Preparation method of chitosan oligosaccharide with narrow molecular weight distribution and high deacetylation degree

Abstract

The present invention provides a method for preparing chitosan oligosaccharides with a narrow molecular weight distribution and a high degree of deacetylation. In the present invention, hydrochloric acid, sulfuric acid, and acetic acid are used to prepare a mixed acid. Under the mixed acid and high temperature conditions, chitosan macromolecular chains in shrimp and crab shells can be broken, especially ether bonds connecting monomers. Since the present invention uses the mixed acid and high temperature to react, the reaction process is relatively intense. In the present invention, a polyol composed of butanediol, isopropyl alcohol, and n-octanol is added to alleviate the violent occurrence of the reaction and simultaneously improve the narrow distribution of product molecular weight. The chitosan oligosaccharide prepared by the present invention has a molecular weight distribution index of 1.005-1.15 and a degree of deacetylation of 93-99%.

Description

Technical Field

[0001] The invention relates to the technical field of chitosan oligosaccharide preparation, in particular to a method for preparing chitosan oligosaccharide with narrow molecular weight distribution and high deacetylation degree. Background Art

[0002] Chitosan oligosaccharides are low-molecular-weight, alkaline natural oligosaccharides composed of 2 to 10 glucosamines linked by β-1,4 glycosidic bonds. Chitosan oligosaccharides possess numerous important biological activities, including preventing and treating metabolic-related diabetes and hyperlipidemia, combating tumors, alleviating degenerative diseases such as Alzheimer's disease and osteoarthritis, and enhancing immunity. Chitosan oligosaccharides of varying degrees of polymerization have different absorption and distribution patterns in the human body, resulting in distinct functions.

[0003] Common methods for preparing chitosan oligosaccharides include enzymatic, chemical, and physical methods. Enzymes used in enzymatic methods can be divided into specific and nonspecific enzymes. Chitosanase and chitinase are specific enzymes, while cellulase, xylanase, α-amylase, pectinase, papain, β-glucosidase, lipase, and pepsin are nonspecific enzymes. Chemical methods use acids and oxidants to break down hydrogen bonds within or between polymer molecules, achieving degradation. Commonly used acids for the chemical conversion of chitin include hydrochloric acid, sulfuric acid, acetic acid, lactic acid, trichloroacetic acid, and formic acid, while commonly used oxidants include hydrogen peroxide, ozone, sodium perborate, and potassium persulfate. Physical methods use ultrasound, gamma rays, microwave radiation, or ball milling to break chemical bonds within chitin molecules, yielding molecules with varying degrees of polymerization.

[0004] Because the degradation degree of the above method is difficult to control, the chitosan oligosaccharide prepared has a wide molecular weight distribution and is difficult to separate to obtain a product with a narrow molecular weight distribution. Summary of the Invention

[0005] In view of this, the present invention proposes a method for preparing chitosan oligosaccharides with narrow molecular weight distribution and high deacetylation degree to solve the above problems.

[0006] The technical solution of the present invention is achieved as follows:

[0007] A method for preparing chitosan oligosaccharide with a narrow molecular weight distribution and a high degree of deacetylation comprises the following steps:

[0008] (1) heating and mixing the mixed polyol, adding shrimp and crab shell powder, and then adding mixed acid to prepare liquefied shrimp and crab shell;

[0009] (2) diluting and cleaning the liquefied shrimp and crab shells with an ethanol solution, vacuum filtering, removing the residue, and rotary evaporating the filtrate to obtain cleaned liquefied chitosan. The liquefied chitosan is added to water and preliminarily purified by filtration with a filter membrane. The resulting liquefied product is dissolved in water and then dropwise added to a sufficient amount of anhydrous ethanol to precipitate. The product is centrifuged and dried to obtain a crude chitosan oligosaccharide.

[0010] (3) The crude chitosan oligosaccharide is dissolved in a solvent, and the molecular weight range of the obtained liquefied product is quantified using a GPC instrument (standard sample is 500), and then the crude chitosan oligosaccharide is dissolved in pure water, first filtered with a nanofiltration membrane, and then separated using a dialysis bag, and freeze-dried to obtain a finished product.

[0011] Furthermore, in step (1), the mixed polyol comprises butanediol, isopropanol and n-octanol, and the mass ratio of the shrimp and crab shell powder, butanediol, isopropanol and n-octanol is 2-4:5-7:4-6:2-4.

[0012] Furthermore, in step (1), the mixed acid comprises sulfuric acid, hydrochloric acid and acetic acid in a mass ratio of 1-2.5:0.5-2:0.5-2, the mass ratio of the shrimp and crab shell powder to the mixed acid is 2.8-3.2:1.8-2.2, the mass concentration of the sulfuric acid is 70-98.5%, the mass concentration of the hydrochloric acid is 30-38%, and the mass concentration of the acetic acid is 2-10%.

[0013] Furthermore, in step (1), the mixed polyol is heated to 100-180° C., shrimp and crab shell powder is added and the reaction time is 5-30 minutes, and then the mixed acid is added and the reaction time is 0.5-4 hours.

[0014] Furthermore, in step (2), the concentration of the ethanol solution is 98-100%, and the mass ratio of the liquefied chitosan to the ethanol solution is 1:0.5-10.

[0015] Furthermore, in step (2), the liquefied crab shells are added to water to obtain a liquefied shrimp and crab shell suspension with a concentration of 10-30%, centrifuged, and the supernatant is collected. The supernatant is added to anhydrous ethanol for precipitation to obtain a solid, and the solid is washed with anhydrous ethanol to obtain a crude chitosan oligosaccharide.

[0016] Furthermore, in step (2), the filter membrane size is 0.25 micrometers and the drying temperature can be 30-70°C.

[0017] Furthermore, in step (3), the molecular weight cut-off of the dialysis bag is 900-2000, and the solvent used in the GPC measurement can be water or DMF.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention adopts hydrochloric acid, sulfuric acid and acetic acid to prepare a mixed acid. Under the conditions of mixed acid and high temperature, the chitin macromolecular chains in shrimp and crab shells can be broken, especially the ether bonds connecting monomers. Since the present invention adopts mixed acid and high temperature to react, the reaction process is relatively violent. The addition of a polyol composed of butanediol, isopropyl alcohol and n-octanol can alleviate the violent occurrence of the reaction and improve the narrow distribution of the product molecular weight. During the reaction process, the amide group will also undergo acidolysis. The carbon atoms on the amide group will be attacked by the lone electron pairs of the oxygen atoms on the polyol, destroying the amide bond and converting it into an amino group under the action of the acid. The chitosan oligosaccharide prepared by the preparation method of the present invention has the advantages of narrow molecular weight distribution and high deacetylation degree. The molecular weight distribution index of the chitosan oligosaccharide prepared by the present invention is 1.005-1.15, and the deacetylation degree is 93-99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 C NMR spectrum of chitosan oligosaccharide prepared in Example 1

[0021] Figure 2 H NMR spectrum of chitosan oligosaccharide prepared in Example 1 DETAILED DESCRIPTION

[0022] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0023] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0024] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0025] Example 1

[0026] A method for preparing chitosan oligosaccharide with a narrow molecular weight distribution and a high degree of deacetylation comprises the following steps:

[0027] (1) Butanediol, isopropanol and n-octanol are prepared into a mixed polyol, the mixed polyol is poured into a three-necked flask, and the mixture is heated to reflux in an oil bath pot under nitrogen protection, and the temperature is raised to 100° C., shrimp and crab shell powder is added and reacted for 30 minutes, the mass ratio of shrimp and crab shell powder, butanediol, isopropanol and n-octanol is 3:6:5:3, and then a mixed acid prepared by 70wt% sulfuric acid, 30wt% hydrochloric acid and 2wt% acetic acid in a mass ratio of 2:1:1 is added and reacted for 4 hours, the mass ratio of shrimp and crab shell powder to mixed acid is 3:2, and liquefied shrimp and crab shell is obtained;

[0028] (2) The liquefied shrimp and crab shells were washed with a 98 wt% ethanol solution with a mass ratio of 1:1, vacuum filtered to remove the residue, and the filtrate was rotary evaporated to obtain washed liquefied chitin. The liquefied shrimp and crab shells were added to water to obtain a liquefied shrimp and crab shell liquid with a concentration of 20% by mass. After that, it was preliminarily purified with a filter membrane, and the supernatant was dropped into anhydrous ethanol to obtain a precipitate. The solid was filtered to obtain a solid, and then washed with anhydrous ethanol three to four times and dried at 40°C to obtain a crude chitosan oligosaccharide.

[0029] (3) The crude chitosan oligosaccharide and distilled water were mixed and filtered using a 0.25 micron filter membrane, and the filtrate was collected. The filtrate was separated using a dialysis bag with a molecular weight of 1700, and freeze-dried to obtain a finished product.

[0030] Example 2

[0031] A method for preparing chitosan oligosaccharide with a narrow molecular weight distribution and a high degree of deacetylation comprises the following steps:

[0032] (1) Butanediol, isopropanol and n-octanol are prepared into a mixed polyol, the mixed polyol is poured into a three-necked flask, and the mixture is heated to reflux in an oil bath pot under nitrogen protection, and the temperature is raised to 180° C., shrimp and crab shell powder is added and reacted for 5 minutes, the mass ratio of shrimp and crab shell powder, butanediol, isopropanol and n-octanol is 2:5:4:2, and then a mixed acid prepared by 98.5wt% sulfuric acid, 38wt% hydrochloric acid and 10wt% acetic acid in a mass ratio of 2:1:1 is added and reacted for 1 hour, the mass ratio of shrimp and crab shell powder and mixed acid is 2.8-3.2:1.8-2.2, to obtain liquefied shrimp and crab shells;

[0033] (2) washing the liquefied shrimp and crab shells with a 98 wt% ethanol solution at a mass ratio of 1:2, vacuum filtering to remove the residue, and rotary evaporating the filtrate to obtain washed liquefied chitosan. The liquefied chitosan was added to water to obtain a liquefied product with a concentration of 20% by mass, and the product was filtered with a filter membrane. The supernatant was added dropwise into anhydrous ethanol under stirring to obtain a precipitate, and the solid was filtered to obtain a solid. The solid was then rinsed with anhydrous ethanol three to four times and then dried at 30° C. to obtain a crude chitosan oligosaccharide.

[0034] (3) The crude chitosan oligosaccharide and distilled water were mixed and filtered using a 0.25 micron filter membrane, and the filtrate was collected. The filtrate was separated using a dialysis bag with a molecular weight of 900, and freeze-dried to obtain a finished product.

[0035] Example 3

[0036] A method for preparing chitosan oligosaccharide with a narrow molecular weight distribution and a high degree of deacetylation comprises the following steps:

[0037] (1) Butanediol, isopropanol and n-octanol are prepared into a mixed polyol, the mixed polyol is poured into a three-necked flask, heated to reflux in an oil bath, protected by nitrogen, and heated to 140° C., shrimp and crab shell powder is added and reacted for 20 minutes, the mass ratio of shrimp and crab shell powder, butanediol, isopropanol and n-octanol is 4:7:6:4, and then a mixed acid prepared by 80wt% sulfuric acid, 35wt% hydrochloric acid and 8wt% acetic acid in a mass ratio of 2:1.5:0.5 is added and reacted for 2 hours, the mass ratio of shrimp and crab shell powder to mixed acid is 2.8:1.8, to obtain liquefied shrimp and crab shell;

[0038] (2) washing the liquefied shrimp and crab shell powder with a 98 wt% ethanol solution at a mass ratio of 1:5, vacuum filtering to remove the residue, and rotary evaporating the filtrate to obtain washed liquefied chitosan. The liquefied chitosan was added to water to obtain a liquefied chitosan solution with a concentration of 10% by mass. After filtering with a filter membrane, the supernatant was dropped into anhydrous ethanol under stirring to obtain a precipitate, and the solid was filtered to obtain a solid. The solid was then rinsed with anhydrous ethanol three to four times and then dried at 50° C. to obtain a crude chitosan oligosaccharide.

[0039] (3) The crude chitosan oligosaccharide and distilled water were mixed and filtered using a 0.25 micron filter membrane, and the filtrate was collected. The filtrate was separated using a dialysis bag with a molecular weight of 1600, and freeze-dried to obtain a finished product.

[0040] Example 4

[0041] A method for preparing chitosan oligosaccharide with a narrow molecular weight distribution and a high degree of deacetylation comprises the following steps:

[0042] (1) Butanediol, isopropanol and n-octanol are prepared into a mixed polyol, the mixed polyol is poured into a three-necked flask, heated to reflux in an oil bath, protected by nitrogen, and heated to 150° C., shrimp and crab shell powder is added and reacted for 30 minutes, the mass ratio of shrimp and crab shell powder, butanediol, isopropanol and n-octanol is 3:6:5:3, and then a mixed acid prepared by 70wt% sulfuric acid, 30wt% hydrochloric acid and 2wt% acetic acid in a mass ratio of 3:0.5:0.5 is added and reacted for 2 hours, the mass ratio of shrimp and crab shell powder to mixed acid is 3:2, and liquefied shrimp and crab shell powder is obtained;

[0043] (2) washing the liquefied shrimp and crab shells with a 98 wt% ethanol solution at a mass ratio of 1:1, vacuum filtering to remove the residue, and rotary evaporating the filtrate to obtain washed liquefied chitosan. The liquefied chitosan was added to water to obtain a liquefied chitosan solution with a concentration of 20% by mass, and filtered through a filter membrane. The supernatant was added dropwise into anhydrous ethanol under stirring to obtain a precipitate, and the solid was filtered to obtain a solid. The solid was then rinsed with anhydrous ethanol three to four times and dried at 40° C. to obtain a crude chitosan oligosaccharide.

[0044] (3) The crude chitosan oligosaccharide and distilled water were mixed and filtered using a 0.25 micron filter membrane, and the filtrate was collected. The filtrate was separated using a dialysis bag with a molecular weight of 1700, and freeze-dried to obtain a finished product.

[0045] Test Example 1

[0046] The finished product of Example 1 was analyzed and tested using NMR carbon spectroscopy, H spectroscopy and GPC.

[0047] See also Figure 1-2 The chitosan oligosaccharide prepared in Example 1 was chitosan undecanose in which no -C=O group signal was detected, indicating that the prepared chitosan oligosaccharide had a high degree of acetylation. Based on the H-NMR spectrum data, the characteristic peak of -CH3 at around 2 ppm and the hydrogen signal at 3.1 ppm was the hydrogen signal on the carbon attached to -NH2. Therefore, the degree of deacetylation can be calculated based on these two characteristic peak signals. The prepared chitosan oligosaccharide has a degree of deacetylation of more than 97%.

[0048] In order to characterize the molecular weight and molecular weight distribution of the prepared chitosan oligosaccharide, GPC test was performed on the sample prepared in Example 1. The molecular weight of the chitosan oligosaccharide unit is used as the basis, i.e. 161, and the weight average molecular weight of the measured sample is used for calculation. If the influence of the residual acetamide group is taken into account, the number of repeating units is about 11. The molecular weight dispersion (PDI) of the obtained material is 1.15.

[0049]

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing chitosan oligosaccharide with a narrow molecular weight distribution and a high degree of deacetylation, characterized in that: The following steps are involved: (1) heating the mixed polyol to 100-180° C., adding shrimp and crab shell powder and reacting for 5-30 minutes, and then adding mixed acid and reacting for 0.5-4 hours to obtain liquefied crab shell; The mixed polyol is composed of butanediol, isopropanol and n-octanol, and the mass ratio of the shrimp and crab shell powder, butanediol, isopropanol and n-octanol is 2-4:5-7:4-6:2-4; The mixed acid is composed of sulfuric acid, hydrochloric acid and acetic acid in a mass ratio of 1-2.5:0.5-2:0.5-2, the mass ratio of the shrimp and crab shell powder to the mixed acid is 2.8-3.2:1.8-2.2, the mass concentration of the sulfuric acid is 70-98.5%, the mass concentration of the hydrochloric acid is 30-38%, and the mass concentration of the acetic acid is 2-10%; (2) washing the liquefied shrimp and crab shells with an ethanol solution, vacuum filtering to remove the residue, and rotary evaporating the filtrate to obtain washed liquefied chitosan. The liquefied chitosan was added to water, centrifuged, and the supernatant was taken. The supernatant was added to anhydrous ethanol and purified to obtain crude chitosan oligosaccharide. (3) The crude chitosan oligosaccharide was filtered through a 0.25 μm filter membrane, and the filtrate was collected. The filtrate was separated using a dialysis bag with a molecular weight cut-off of 900-2000, and freeze-dried to obtain a finished product.

2. the preparation method of the chitosan oligosaccharide with narrow molecular weight distribution high deacetylation degree as claimed in claim 1, is characterized in that: In step (2), the mass concentration of the ethanol solution is 98-100%, and the mass ratio of the liquefied chitosan to the ethanol solution is 1:0.5-10.

3. the preparation method of the chitosan oligosaccharide with narrow molecular weight distribution high deacetylation degree as claimed in claim 1, is characterized in that: In step (2), liquefied chitosan is added to water to obtain a liquefied chitosan suspension with a concentration of 10-30%, centrifuged, and the supernatant is collected. The supernatant is added to anhydrous ethanol for precipitation to obtain a solid, and the solid is washed with anhydrous ethanol to obtain a crude chitosan oligosaccharide.

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