CN108997512A - A kind of low molecular weight chitosan oligosaccharide and preparation method thereof - Google Patents
A kind of low molecular weight chitosan oligosaccharide and preparation method thereof Download PDFInfo
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- CN108997512A CN108997512A CN201810950138.6A CN201810950138A CN108997512A CN 108997512 A CN108997512 A CN 108997512A CN 201810950138 A CN201810950138 A CN 201810950138A CN 108997512 A CN108997512 A CN 108997512A
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
The invention belongs to oligosaccharide preparation method fields, and in particular to a kind of low molecular weight chitosan oligosaccharide and preparation method thereof.Chitosan oligosaccharide preparation method provided by the invention, including dissolving the chitosan in NaAc_HAc buffer solution, prepare chitosan solution, a kind of complex enzyme liquid enzymatic hydrolysis is added into chitosan solution, it is dialysed filtrate after the completion of enzymatic hydrolysis using dialysis, will be spray-dried after the concentration of gained dialyzate and etc..The present invention, which provides preparation method, has the advantages such as low molecular weight chitosan oligosaccharide yield is high, condition is easy, short the time required to preparation, and enzyme used is cheap and easy to get, and production cost is low, is suitable for a wide range of promote and apply.
Description
Technical field
The invention belongs to oligosaccharide preparation method fields, and in particular to a kind of low molecular weight chitosan oligosaccharide and preparation method thereof.
Background technique
Chitosan oligosaccharide be in presently found nature it is unique it is positively charged, in alkalinity, water-soluble polysaccharide, have superior
Bioactivity, can by chitin, chitosan prepare.The chitosan oligosaccharide of different molecular weight has the function of different, molecular weight
The chitosan oligosaccharide of 5000Da or so has the function of reducing blood lipid, and the chitosan oligosaccharide of molecular weight 3000-2000Da or so, which has, inhibits tumour
The effect of growth and the transfer of cell；It is worth noting that molecular weight is living with unique physiology in 1000Da chitosan oligosaccharide below
Property and function, including enhancing humoral immunity and cellular immunity；Promote absorption and the bone health of calcium；It is antibacterial, antibacterial and significant
Moisturizing wettability power etc..
Chitosan is mainly degraded to prepare chitosan oligosaccharide, enzymatic isolation method strong, reaction condition with specificity by enzymatic isolation method at present
Mildly, the advantages that process is easy to control and low in the pollution of the environment.And currently used enzyme can divide specificity enzyme and non-specific enzyme, it is single-minded
Property enzyme hydrolysis result is good but expensive, and the prices such as the non-specific enzyme in part such as papain and bromelain are lower, but
Hydrolysis result is bad, makes as the Chinese patent of publication number CN107739418A discloses a kind of be freeze-dried using papain
The method of standby chitosan oligosaccharide, although this method enzymolysis time is shorter, molecular weight of product is mostly in 3000Da or so；Publication number
The Chinese patent of CN107988287A discloses a kind of method for preparing chitosan oligosaccharide using cellulase spray drying, this method institute
The problems such as cheap and easy to get with enzyme, but long there are enzymolysis time.
Tannase is also known as tannase, is a kind of tannin Acyl- hydrolase.The enzyme can be by mould, as aspergillus niger, aspergillus oryzae are raw
It produces, is currently used primarily in tannin, protein in processing beer, makes its clear.But it is not directed in enzymatic hydrolysis chitosan
The effect of aspect.
Not yet occur at present using ficin and tannase as complex enzyme zymohydrolysis chitosan production chitosan oligosaccharide
Report.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of chitosan oligosaccharides and preparation method thereof.The present invention
A kind of chitosan oligosaccharide preparation method mild condition provided, short, the chitosan oligosaccharide of molecular weight 1000Da or so the time required to easy, preparation
Yield is higher, and does not need that a large amount of reaction reagent is added, and environmental pollution is less, ficin and tannase valence used
It is honest and clean to be easy to get.
The technical scheme is that a kind of low molecular weight chitosan oligosaccharide preparation method, comprising the following steps:
S1, it dissolves the chitosan in NaAc_HAc buffer solution, is configured to the shell that mass concentration is 0.5~1.5%
Glycan solution；
S2, complex enzyme is added into chitosan solution obtained by step S1, the quality of complex enzyme and the mass ratio of chitosan are
2.5~25%, 1~3h is reacted at 65 DEG C, obtains enzymolysis liquid, and wherein complex enzyme presses 1:(0.1 by ficin and tannase
~1) weight ratio composition；
S3, the lower NaOH solution that dropwise addition mass concentration is 5% into step S2 gained enzymolysis liquid of stirring, adjusting pH is 7.0
~8.0, filtering separates water-insoluble chitosan, and filtrate is fitted into bag filter, is dialysed for 24 hours in distilled water, obtain dialyzate；
S4, dialyzate obtained by step S3 is concentrated, is concentrated into the concentrate that solid quality concentration is 9~15%,
It is spray-dried.
Further, complex enzyme described in the step S1 is pressed the weight ratio of 1:0.3 by ficin and tannase
Composition.
Further, the pH value of NaAc_HAc buffer solution described in the step S1 is 5.0, concentration 0.2mol/
L。
Further, the mass ratio of the quality of complex enzyme described in the step S2 and chitosan is 5~10%.
Further, the mass ratio of the quality of complex enzyme described in the step S2 and chitosan is 7.5%.
Further, in 65 DEG C of reaction 2h in the step S2.
Further, bag filter described in the step S3 is regenerated cellulose bag filter, molecular cut off 1000Da.
Further, the solid quality concentration of the concentrate of spray drying described in the step S4 is 10%.
Further, the process conditions of spray drying described in the step S4 are as follows: 180 DEG C of inlet air temperature, charging rate is
700mL/h。
The present invention using ficin and tannase enzymatic hydrolysis chitosan can in a relatively short period of time, digest obtain compared with
Polymolecular amount is in the small-molecular-weight chitosan oligosaccharide of 1000Da or so, and average yield can reach 39.47%, when significantly shortening enzymatic hydrolysis
Between, it reduces costs.And when individually can not obtaining same as described above or class using ficin or while being tannase
As effect.
Compared with prior art, the invention has the following advantages:
Method mild condition provided by the invention, complex enzyme used is cheap and easy to get, and production cost is low, the time required to preparation
It is short, it is often more important that low molecular weight chitosan oligosaccharide yield is higher, achieves significant progress compared with prior art.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
Chitosan (lot number: 171112A, DD85%, Shandong aokang Biotechnology Co., Ltd)；Ficin (ladder
Xi Ai chemical conversion industry Development Co., Ltd)；Tannase (Henan Xi He Chemical Co., Ltd.)；Chitosan oligosaccharide (the Qingdao ocean Xiang Sheng section
Skill Co., Ltd)；Regenerated cellulose bag filter (molecular cut off 1000Da, MYM biotech company of the U.S.).
The preparation of embodiment 1, low molecular weight chitosan oligosaccharide
S1, it dissolves the chitosan in the 0.2mol/L NaAc_HAc buffer solution of pH value 5.0, it is dense to be configured to quality
The chitosan solution that degree is 1.0%；
S2, the complex enzyme liquid that mass concentration is 0.5% is added into chitosan solution obtained by step S1, complex enzyme is by no flower
Fruit protease and tannase are formed by the weight ratio of 1:0.3, and the mass ratio of complex enzyme quality and chitosan is 7.5%, in 65 DEG C
Lower reaction 2h, obtains enzymolysis liquid；
The NaOH solution that mass concentration is 5% is added dropwise under S3, stirring into enzymolysis liquid obtained by step S2, adjusting pH is 7.0
~8.0, filtering separates water-insoluble chitosan, and it is saturating that filtrate is respectively charged into the regenerated cellulose that molecular cut off is 1000Da
It analyses in bag, dialyses in distilled water for 24 hours, obtain dialyzate；
S4, dialyzate obtained by step S3 is concentrated, is concentrated into the concentrate that solid quality concentration is 10%, carried out
Spray drying, the process conditions of spray drying are as follows: 180 DEG C of inlet air temperature, charging rate 700mL/h.
The complex enzyme liquid is formulated by the NaAc_HAc buffer solution of the 0.2mol/L of pH value 5.0.
The preparation of embodiment 2, low molecular weight chitosan oligosaccharide
Embodiment 2 the difference from embodiment 1 is that, in step S1, the mass concentration of chitosan solution is 1.2%, S2 step
The mass ratio of the quality of complex enzyme and chitosan is 5% in rapid, and complex enzyme is pressed the weight of 1:0.1 by ficin and tannase
Amount is than composition, remaining is the same as embodiment 1.
The preparation of embodiment 3, low molecular weight chitosan oligosaccharide
Embodiment 3 the difference from embodiment 1 is that, in step S1, the mass concentration of chitosan solution is 1.2%, S2 step
The mass ratio of the quality of complex enzyme and chitosan is 7.5% in rapid, and complex enzyme is pressed the weight of 1:1 by ficin and tannase
Amount is than composition, remaining is the same as embodiment 1.
The preparation of comparative example 1, low molecular weight chitosan oligosaccharide
This comparative example difference compared with Example 1 is: enzyme used is ficin, remaining is the same as embodiment 1.
The preparation of comparative example 2, low molecular weight chitosan oligosaccharide
This comparative example difference compared with Example 1 is: enzyme used is tannase, remaining is the same as embodiment 1.
The preparation of comparative example 3, low molecular weight chitosan oligosaccharide
This comparative example difference compared with Example 1 is: replacing tannase with cellulase, remaining is the same as embodiment 1.
Test example one, properties of product measurement
The moisture content of the chitosan oligosaccharide sample that Examples 1 to 3 and comparative example 1~3 are prepared, content of reducing sugar,
The yield of 5% solution viscosity and 1000Da chitosan oligosaccharide is detected, and as a control group with corresponding chitosan oligosaccharide bulk pharmaceutical chemicals,
Testing result is as shown in table 1 below.
1 testing result of table
| Group | Moisture (%) | Content of reducing sugar (%) | 5% solution viscosity | Yield (%) |
| Embodiment 1 | 5.02 | 62.71 | 1.38 | 39.47% |
| Embodiment 2 | 5.15 | 61.03 | 1.41 | 37.36% |
| Embodiment 3 | 5.29 | 64.69 | 1.35 | 38.54% |
| Comparative example 1 | 5.18 | 43.74 | 1.42 | 20.06% |
| Comparative example 2 | 5.00 | 30.65 | 1.43 | 11.57% |
| Comparative example 3 | 5.23 | 56.53 | 1.40 | 25.36% |
| 1000Da chitosan oligosaccharide bulk pharmaceutical chemicals | 4.96 | 57.66 | 1.42 | - |
As shown in Table 1, compared with Example 1, difference, which is only that, replaces composite degradation enzyme for comparative example 1 and comparative example 2
It is far below the yield of composite degradation enzyme in embodiment 1 for the yield of the single enzyme of the two, but 1000Da chitosan oligosaccharide, especially adopts
It is digested with tannase, yield is only 11.57%；In addition, comparative example 3 is compared with embodiment 1, although both using complex enzyme enzyme
Solve chitosan, but the enzymatic hydrolysis performance of cradin used in comparative example 3 and cellulase complex enzyme nothing not as good as described in Examples 1 to 3
Ficin and tannase complex enzyme to sum up illustrate that the present invention achieves unexpected effect.
The above disclosure is only a preferred embodiment of the invention, cannot limit the right of the present invention with this certainly
Range, therefore according to equivalent variations made by scope of the present invention patent, it is still within the scope of the present invention.
Claims (10)
1. a kind of preparation method of low molecular weight chitosan oligosaccharide, which comprises the following steps:
S1, it dissolves the chitosan in NaAc_HAc buffer solution, is configured to the chitosan that mass concentration is 0.5~1.5%
Solution；
S2, it is added complex enzyme into chitosan solution obtained by step S1, the quality of complex enzyme and the mass ratio of chitosan be 2.5~
25%, 1~3h is reacted at 65 DEG C, obtains enzymolysis liquid, wherein complex enzyme presses 1:(0.1~1 by ficin and tannase)
Weight ratio composition；
S3, the NaOH solution that mass concentration is 5% is added dropwise into step S2 gained enzymolysis liquid under stirring conditions, adjusting pH is
7.0~8.0, filtering separates water-insoluble chitosan, and filtrate is fitted into bag filter, is dialysed for 24 hours in distilled water, obtain dialyzate；
S4, dialyzate obtained by step S3 is concentrated, is concentrated into the concentrate that solid quality concentration is 9~15%, carried out
Spray drying.
2. low molecular weight chitosan oligosaccharide preparation method as described in claim 1, which is characterized in that compound described in the step S1
Enzyme is made of ficin and tannase by the weight ratio of 1:0.3.
3. low molecular weight chitosan oligosaccharide preparation method as described in claim 1, which is characterized in that vinegar described in the step S1
Acid-sodium acetate buffer pH value is 5.0, concentration 0.2mol/L.
4. low molecular weight chitosan oligosaccharide preparation method as described in claim 1, which is characterized in that compound described in the step S2
The quality of enzyme and the mass ratio of chitosan are 5~10%.
5. low molecular weight chitosan oligosaccharide preparation method as claimed in claim 4, which is characterized in that the quality and shell of the complex enzyme
The mass ratio of glycan is 7.5%.
6. low molecular weight chitosan oligosaccharide preparation method as described in claim 1, which is characterized in that anti-in 65 DEG C in the step S2
Answer 2h.
7. low molecular weight chitosan oligosaccharide preparation method as described in claim 1, which is characterized in that dialyse described in the step S3
Bag is regenerated cellulose bag filter, molecular cut off 1000Da.
8. low molecular weight chitosan oligosaccharide preparation method as described in claim 1, which is characterized in that the step S4 is concentrated into solid
The concentrate that amount of substance concentration is 10%.
9. low molecular weight chitosan oligosaccharide preparation method as described in claim 1, which is characterized in that spraying described in the step S4
Dry process conditions are as follows: 180 DEG C of inlet air temperature, charging rate 700mL/h.
10. a kind of chitosan oligosaccharide that the method as described in claim 1~9 is any is prepared.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810950138.6A CN108997512B (en) | 2018-08-20 | 2018-08-20 | Low molecular weight chitosan oligosaccharide and preparation method thereof |
| PCT/CN2018/118048 WO2020037866A1 (en) | 2018-08-20 | 2018-11-29 | Low molecular weight chitooligosaccharide and preparation method therefor |
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|---|---|---|---|
| CN201810950138.6A CN108997512B (en) | 2018-08-20 | 2018-08-20 | Low molecular weight chitosan oligosaccharide and preparation method thereof |
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| CN108997512A true CN108997512A (en) | 2018-12-14 |
| CN108997512B CN108997512B (en) | 2019-12-06 |
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Cited By (2)
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| CN109136307A (en) * | 2018-09-06 | 2019-01-04 | 广东药科大学 | A kind of method and application thereof preparing chitosan oligosaccharide with glusulase |
| CN109438103A (en) * | 2019-01-10 | 2019-03-08 | 江苏心实肥业集团有限公司 | A kind of chitosan oligosaccharide crop nutrition spray fertilizer and preparation method thereof |
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| CN101240040A (en) * | 2008-03-11 | 2008-08-13 | 广西大学 | Method for preparing active chitosan oligosaccharide by using ozone to degrade chitosan |
| CN101805409A (en) * | 2009-05-15 | 2010-08-18 | 董平 | Polyesterase polymerization process and device |
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| CN1177856C (en) * | 2001-08-14 | 2004-12-01 | 华东理工大学 | Process for industrial production of oligochitose and chitooligose |
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| CN107739418A (en) * | 2017-11-08 | 2018-02-27 | 广东药科大学 | A kind of method that chitosan oligosaccharide is prepared using papain freeze-drying |
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- 2018-08-20 CN CN201810950138.6A patent/CN108997512B/en active Active
- 2018-11-29 WO PCT/CN2018/118048 patent/WO2020037866A1/en active Application Filing
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| CN1746193A (en) * | 2005-09-15 | 2006-03-15 | 武汉大学 | Preparation of low-molecular weight chitoglycan or chitooligose |
| CN101240040A (en) * | 2008-03-11 | 2008-08-13 | 广西大学 | Method for preparing active chitosan oligosaccharide by using ozone to degrade chitosan |
| CN101805409A (en) * | 2009-05-15 | 2010-08-18 | 董平 | Polyesterase polymerization process and device |
| CN104829749A (en) * | 2015-05-08 | 2015-08-12 | 中国科学院理化技术研究所 | Method for preparing chitosan oligosaccharide by taking shrimp and crab shells as raw materials through one step by adopting enzymolysis method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136307A (en) * | 2018-09-06 | 2019-01-04 | 广东药科大学 | A kind of method and application thereof preparing chitosan oligosaccharide with glusulase |
| CN109438103A (en) * | 2019-01-10 | 2019-03-08 | 江苏心实肥业集团有限公司 | A kind of chitosan oligosaccharide crop nutrition spray fertilizer and preparation method thereof |
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| WO2020037866A1 (en) | 2020-02-27 |
| CN108997512B (en) | 2019-12-06 |
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