1. A method for preparing chitosan oligosaccharide by a membrane separation process is characterized by comprising the following steps: the method specifically comprises the following steps:

s1, selecting a proper amount of animal shell raw materials, and cleaning, wherein the animal shell comprises shrimp shells and crab shells;

s2, decalcifying, deproteinizing and deacetylating the animal shell raw material;

s3, carrying out enzymolysis treatment on the raw material treated in the step S2 to obtain a stock solution;

s4, introducing the stock solution into an ultrafiltration membrane system, and performing ultrafiltration membrane filtration treatment to obtain a treatment solution;

s5, introducing the treatment solution into a nanofiltration membrane system, filtering by using a nanofiltration membrane to obtain concentrated solution and clear solution, introducing the clear solution into a reverse osmosis membrane system, further filtering, and recycling;

s6, drying the concentrated solution obtained in the step S5.

2. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 1, wherein: the decalcification processing in step S2 specifically includes the following steps: soaking the raw materials in 1-5% HCl solution at 10-40 deg.C for 5-15 hr until no bubbles are generated, taking out, and cleaning.

3. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 1, wherein: the deproteinization processing in step S2 specifically includes the following steps: soaking the decalcified raw materials in 2-5% NaOH solution at 60-90 deg.C for 3-8 hr, and cleaning.

4. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 1, wherein: the deacetylation treatment in the step S2 specifically includes the following steps: putting the deproteinized raw material into 32-40% NaOH solution for 10-15h, soaking at 70-90 deg.C, taking out, and cleaning.

5. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 1, wherein: the enzymolysis treatment in the step S3 specifically includes the following steps: soaking the raw materials in acetic acid solution, adding degrading enzyme, adjusting pH to 3.0-7.0, and reacting at 15-60 deg.C until the raw materials are completely dissolved.

6. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 5, wherein: the degrading enzyme comprises any one of chitinase, cellulase, bromelain, lysozyme, lipase and chitosanase or a compound thereof, and the weight of the degrading enzyme is 0.5-3.0% of that of the raw material.

7. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 1, wherein: the ultrafiltration membrane adopted in the ultrafiltration membrane filtration treatment in the step S4 is a spiral organic membrane or a plate type membrane with the cutting molecular weight of 1000-10000D, the treatment temperature is 5-45 ℃, and the treatment pressure is 3-10 bar.

8. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 1, wherein: the cutting molecular weight of the nanofiltration membrane adopted in the nanofiltration membrane filtration treatment in the step S5 is 150-500D, the treatment temperature is 5-45 ℃, the treatment pressure is 10-30bar, the cutting molecular weight of the reverse osmosis membrane system is less than 100D, the treatment temperature of the reverse osmosis membrane filtration is 5-45 ℃, and the treatment pressure is 10-30 bar.

9. The method for preparing chitosan oligosaccharide by membrane separation process according to claim 1, wherein: the drying process in step S6 is a spray drying process.

Background

Chitosan oligosaccharide, also called chitosan oligosaccharide and oligochitosan, is a micromolecule oligosaccharide with amino groups, which is degraded by chitosan from shrimp and crab shells, is a sugar chain with the polymerization degree of 2-20, has the molecular weight of less than or equal to 3200Da, and is a low molecular weight product with good water solubility, large functional effect and high biological activity. It has high solubility, water solubility and easy absorption by organism. The chitosan oligosaccharide is the only cationic basic amino oligosaccharide with positive charges in the nature, and is animal cellulose. The preparation method of the chitosan oligosaccharide comprises a physical degradation method (an ultrasonic degradation method and a gamma-ray radiation degradation method), a glycosyl transfer reaction, a chemical method (an acid method hydrolysis method and an oxidation method), an enzymatic method (a specific enzyme degradation method and a non-specific enzyme degradation method) and the like. The conventional method is an enzymatic hydrolysis method, but the solution after enzymatic hydrolysis contains a large amount of other components besides chitosan oligosaccharide, which affects the purity of the product and still needs further filtration and purification, but the conventional filtration process is complex in flow or poor in treatment effect.

Disclosure of Invention

The invention aims to provide a method for preparing chitosan oligosaccharide by a membrane separation process, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a method for preparing chitosan oligosaccharide by a membrane separation process specifically comprises the following steps:

s1, selecting a proper amount of animal shell raw materials, and cleaning, wherein the animal shell comprises shrimp shells and crab shells;

s2, decalcifying, deproteinizing and deacetylating the animal shell raw material;

s3, carrying out enzymolysis treatment on the raw material treated in the step S2 to obtain a stock solution;

s4, introducing the stock solution into an ultrafiltration membrane system, and performing ultrafiltration membrane filtration treatment to obtain a treatment solution;

s5, introducing the treatment solution into a nanofiltration membrane system, filtering by using a nanofiltration membrane to obtain concentrated solution and clear solution, introducing the clear solution into a reverse osmosis membrane system, further filtering, and recycling;

s6, drying the concentrated solution obtained in the step S5.

Preferably, the decalcification process in step S2 specifically includes the following steps: soaking the raw materials in 1-5% HCl solution at 10-40 deg.C for 5-15 hr until no bubbles are generated, taking out, and cleaning.

Preferably, the deproteinization process in step S2 specifically includes the following steps: soaking the decalcified raw materials in 2-5% NaOH solution at 60-90 deg.C for 3-8 hr, and cleaning.

Preferably, the deacetylation treatment in step S2 specifically includes the following steps: putting the deproteinized raw material into 32-40% NaOH solution for 10-15h, soaking at 70-90 deg.C, taking out, and cleaning.

Preferably, the enzymolysis treatment in step S3 specifically includes the following steps: soaking the raw materials in acetic acid solution, adding degrading enzyme, adjusting pH to 3.0-7.0, and reacting at 15-60 deg.C until the raw materials are completely dissolved.

Preferably, the degrading enzyme comprises any one of chitinase, cellulase, bromelain, lysozyme, lipase and chitosanase or a compound thereof, and the weight of the degrading enzyme is 0.5-3.0% of that of the raw material.

Preferably, the ultrafiltration membrane used in the ultrafiltration membrane filtration treatment in the step S4 is a spiral organic membrane or a plate membrane with a cut molecular weight of 1000-10000D, the treatment temperature is 5-45 ℃, and the treatment pressure is 3-10 bar.

Preferably, the cutting molecular weight of the nanofiltration membrane adopted in the nanofiltration membrane filtration treatment in the step S5 is 150-500D, the treatment temperature is 5-45 ℃, the treatment pressure is 10-30bar, the cutting molecular weight of the reverse osmosis membrane system is less than 100D, the treatment temperature of the reverse osmosis membrane filtration is 5-45 ℃, and the treatment pressure is 10-30 bar.

Preferably, the drying process in step S6 is a spray drying process.

Compared with the prior art, the invention has the beneficial effects that:

1) high yield, high purity: clear and bright clear solution of the ultrafiltration membrane can remove all insoluble impurities and most macromolecular substances; partial monosaccharide, disaccharide and ash are further removed through a nanofiltration membrane, and the product purity is improved;

2) and (3) recycling produced water: the clear liquid of the nanofiltration membrane is recycled to the production line by a reverse osmosis membrane, so that the water consumption is greatly reduced, and the environmental protection pressure is also reduced;

3) the product quality is high: the membrane concentration section has no phase change, no additive, no chemical change and high product purity;

4) the whole process is simple: the production line has small occupied area, easy operation, low investment cost and low running cost;

5) clean production: the fully-closed pipeline type operation is realized, the external environment is clean, and the FDA production specification requirement is completely met;

6) the system stability and recovery are good: can realize long-term stable continuous industrial production, and has good system recoverability.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

Example one

The invention provides a technical scheme that: a method for preparing chitosan oligosaccharide by a membrane separation process specifically comprises the following steps:

s1, selecting a proper amount of animal shell raw materials, and cleaning, wherein the animal shell comprises shrimp shells and crab shells, and the animal shell with higher chitin content is selected as the raw material;

s2, decalcifying, deproteinizing and deacetylating the animal shell raw material;

s3, carrying out enzymolysis treatment on the raw material treated in the step S2 to obtain a stock solution;

s4, introducing the stock solution into an ultrafiltration membrane system, and performing ultrafiltration membrane filtration treatment to obtain a treatment solution;

s5, introducing the treatment solution into a nanofiltration membrane system, filtering by using a nanofiltration membrane to obtain concentrated solution and clear solution, introducing the clear solution into a reverse osmosis membrane system, further filtering, and recycling;

s6, drying the concentrated solution obtained in the step S5.

The decalcification processing in step S2 specifically includes the following steps: soaking the raw materials in 1% HCl solution at 10 deg.C for 5h until no bubbles are generated, taking out, and cleaning.

The deproteinization processing in step S2 specifically includes the following steps: soaking the decalcified raw materials in 2% NaOH solution at 60 deg.C for 3 hr, taking out, and cleaning to obtain decalcified and deproteinized raw materials containing chitin as main ingredient.

The deacetylation treatment in the step S2 specifically includes the following steps: putting the deproteinized raw materials into 32% NaOH solution for 10h, soaking at 70 deg.C, taking out the raw materials, cleaning, reacting chitin in alkali liquor at a certain temperature, and deacetylating to obtain chitosan.

The enzymolysis treatment in the step S3 specifically includes the following steps: soaking the raw materials in acetic acid solution, adding degrading enzyme into the solution, adjusting pH to 7.0, reacting at 15 deg.C until the raw materials are completely dissolved, and performing enzymolysis on chitosan to obtain mixed solution containing chitosan oligosaccharide, solid impurities, macromolecular protein, etc.

The enzyme for degradation comprises any one of chitinase, cellulase, bromelain, lysozyme, lipase and chitosanase or a compound thereof, and the weight of the enzyme for degradation is 0.5 percent of the weight of the raw material.

The ultrafiltration membrane used in the ultrafiltration membrane filtration treatment in the step S4 is a spiral organic membrane or a plate membrane with cut molecular weight of 10000D, the treatment temperature is 5 ℃, the treatment pressure is 3bar, chitosan oligosaccharide and other small molecular substances in the mixed solution permeate the ultrafiltration membrane to form a treatment solution, and insoluble solid substances, macromolecular proteins and the like are trapped in the ultrafiltration membrane concentrated solution.

The cutting molecular weight of the nanofiltration membrane adopted in the nanofiltration membrane filtration treatment in the step S5 is 500D, the treatment temperature is 5 ℃, the treatment pressure is 10bar, part of monosaccharide, disaccharide, ash and a large amount of water in the treatment solution penetrate through the nanofiltration membrane to form clear solution, and chitosan oligosaccharide is trapped in the nanofiltration membrane concentrated solution; the cutting molecular weight of the reverse osmosis membrane system is less than 100D, the treatment temperature of reverse osmosis membrane filtration is 5 ℃, the treatment pressure is 10bar, the clear liquid of the nanofiltration membrane enters the reverse osmosis membrane system, almost all impurities except water are intercepted in the concentrated liquid of the reverse osmosis membrane, the clear liquid of the reverse osmosis membrane is mainly water, the clear liquid of the reverse osmosis membrane can be recycled in a production line for continuous use, and the production cost is reduced.

The drying treatment in the step S6 is a spray drying method, and chitosan oligosaccharide finished product powder is obtained.

Example two

The difference compared with the first embodiment is that: the decalcification processing in step S2 specifically includes the following steps: soaking the raw materials in 3% HCl solution at 20 deg.C for 10h until no bubbles are generated, taking out, and cleaning;

the deproteinization processing in step S2 specifically includes the following steps: immersing the decalcified raw materials into a 3% NaOH solution for 5h, wherein the immersion treatment temperature is 70 ℃, and then taking out the raw materials and cleaning;

the deacetylation treatment in the step S2 specifically includes the following steps: putting the deproteinized raw material into 35% NaOH solution for 13h, soaking at 80 ℃, and then taking out the raw material and cleaning;

the pH value of the enzymolysis treatment in the step S3 is 5.0, and the reaction is carried out at the temperature of 40 ℃ until the raw materials are completely dissolved;

the weight of the degrading enzyme is 2.0 percent of the weight of the raw materials;

the ultrafiltration membrane adopted in the ultrafiltration membrane filtration treatment in the step S4 is a spiral organic membrane or a plate type membrane with the cut molecular weight of 5000D, the treatment temperature is 30 ℃, and the treatment pressure is 5 bar;

the cutting molecular weight of the nanofiltration membrane adopted in the nanofiltration membrane filtration treatment in the step S5 is 300D, the treatment temperature is 30 ℃, the treatment pressure is 20bar, the cutting molecular weight of the reverse osmosis membrane system is less than 100D, the treatment temperature of the reverse osmosis membrane filtration is 30 ℃, and the treatment pressure is 20 bar.

EXAMPLE III

The difference compared with the first embodiment is that: the decalcification processing in step S2 specifically includes the following steps: soaking the raw materials in 5% HCl solution at 40 deg.C for 15h until no bubbles are generated, taking out, and cleaning;

the deproteinization processing in step S2 specifically includes the following steps: immersing the decalcified raw materials in a 5% NaOH solution for 8h, wherein the immersion treatment temperature is 90 ℃, and then taking out the raw materials and cleaning;

the deacetylation treatment in the step S2 specifically includes the following steps: putting the deproteinized raw material into a 40% NaOH solution for 15h, soaking at 90 ℃, and then taking out the raw material and cleaning;

the pH value of the enzymolysis treatment in the step S3 is 3.0, and the reaction is carried out at the temperature of 60 ℃ until the raw materials are completely dissolved;

the weight of the degrading enzyme is 3.0 percent of the weight of the raw materials;

the ultrafiltration membrane adopted in the ultrafiltration membrane filtration treatment in the step S4 is a roll-type organic membrane or a plate-type membrane with the cut molecular weight of 1000D, the treatment temperature is 45 ℃, and the treatment pressure is 10 bar;

the cutting molecular weight of the nanofiltration membrane adopted in the nanofiltration membrane filtration treatment in the step S5 is 150D, the treatment temperature is 45 ℃, the treatment pressure is 30bar, the cutting molecular weight of the reverse osmosis membrane system is less than 100D, the treatment temperature of the reverse osmosis membrane filtration is 45 ℃, and the treatment pressure is 30 bar.

Examples of the experiments

The content of chitosan oligosaccharide in the finished powder is measured according to the following steps:

1) respectively adding 0, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0ml of chitosan oligosaccharide standard stock solution with the concentration of 4.00mg/ml into a colorimetric tube, then adding 100 mu l of phenol aqueous solution, fixing the volume by distilled water, shaking up, preparing to obtain chitosan oligosaccharide standard solutions with the concentration gradients of 0, 0.40, 0.80, 1.20, 1.60, 2.00 and 2.40mg/ml, measuring the fluorescence intensity F value on a fluorescence spectrophotometer, and establishing a linear relation curve between lnF and the chitosan oligosaccharide concentration X;

2) respectively taking three parts of chitosan oligosaccharide powder prepared in the first to third embodiments in equal amount, adding distilled water in equal amount, and mixing to obtain chitosan oligosaccharide solution with concentration of 3-5 mg/ml;

3) taking 2-6ml of the sample solution prepared in the step 2), measuring according to the detection method in the step 1), recording the fluorescence intensity F value, calculating lnF value, and substituting the value into a linear regression equation to obtain the sample concentration, wherein the result is shown in the following table:

as can be seen from the above table, the content of chitosan oligosaccharide in the finished powder prepared by the embodiments of the invention is stabilized at more than 90%, and the product purity is very high.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.