阅读说明：本技术 一种低聚壳寡糖单体的制备方法 (Preparation method of oligo-chitosan oligosaccharide monomer ) 是由 夏文水 王斌 毛水芳 刘晓丽 姜启兴 许艳顺 于 2019-11-15 设计创作，主要内容包括：本发明公开了一种低聚壳寡糖单体的制备方法。本发明对壳聚糖溶液进行预处理,使壳聚糖溶液充分溶胀分散,扩大了酶与底物的接触范围,得到分布较为集中的壳寡糖产品,大大降低了单糖的生成。在制备壳寡糖的过程中与膜分离相结合,使活性壳寡糖及时得到分离,有效的控制了壳寡糖在酶作用下的进一步降解。超滤之后纳滤,除去低聚合度的无活性的寡糖和大量的水,使获得生理活性高的某聚合度范围内的壳寡糖产品。再进行凝胶排阻色谱分离,将相同组分合并收集,冷冻干燥得壳寡糖单体,得到的低聚壳寡糖单体的纯度达到40％以上。(The invention discloses a preparation method of an oligo-chitosan oligosaccharide monomer. The invention pretreats the chitosan solution, so that the chitosan solution is fully swelled and dispersed, the contact range of enzyme and substrate is enlarged, chitosan oligosaccharide products with concentrated distribution are obtained, and the generation of monosaccharide is greatly reduced. The process of preparing the chitosan oligosaccharide is combined with membrane separation, so that the active chitosan oligosaccharide is separated in time, and the further degradation of the chitosan oligosaccharide under the action of enzyme is effectively controlled. And (3) nanofiltration is carried out after ultrafiltration, and inactive oligosaccharide with low polymerization degree and a large amount of water are removed, so that a chitosan oligosaccharide product with high physiological activity and a certain polymerization degree range is obtained. And performing gel exclusion chromatography separation, separating and collecting the same components, and freeze-drying to obtain the chitosan oligosaccharide monomer, wherein the purity of the obtained chitosan oligosaccharide monomer reaches more than 40%.)

1. The preparation method of the oligo-chitosan oligosaccharide monomer is characterized by comprising the following steps:

(1) dissolving chitosan, adding enzyme for hydrolysis to obtain chitosan hydrolysate;

(2) performing microfiltration and impurity removal on the chitosan hydrolysate obtained in the step (1), and performing ultrafiltration and nanofiltration treatment to obtain a crude product of the oligochitosan oligosaccharide;

(3) and (3) separating the crude product of the oligochitosan oligosaccharide obtained in the step (2) by gel exclusion chromatography, and combining the same components to obtain the oligochitosan oligosaccharide monomer.

2. The method of claim 1, wherein the dissolving step comprises adding chitosan to water to disperse the chitosan uniformly, and adding an acetic acid buffer to dissolve the chitosan.

3. The method of claim 1, wherein the enzyme is one or more of chitosan hydrolase, cellulase, papain, and lipase.

4. The method as claimed in claim 1, wherein the enzyme is added in an amount of 5-10U/mL, and the enzymatic hydrolysis is carried out at 40-50 ℃ under stirring at 200-400r/min for 1-3 h.

5. The method according to claim 4, wherein after the enzymatic reaction, the enzymatic hydrolysate is heated at 80-100 ℃ for 40-50min to terminate the reaction.

6. The method of claim 1, wherein the microfiltration is performed using a 0.1-10 μm microfiltration membrane.

7. The method of claim 1, wherein the ultrafiltration is performed at an operating pressure of 0.2 to 0.3MPa and a molecular weight cut-off of the ultrafiltration membrane is 2 to 12 KDa.

8. The method as claimed in claim 1, wherein the operating pressure of nanofiltration is 0.4-0.6MPa, and the molecular weight cut-off of the nanofiltration membrane is 400-1000 Da.

9. The method of claim 1, wherein the gel exclusion chromatography is performed using 0.08-0.12mol/LNH₄HCO₃The polyacrylamide Gel Bio Gel P-2 resin was washed and 1 set of samples were collected every 5-15min at a flow rate of 0.10-0.20mL/min for the crude oligo-chitosan oligosaccharide.

10. The method of claim 1, wherein the preparation method further comprises freeze-drying the oligo-chitosan oligosaccharide monomer.

Technical Field

The invention relates to a preparation method of an oligo-chitosan oligosaccharide monomer, belonging to the technical field of chitosan oligosaccharide preparation.

Background

Chitosan is a chitosan deacetylation derivative, the chitosan source is rich and has various physiological activities, but the chitosan has large molecular weight and is limited to be absorbed by human bodies, animals and plants, so the preparation of chitosan oligosaccharide with small molecular weight becomes a hot point of the neighborhood, and the preparation method of chitosan oligosaccharide mainly comprises a chemical method, a physical method and an enzymatic method. The chemical method has low cost and simple and convenient operation, but the molecular weight distribution of the chitosan oligosaccharide is difficult to control, the structure of the chitosan oligosaccharide is easy to damage, and the safety is greatly questioned. Physical method has little pollution and simpler operation, but the product has high polymerization degree and wider molecular weight distribution, and the single use effect is not good. The enzymatic method is a method for degrading chitosan by using specific enzyme or non-specific enzyme to obtain chitosan oligosaccharide, has the advantages of small pollution, mild reaction conditions and the like, but often causes too low purity of an extract due to too many magazines which cannot be processed in time, and the extracted chitosan oligosaccharide is not classified due to different molecular sizes, so that the product quality is too low. Therefore, how to prepare the oligomeric narrow-distribution chitosan oligosaccharide with uniform molecular weight and properties is an important problem in the preparation of the chitosan oligosaccharide.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of an oligo-chitosan oligosaccharide monomer. The method comprehensively uses an enzyme method, membrane separation and gel exclusion chromatography to separate and prepare the oligochitosan monomer, and the purity of the obtained oligochitosan monomer reaches more than 40 percent.

The first object of the present invention is to provide a method for preparing oligo-chitosan oligosaccharide monomer, comprising the following steps:

(1) dissolving chitosan, adding enzyme for hydrolysis to obtain chitosan hydrolysate;

(2) performing microfiltration and impurity removal on the chitosan hydrolysate obtained in the step (1), and performing ultrafiltration and nanofiltration treatment to obtain a crude product of the oligochitosan oligosaccharide;

(3) and (3) separating the crude product of the oligochitosan oligosaccharide obtained in the step (2) by gel exclusion chromatography, and combining the same components to obtain the oligochitosan oligosaccharide monomer.

Further, the dissolving is to add chitosan into water to be uniformly dispersed, and then add acetic acid buffer solution to dissolve.

Further, the dissolving is specifically to dissolve the chitosan by adding a small amount of water, then adding NaAc-HAc buffer solution with pH of 4-5 into each g of chitosan according to 40-60mL, and stirring for 4-6h at normal temperature under the condition of 600-700 r/min.

Further, the enzyme is one or more of chitosan hydrolase, cellulase, papain and lipase. Chitosan hydrolases are preferred.

Further, the addition amount of the enzyme is 5-10U/mL, and the enzymolysis reaction is stirred for 1-3h at 40-50 ℃ and 400 r/min.

Further, after the enzymolysis reaction, the enzymolysis liquid is heated at 80-100 ℃ for 40-50min to terminate the reaction.

Further, the microfiltration is carried out by adopting a microfiltration membrane with the diameter of 0.1-10 mu m. Preferably, the filtration is carried out by using filter paper, and then the filtration is carried out to remove impurities by using microfiltration membranes with the diameter of 0.45 μm and the diameter of 0.22 μm respectively.

Furthermore, the operating pressure of the ultrafiltration is 0.2-0.3MPa, and the molecular weight cut-off of the ultrafiltration membrane is 2-12 KDa. Preferably, a 10KDa ultrafiltration membrane is adopted for ultrafiltration, then a 3KDa ultrafiltration membrane is adopted for ultrafiltration, in the ultrafiltration process, the ultrafiltration constant volume water adding amount is 2 times of the volume of the stock solution, the operation temperature is 37 ℃, and no requirement is imposed on pH.

Furthermore, the operating pressure of the nanofiltration is 0.4-0.6MPa, and the molecular weight cut-off of the nanofiltration membrane is 400-1000 Da. Nanofiltration is preferably carried out by adopting a nanofiltration membrane of 500Da, the nanofiltration constant volume water addition amount is 4 times of the volume of the stock solution, the operation temperature is 37 ℃, and no requirement is imposed on the pH value.

Further, the gel exclusion chromatography is performed by adopting 0.08-0.12mol/LNH₄HCO₃Rinsing Polyacrylamide Gel Bio GelP-2 resin, collecting 1 group of samples of the crude oligochitosan oligosaccharide according to the flow rate of 0.10-0.20mL/min every 5-15 min.

Further, the preparation method also comprises freeze drying the oligo-chitosan oligosaccharide monomer.

The invention has the beneficial effects that:

the invention pretreats the chitosan solution, so that the chitosan solution is fully swelled and dispersed, the contact range of enzyme and substrate is enlarged, chitosan oligosaccharide products with concentrated distribution are obtained, and the generation of monosaccharide is greatly reduced. The process of preparing the chitosan oligosaccharide is combined with membrane separation, so that the active chitosan oligosaccharide is separated in time, and the further degradation of the chitosan oligosaccharide under the action of enzyme is effectively controlled. And (3) nanofiltration is carried out after ultrafiltration, and inactive oligosaccharide with low polymerization degree and a large amount of water are removed, so that a chitosan oligosaccharide product with high physiological activity and a certain polymerization degree range is obtained. And performing gel exclusion chromatography separation, separating and collecting the same components, and freeze-drying to obtain the chitosan oligosaccharide monomer, wherein the purity of the obtained chitosan oligosaccharide monomer reaches more than 40%.

Drawings

FIG. 1 is a TLC analysis diagram of a crude chitosan oligosaccharide product after nanofiltration, in which Std is a mixed standard, 1 is 2% enzymolysis liquid, 2 is a microfiltration membrane-permeated concentrated solution, 3 is a 10 KDa-permeated concentrated solution, and 4-6 is a 500 Da-intercepted concentrated solution;

FIG. 2 is an HPLC analysis chart of crude chitosan oligosaccharide after nanofiltration;

FIG. 3 is a TLC analysis chart of the oligo-chitosan oligosaccharide monomer after chromatographic separation, wherein 51-56 are the collection tube number of chitotetraose;

FIG. 4 is a HPLC analysis of chitotetraose monomers.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

TLC analysis: preparing a 1% solution from a chitosan oligosaccharide sample, sampling 2 mu L of the solution on a high-efficiency silica gel plate by using a capillary pipette, wherein the distance between each point is 0.5cm, the distance between each point and the bottom end is 1.0cm, and the system of the well-sampled silica gel plate as an expansion system is isopropanol: ammonia water: water 15: 7.5: 1, taking out the chromatographic cylinder after upward development, and drying by using an electric blower. Spraying 0.5% ninhydrin color-developing agent on silica gel plate, placing in oven at 100 deg.C for 5min, and heating for developing to obtain purple color.

HPLC analysis: chromatographic conditions are as follows: an agent 1260 high performance liquid chromatograph; a differential refractive detector; a ZoRBAXNH2 Analytical (4.6 mmID. times.250 mmL) column; column temperature: 30 ℃; the mobile phase is acetonitrile: water 7: 4; flow rate: 1.0 mL/min; sample introduction amount: 10 mu L of the solution; sample concentration: 5 percent. (Mixed standard: 20mg/mL)