CN105154496B - A method of specified molecular weight water soluble chitosan is prepared using enzyme process - Google Patents
A method of specified molecular weight water soluble chitosan is prepared using enzyme process Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, specifically disclose a kind of method for preparing specified molecular weight water soluble chitosan using enzyme process, chitosan of this method using deacetylation higher than 80% prepares the water soluble chitosan of specified molecular weight as raw material through processes such as specificity enzyme hydrolysis, control reaction end, hydrolyzate concentration, freeze-dryings after swelling.This method can get the chitosan of content height, good water solubility, specified molecular weight by control enzyme additive amount, reaction time and solution viscosity, and have many advantages, such as that simple process, reaction speed are fast, specificity is strong, at low cost, safety and environmental protection.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of to prepare specified molecular weight water soluble chitosan using enzyme process
Method.
Background technique
Chitosan (chitosan) is also known as chitosan, is unique alkaline polysaccharide in nature, from a wealth of sources.Shell
Glycan have many excellent performances and bioactivity, as biocompatibility, biodegradability, sustained and controlled release, at fibre
Property, can spinnability, cancer resistance, hemostatic, antibiotic property etc., it is therefore, many in medicine, agricultural, environmental protection, food, cosmetics etc.
Field has very big application value, especially can be used as medical dressing, pharmaceutical carrier, dental material etc., city in medical domain
Field has a high potential.But common molecular weight of chitosan is mostly that hundreds of thousands is even up to a million, has stronger hydrogen bond and higher
Crystallinity is insoluble in water and general organic solvent, and then causes it to study and promote and apply and be limited by very large.Research
Show that, when molecular weight of chitosan drops to a certain degree, many specific functions just show.Therefore, water soluble chitosan
Or even the chitosan of different molecular weight, different function becomes the hot spot of Recent study.
According to the literature, the preparation method of water soluble chitosan can be summarized as three kinds: first is that passing through the de- of control chitin
Acetyl degree and deacetylated condition, obtain the water soluble chitosan of higher molecular weight；Second is that being introduced in chitosan molecule chain hydrophilic
Property group is grafted, and the water soluble chitosan of different structure is obtained；Third is that under proper condition, chitosan depolymerization obtains
Obtain the lower water soluble chitosan of molecular weight.Currently, first two method using in the majority, but there are processes complicated, reagent consumption
The disadvantages of amount is big, molecular weight is uncontrollable.The third method is that degradation of chitosan is obtained water soluble chitosan.Degradation of chitosan
Method mainly have chemical method, physical method and enzyme process, wherein chemical degradation method is a kind of side for being widely used in large-scale production
Method mainly has acid degradation method and oxidation degradation method, but this method equally exists more disadvantage, if reagent consumption is big, hydrolysis degree
Not easy to control, molecular weight of product is not concentrated, and product not readily dissolves, and easily causes environmental pollution etc..In comparison, enzyme process has obvious
Advantage, such as simple process, not high to equipment requirement, reaction condition is mild, and reaction speed is fast, specificity is strong, hydrolysis degree and
Hydrolysate relative molecular mass is easily controllable, product purity height, good water solubility, safety and environmental protection etc., but phase in the prior art
Answer the report of enzyme process solution chitosan also seldom, how to develop enzyme process that is more preferable and being more conducive to control becomes to solve chitosan
One of urgent problem to be solved.
Currently, the water soluble chitosan or chitosan of the preparation of the chitosan oligosaccharide product for having enzyme process to prepare in the market, chemical method spread out
Biological product (also referred to as water soluble chitosan).From the point of view of product characteristic, they all have the defects that different degrees of or deficiency or water
Dissolubility is good, bioactivity is not high, application effect is not significant, or it is water-soluble it is poor, be of limited application etc..
Summary of the invention
In view of the problems of the existing technology the present invention, provides and a kind of prepares specified molecular weight water soluble shells using enzyme process
The method of glycan, chitosan of this method using deacetylation higher than 80% are anti-through specificity enzyme hydrolysis, control after swelling as raw material
The processes such as terminal, hydrolyzate concentration, freeze-drying are answered to prepare the water soluble chitosan of specified molecular weight.This method passes through control
Enzyme additive amount, reaction time and solution viscosity can get the chitosan of content height, good water solubility, specified molecular weight, and have work
The advantages that skill is simple, reaction speed is fast, specificity is strong, at low cost, safety and environmental protection.
The specific technical solution of the present invention is:
Chitosan using deacetylation higher than 80% prepares specified molecular weight water soluble chitosan using enzyme process as raw material,
Specific step is as follows:
(1) acquisition of viscosity average molecular weigh curve
The chitosan solution for first preparing w/v 2%, is hydrolyzed using known enzyme and technology be prepared into a series of spies later
Determine the chitosan of molecular weight, measurement reaction fluid viscosity and viscosity average molecular weigh, draws curve to react fluid viscosity and viscosity average molecular weigh；
(2) it is swollen: taking chitosan raw material in reaction vessel, the chitosan solution of w/v 2%~8% is configured to water,
Adding the pH of acid-conditioning solution is 4~6, is stirred continuously, is swollen 60~120 minutes in 40 DEG C~60 DEG C water-baths；
(3) it hydrolyzes: enzyme being added in the reaction vessel with the ratio of 0.1U/g~6U/g according to chitosan mass, is started anti-
It answers, until starting at interval of sampling in 5 minutes once, sampling is diluted to reaction solution 10~15 minutes before the theoretical enzymatic hydrolysis complete time
Concentration is w/v 2%, measures its solution viscosity, until hydrolytic process terminates after predetermined viscosity, utilizes boiling water bath enzyme deactivation 10-30 later
Minute, reaction was completed to get specified molecular weight chitosan solution；Above-mentioned hydrolytic process control was at 60~150 minutes；
(4) dry: above-mentioned chitosan solution is cooling, through the centrifugation of 4000r/min~12000r/min centrifuge or filter cloth
The chitosan clear solution Direct spraying of filtering, acquisition is dry, or freezes after rotary evaporator is concentrated into 1/2~1/4 volume
Drying is to get solid, water soluble chitosan；
The solid, water soluble chitosan finally obtained, content are 91.5-98.5% (w/w)；
In above-mentioned swelling process, the chitosan solution concentration of preparation should be according to the viscous of the target water soluble chitosan of preparation
Depending on average molecular weight.If preparing the target product of 5kDa~150kDa, the chitosan solution concentration of preparation should be w/v 6%~
8%；If preparing the target product of 150kDa~350kDa, the chitosan solution concentration of preparation should be w/v 2%~4%.
In the hydrolytic process of above-mentioned chitosan, liquid enzymes can be used, immobilised enzymes can also be used, can reach efficient drop
The purpose for solving chitosan, if preparing the target product of 5kDa~150kDa, according to chitosan mass with the ratio of 0.5U/g~6U/g
Enzyme is added in example；If preparing the target product of 150kDa~350kDa, according to chitosan mass with the ratio of 0.1U/g~0.5U/g
Enzyme is added.The enzyme is selected generally from the thick enzyme powder of solid fermentation chitosan enzyme, the thick enzyme of liquid fermentation chitosan enzyme, contains chitosan
The shell that fermentation liquid, the resin-immobilized chitosan enzyme of absorption, chitin incision enzyme of the thick enzyme of enzyme etc. can directly be bought from the market is poly-
Carbohydrase.
In the control of above-mentioned reaction process, the control of enzyme concentration and reaction time: the dosage of enzyme is bigger, and the reaction time is shorter,
For the chitosan (the especially chitosan of macromolecule) for preparing specified molecular weight, react more not easy to control.Therefore, water
The solution reaction time is preferably controlled in 60~150 minutes, and according to reaction time and enzyme activity, enzyme is added by described above；
The control of solution viscosity: first preparing certain density chitosan solution, and a series of shell for preparing specified molecular weights is poly-
Sugar measures and reacts fluid viscosity and viscosity average molecular weigh under the concentration, draws curve to react fluid viscosity and viscosity average molecular weigh, further according to
Reaction fluid viscosity can prepare the chitosan of any specific molecular weight.
In specifically measurement real time viscosity, the concentration of chitosan material solution should be with chitosan solution when measurement curve
Concentration is identical, then needs to be adjusted it when different.
The chitosan of the specific viscosity average molecular weigh of 5kDa~350kDa of above-mentioned preparation and in the market water soluble chitosan are (actually
Chitosan derivatives, and molecular weight and be of limited application) the difference is that, be dissolved in water, and good water solubility (dissolution
Property be shown in Table 1), for water soluble chitosan truly, molecular weight product is different, and function is also different, can meet various applications
Demand.Meanwhile compared with the chitosan oligosaccharide in patent CN201210300863, reaction process enzyme concentration will reduce 2.4-40 times, and
Reaction time is short, energy consumption and at low cost, and bioactivity is good, and product multiplicity is widely used.
And compared with prior art, enzyme of the present invention is the common enzyme that routine is commercially available in the market, invention
People is mainly, using the control to enzymatic hydrolysis condition, the final product of enzymatic hydrolysis to be controlled from chitosan oligosaccharide using the achievement to study for a long period of time
For chitosan, water soluble chitosan truly is obtained, and can according to need STOCHASTIC CONTROL water soluble chitosan
Molecular weight.
In conclusion method provided by the invention can get and be contained by control enzyme additive amount, reaction time and solution viscosity
Measure height, good water solubility, specified molecular weight chitosan, and have simple process, reaction speed is fast, specificity is strong, it is at low cost, pacify
The advantages that loopful is protected.
Detailed description of the invention
The chitosan viscosity average molecular weigh and solution viscosity curve that Fig. 1 is molecular weight > 50kDa；
Fig. 2 is molecular weight≤50kDa chitosan viscosity average molecular weigh and solution viscosity curve.
Specific embodiment
Embodiment 1
The chitosan solution that w/v is 2% is prepared first, is hydrolyzed preparation using immobilization chitosan enzyme and technology later
At a series of chitosan of specified molecular weights, measurement reaction fluid viscosity and viscosity average molecular weigh, to react fluid viscosity and glue equal molecule
Amount draws curve, and the curve is as shown in Figure 2；
Chitosan 30g (deacetylation 93%) is added in reaction vessel, it is poly- to be configured to the shell that 1.5L w/v is 2% with water
Sugar juice, adding the pH value of vinegar acid-conditioning solution is 4.5, and 60min is kept the temperature in 40 DEG C~60 DEG C water-baths and is swollen；
After swelling sufficiently, the immobilization chitosan enzyme of 6U/g chitosan mass is added, starts under the conditions of 40 DEG C~60 DEG C anti-
It answers, using viscosity as control point, until stopping reaction, hydrolysis time 125min, boiling water bath enzyme deactivation when solution viscosity is 3.1mPas
10min, 60 DEG C are concentrated under reduced pressure, are freeze-dried, and chitosan 26g are obtained, according to reaction fluid viscosity and viscosity average molecular weigh curve calculating
Chitosan viscosity average molecular weigh is 10kDa.This chitosan 0.2g is weighed, adds water 1mL instant, it is seen that obtained is water soluble shells
Glycan.
Embodiment 2
The chitosan solution that w/v is 2% is prepared first, is hydrolyzed preparation using immobilization chitosan enzyme and technology later
At a series of chitosan of specified molecular weights, measurement reaction fluid viscosity and viscosity average molecular weigh, to react fluid viscosity and glue equal molecule
Amount draws curve, and the curve is as shown in Figure 2；
Chitosan 30g (deacetylation 93%) is added in reaction vessel, the chitosan for being configured to 1.5L2% with water is molten
Liquid, adding the pH value of vinegar acid-conditioning solution is 4.5, and 60min is kept the temperature in 40 DEG C~60 DEG C water-baths and is swollen；
After swelling sufficiently, the immobilization chitosan enzyme of 4U/g chitosan mass is added, starts under the conditions of 40 DEG C~60 DEG C anti-
It answers, using viscosity as control point, until stopping reaction when solution viscosity is 10.6mPas, hydrolysis time 116min, boiling water bath goes out
Enzyme 10min, 60 DEG C are concentrated under reduced pressure, are freeze-dried, and obtain chitosan 25g, are pushed away according to reaction fluid viscosity and viscosity average molecular weigh curve
Calculation chitosan viscosity average molecular weigh is 50kDa.This chitosan 0.2g is weighed, adds water 1mL instant, it is seen that obtained is water solubility
Chitosan.
Embodiment 3
The chitosan solution that w/v is 2% is prepared first, is hydrolyzed preparation using immobilization chitosan enzyme and technology later
At a series of chitosan of specified molecular weights, measurement reaction fluid viscosity and viscosity average molecular weigh, to react fluid viscosity and glue equal molecule
Amount draws curve, and the curve is as shown in Figure 1；
Chitosan 30g (deacetylation 93%) is added in reaction vessel, the chitosan for being configured to 1.5L2% with water is molten
Liquid, adding the pH value of vinegar acid-conditioning solution is 4.5, and 60min is kept the temperature in 40 DEG C~60 DEG C water-baths and is swollen；
After swelling sufficiently, the immobilization chitosan enzyme of 1U/g chitosan mass is added, starts under the conditions of 40 DEG C~60 DEG C anti-
It answers, using viscosity as control point, until stopping reaction, hydrolysis time 108min, boiling water bath enzyme deactivation when solution viscosity is 196mPas
10min, 60 DEG C are concentrated under reduced pressure, are freeze-dried, and chitosan 25g are obtained, according to reaction fluid viscosity and viscosity average molecular weigh curve calculating
Chitosan viscosity average molecular weigh is 100kDa.This chitosan 0.2g is weighed, adds water 2mL instant, it is seen that obtained is water solubility
Chitosan.
1 chitosan water solubility of table compares
Aqueous solution chitosan solubility prepared by the present invention and the prior art maintain an equal level as seen from the above table, but dissolution time is bright
The aobvious shorter than prior art, can get the chitosan of content height, good water solubility, specified molecular weight, and have simple process, reaction speed
The advantages that degree is fast, specificity is strong, at low cost, safety and environmental protection.
Claims (1)
1. a kind of method for preparing specified molecular weight water soluble chitosan using enzyme process, it is characterised in that: specific step is as follows:
(1) acquisition of viscosity average molecular weigh curve
The chitosan solution for first preparing w/v 2%, is hydrolyzed using known enzyme and technology be prepared into a series of specific moleculars later
The chitosan of amount, measurement reaction fluid viscosity and viscosity average molecular weigh draw curve to react fluid viscosity and viscosity average molecular weigh；
(2) it is swollen: taking chitosan raw material in reaction vessel, the chitosan solution of w/v 2%~8%, acid adding tune are configured to water
The pH for saving solution is 4~6, is stirred continuously, is swollen 60~120 minutes in 40 DEG C~60 DEG C water-baths；
(3) it hydrolyzes: enzyme being added in the reaction vessel with the ratio of 0.1U/g~6U/g according to chitosan mass, starts to react, until
Theory digests before the complete time 10~15 minutes, starts at interval of sampling in 5 minutes once, sampling is diluted to reaction solution concentration and is
W/v 2% measures its solution viscosity, until hydrolytic process terminates after predetermined viscosity, utilizes boiling water bath enzyme deactivation 10-30 minutes later, knot
Shu Fanying to get specified molecular weight chitosan solution；Above-mentioned hydrolytic process control was at 60~150 minutes；
(4) dry: above-mentioned chitosan solution is cooling, through the centrifugation of 4000r/min~12000r/min centrifuge or filter-cloth filtering,
The chitosan clear solution Direct spraying of acquisition is dry, or is freeze-dried after rotary evaporator is concentrated into 1/2~1/4 volume,
Up to solid, water soluble chitosan；
Enzyme described in step (2) hydrolytic process be chitosan enzyme or chitinase or chitin deacetylase it is one such or
Two kinds；Prepare the target product of 5kDa~150kDa, the chitosan solution concentration of preparation is w/v 2%, according to chitosan mass with
Enzyme is added in the ratio of 1U/g~6U/g；
Chitosan raw material in the step (1) is the chitosan that deacetylation is greater than 80%；
The acid for adjusting pH in the step (2) described in swelling process is acetic acid.
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| CN111375067B (en) * | 2020-03-18 | 2022-02-11 | 四川大学 | Chitosan scaffold and preparation method and application thereof |
| CN111518856A (en) * | 2020-05-07 | 2020-08-11 | 山东肽和生物科技有限公司 | Production process of high-concentration low-molecular-weight liquid chitosan oligosaccharide |
| CN113069591A (en) * | 2021-03-29 | 2021-07-06 | 黄河三角洲京博化工研究院有限公司 | Chitosan-calcium polyglutamate biological dressing and preparation method thereof |
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| 壳聚糖的酶法降解;戴大章等;《功能高分子学报》;20051231;第687-691页 |
| 窦屾等.酶法降解壳聚糖及产物应用研究进展.《食品工业科技》.2011,第32卷(第12期),第537-543页. |
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