrane, uses 1kDa ultra-filtration membrane separation of polymeric degree less than 8 oligosaccharides at last.If obtain chitooligosaccharide-salt after the solution lyophilize with each component.If transfer pH8-9 with NaOH after the solution for vacuum concentration with each component, the ethanol sedimentation, washing with alcohol, vacuum-drying obtains chitooligosaccharide-(free amino group form) respectively.
Above-mentioned enzyme is chitoanase, hemicellulase or N,O-Diacetylmuramidase, and the mixture of aforementioned two kinds of enzymes.
Above-mentioned dilute acid soln is acetic acid or lactic acid.
Above-mentioned ultra-filtration membrane refers to the various ultra-filtration membranes that use in the industry.
The present invention utilizes enzyme that the recognition capability of different sugar glycosidic bond and the difference and the uneven distribution of kharophen in the chitosan raw material of the ability of cut-out are prepared the water soluble shells oligose.Thereby technology is fairly simple, and in the preparation oligochitosan, obtaining the polymerization degree by the isolating method of ultra-filtration membrane is 8 to 200 water soluble shells oligose.Experiment shows that products obtained therefrom has antitumor action and immunologic enhancement.Inhibition experiment to the Sacroma180 solid tumor shows that the water soluble shells oligose that the present invention makes has better tumor-inhibiting activity, and the abdominal injection tumour inhibiting rate is up to 64% greater than 40%.Oral inhibitory rate 33.7%.
Embodiment
Embodiment 1: get crab shell chitosan 100 grams of deacetylation 80.2%, after 2 liter 2% acetum dissolving, transfer pH value of solution 5.5 with saturated sodium hydroxide, 47 ℃ of temperature controls add 10 milliliters of hemicellulase crude enzyme liquid catalyzed degradation chitosans.When reaction solution splashes in 2% sodium hydroxide solution after the no muddiness, dezymotize with the ultrafiltration of NMWL 10kDa ultra-filtration membrane, further obtain the solution of three components with 3kDa and the classification of 1kDa ultra-filtration membrane by the solution of 10kDa ultra-filtration membrane.CHU10-3 is by the 10kDa film, does not pass through the component of 3kDa film; CHU3-1 has passed through the 3kDa film, does not pass through the component of 1kDa film.Behind the filtrate vacuum concentration, transfer pH liquid 8-9 with NaOH, the ethanol sedimentation, washing with alcohol, vacuum-drying obtains CHU10-3 respectively, CHU3-1 and CHU1 water soluble shells oligose.
Table 1.10kDa, 3kDa and 1kDa ultra-filtration membrane classification product
| Chitooligosaccharide- | Mean polymerisation degree | Dispersity | Deacetylation | Yield (%) |
| CHU10-3 CHU3-1 | 43 16 | 2.3 3.2 | 57.9 71.5 | 12.2 6.8 |
When adopting the differential detector to detect, can detect the oligochitosan of the polymerization degree below 8.The oligochitosan standard specimen of the hexa polyose and the lower polymerization degree all can detect in the experiment test.Do not detect six aggressiveness and lower oligosaccharides among the sample CHU10-3, and only contain the oligochitosan of the polymerization degree of a spot of six aggressiveness and Geng Gao among the CHU3-1.
Embodiment 2: shrimp shell chitosan 50 grams of deacetylation 71.2% add with stirring 8 hours after-filtration in 2 liter 2% the acetum, transfer pH value of solution 5.0, and 40 ℃ of temperature controls add bacteriolyze enzymatic degradation chitosan.After 24 hours, first micro-filtration is used NMWL 1kDa ultra-filtration membrane ultra-filtration and separation oligosaccharides part again.Unsanctioned component solution is transferred pH value of solution 8-9 with saturated sodium hydroxide, centrifugation, and behind the clear liquid vacuum concentration, back ethanol sedimentation, washing with alcohol gets water soluble shells oligose product, yield 38.7%, mean polymerisation degree 21, dispersity 2.65 after the drying.
Embodiment 3: shrimp shell chitosan 25 grams of getting deacetylation 73% add with stirring 8 hours after-filtration in 1 liter 2% the lactic acid solution, transfer pH value of solution 5.5,45 ℃ of temperature controls, the prozyme catalyzed degradation chitosan that adds chitosan-containing enzyme and hemicellulase, when reaction solution splashes in 2% sodium hydroxide solution after the no muddiness, dezymotize with the ultrafiltration of NMWL 10kDa ultra-filtration membrane, by the further NMWL 1kDa of the solution of 10kDa ultra-filtration membrane ultra-filtration membrane ultra-filtration and separation oligosaccharides part.Passed through the 10kDa film,, do not obtained chitooligosaccharide-lactic acid salt sample after the lyophilize, yield 23%, mean polymerisation degree 25, dispersity 2.85 by the component of 1kDa film; After the solution by the 10kDa ultra-filtration membrane does not boil extremely enzyme, after 50 ℃ of vacuum-dryings chitooligosaccharide-lactic acid salt sample, yield 5.3%, mean polymerisation degree 82, dispersity 2.1.
Claims (6)
1. the preparation method of a water soluble shells oligose, get deacetylation and be 50～85% chitosan, dissolve with dilute acid soln, transferring pH value of solution with alkali is 5-6, and the control solution temperature is 40-55 ℃, enzyme-added liquid catalyzed degradation chitosan, filtering insolubles then, with the ultra-filtration membrane ultrafiltration of filtering insolubles gained filtrate with 10kDa, after unsanctioned solution boiled and dezymotizes, obtaining the polymerization degree was the water soluble shells oligose of 60-200; Solution by the 10kDa ultra-filtration membrane further obtains the water soluble shells oligose that the polymerization degree is 30-60 with the classification of 3kDa ultra-filtration membrane, solution by the 3kDa ultra-filtration membrane obtains the water soluble shells oligose that the polymerization degree is 8-30 with the classification of 1kDa ultra-filtration membrane, uses 1kDa ultra-filtration membrane separation of polymeric degree less than 8 oligosaccharides at last.
2. method according to claim 1 is characterized in that: obtain chitooligosaccharide-salt after the solution lyophilize of each component that will obtain with the ultra-filtration membrane classification.
3. method according to claim 1 is characterized in that: transfer pH8-9 with NaOH after the solution for vacuum concentration of each component that will obtain with the ultra-filtration membrane classification, and the ethanol sedimentation, washing with alcohol, vacuum-drying obtains the chitooligosaccharide-of free amino group form respectively.
4. according to claim 1,2 or 3 described methods, it is characterized in that: described enzyme is chitoanase, hemicellulase or N,O-Diacetylmuramidase, and the mixture of aforementioned two kinds of enzymes.
5. according to claim 1,2 or 3 described methods, it is characterized in that: described dilute acid soln is acetic acid or lactic acid.
6. according to claim 1,2 or 3 described methods, it is characterized in that: used ultra-filtration membrane is the various ultra-filtration membranes that use in the industry.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868076A (en) * | 2017-03-02 | 2017-06-20 | 杭州垚信生物科技有限公司 | A kind of method that bacteria residue prepares chitosan oligosaccharide |
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| CN100391981C (en) * | 2005-12-16 | 2008-06-04 | 武汉大学 | Method for preparing complete water soluble low molecular weight chitosan/chitooligosaccharace |
| CN100386345C (en) * | 2006-06-05 | 2008-05-07 | 孝感学院 | Preparation process of chito oligosaccharace hydrochloride |
| CN100441692C (en) * | 2006-08-17 | 2008-12-10 | 孝感学院 | Chitosan oligosaccharide sulphate and preparation method thereof |
| CN1975419B (en) * | 2006-12-11 | 2012-05-09 | 武汉大学 | Water soluble chitin pH probe and producing method thereof |
| CN102899308B (en) * | 2012-10-17 | 2015-01-07 | 东华大学 | Cellulose thermostable additive |
| CN103695399A (en) * | 2013-01-06 | 2014-04-02 | 东华大学 | Cellulose activator |
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| CN109536551A (en) * | 2018-12-07 | 2019-03-29 | 扬州日兴生物科技股份有限公司 | A kind of chitosan oligosaccharide preparation method based on UF membrane |
| CN110305916A (en) * | 2019-07-22 | 2019-10-08 | 厦门大学 | A kind of preparation method of the fluorescent material based on chitosan oligosaccharide Maillard reaction |
| CN111154011B (en) * | 2020-02-25 | 2021-10-08 | 江西师范大学 | Method for preparing chitosan hydrochloride with extremely low viscosity |
| CN113293185A (en) * | 2021-04-02 | 2021-08-24 | 上海应用技术大学 | Preparation method of chitosan oligosaccharide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868076A (en) * | 2017-03-02 | 2017-06-20 | 杭州垚信生物科技有限公司 | A kind of method that bacteria residue prepares chitosan oligosaccharide |
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