A fully deacetylated chitosan oligosaccharide, and a preparation method and application thereof

Abstract

This invention belongs to the field of biomedicine and discloses a fully deacetylated chitosan oligosaccharide, its preparation method, and its applications. The preparation method involves first mixing a 0.1%-0.2% chitosan oligosaccharide solution with a hydrogen-form strong acid ion exchange resin at a volume ratio of 1:(0.5-1), and stirring for adsorption for 5-10 hours. Then, a dilute hydrochloric acid solution is added, and the mixture is heated at 60-80°C for 5-10 hours. Next, ammonia water is added, and the mixture is stirred for desorption for 2-5 hours. Finally, the resulting eluent is freeze-dried to obtain the fully deacetylated chitosan oligosaccharide. This preparation method is simple to operate, uses low-cost raw materials, and eliminates the need for high-concentration inorganic acids as catalysts, reducing experimental risks and equipment wear. It is a safe, efficient, and environmentally friendly preparation method. The fully deacetylated chitosan oligosaccharide obtained using this method has a high yield and can be widely used in antibacterial, preservative, and antioxidant applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a fully deacetylated chitosan oligosaccharide, its preparation method, and its application. Background Technology

[0002] Chitosan oligosaccharides, also known as chitosan oligosaccharides or chitosan oligomers, are degradation products of chitosan. They are low molecular weight products with good water solubility, significant functional effects, and high biological activity. Chitosan oligosaccharides possess various biological activities, such as anti-tumor, antioxidant, antibacterial, and immunomodulatory effects.

[0003] However, previous literature reports have all used chitosan with different degrees of deacetylation as raw materials, employing enzymatic hydrolysis, oxidative degradation, or concentrated inorganic acid-catalyzed hydrolysis to prepare chitosan oligosaccharides. Enzymatic hydrolysis has been widely used to prepare chitosan oligosaccharides with different degrees of deacetylation, but the resulting product is a complex mixture of chitosan oligosaccharides with varying degrees of polymerization and residual N-acetyl groups, resulting in low purity. Oxidative degradation suffers from the loss of amino groups due to oxidation and has been phased out. While concentrated inorganic acid-catalyzed hydrolysis can yield fully deacetylated chitosan oligosaccharides, it requires concentrated inorganic acids as catalysts, easily causing equipment damage and environmental risks, and the process is outdated with low yields. Therefore, how to efficiently, environmentally friendly, and safely prepare fully deacetylated chitosan oligosaccharides has become a crucial issue. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fully deacetylated chitosan oligosaccharide, its preparation method, and its application. Specifically, the following technical solution is adopted:

[0005] A method for preparing fully deacetylated chitosan oligosaccharide includes the following steps:

[0006] S1. Add the hydrogen-form strong acid ion exchange resin to a chitosan oligosaccharide solution with a concentration of 0.1%-0.2%, and the volume ratio of the chitosan oligosaccharide solution to the hydrogen-form strong acid ion exchange resin is 1:(0.5-1). Stir and adsorb for 5-10 hours.

[0007] S2. Take out the hydrogen-form strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in S1 and wash it to remove impurities. Then add dilute hydrochloric acid solution and heat it at 60℃-80℃ for 5h-10h.

[0008] S3. Filter out the hydrogen-form strong acid ion exchange resin that has adsorbed fully deacetylated chitosan oligosaccharide obtained in S2, wash it, add ammonia water, stir and desorb for 2-5 hours, and finally freeze-dry the obtained eluent to obtain fully deacetylated chitosan oligosaccharide with a degree of deacetylation >99.5%.

[0009] This invention sets the chitosan oligosaccharide solution to a suitable concentration (0.1%-0.2%) and sets the volume ratio of the chitosan oligosaccharide solution to a hydrogen-form strong acid ion exchange resin to 1 mL:(0.5-1) mL. This ensures that the number of sulfonic acid groups in the hydrogen-form strong acid ion exchange resin is much higher than the number of amino and N-acetylamino groups in the chitosan oligosaccharide to be adsorbed. This allows the hydrogen-form strong acid ion exchange resin to act as both an ion exchange adsorbent for chitosan oligosaccharide and a catalyst for its deacetylation. As an ion exchange adsorbent, this preparation method ensures that the resin completes the adsorption of chitosan oligosaccharide in a short time (5h-10h). As a catalyst, the adsorbed chitosan oligosaccharide molecules have a large number of sulfonic acid groups in their microenvironment, which can efficiently catalyze the deacetylation of N-acetylamino groups under suitable temperature conditions (60℃-80℃) to obtain fully deacetylated chitosan oligosaccharide. Furthermore, this invention employs a hydrogen-form strong acid ion exchange resin as both an ion exchange adsorbent and a catalyst, instead of using high-concentration inorganic acids that can only act as catalysts. This reduces process risks and equipment wear. The hydrogen-form strong acid ion exchange resin is not only inexpensive, but also, under suitable temperature conditions (60℃-80℃; too low a temperature reduces catalytic efficiency, while too high a temperature accelerates resin aging and deterioration), it can complete the deacetylation of chitosan oligosaccharides in a relatively short time (5h-10h). Moreover, the resin can be reused after regeneration. In summary, the preparation method of this invention has advantages such as safety, high efficiency, greenness, and environmental friendliness, and can be widely applied in the biomedical field.

[0010] Preferably, the hydrogen-form strong acid ion exchange resin has a particle size of 40-400 mesh. If the particle size is too small, the filtration efficiency will be reduced, while if the particle size is too large, the ion exchange adsorption rate will be reduced. More preferably, the hydrogen-form strong acid ion exchange resin is a macroporous strong acid ion exchange resin with a particle size of 100-200 mesh, which can effectively balance the filtration efficiency and the ion exchange adsorption rate.

[0011] Preferably, in step S1, the degree of deacetylation of chitosan in the chitosan oligosaccharide solution is 55%-99%. The preparation method of this invention uses chitosan oligosaccharide as a raw material, which is obtained through chitosanase-catalyzed hydrolysis. If the deacetylation is too low, it will affect complete deacetylation; if the degree of deacetylation is greater than 99%, the chitosan oligosaccharide raw material is difficult to obtain. More preferably, in step S1, the degree of deacetylation of chitosan in the chitosan oligosaccharide solution is 90%-99%, which is high-degree-of-deacetylation chitosan oligosaccharide.

[0012] Preferably, the concentration of the dilute hydrochloric acid solution is 0.001 mol / L to 0.01 mol / L, and the concentration of the ammonia solution is 0.5 mol / L. Adding the 0.001 mol / L to 0.01 mol / L dilute hydrochloric acid solution (pH range between 2 and 3) to the hydrogen-form strong acid ion exchange resin (after impurity removal) at a volume ratio of (0.5-1) mL:1 mL allows for complete dissociation of the amino groups on the chitosan oligosaccharide molecules, resulting in tighter adsorption onto the hydrogen-form strong acid ion exchange resin. After deacetylation of the chitosan oligosaccharide, washing removes the dilute hydrochloric acid and the acetate ions generated during the deacetylation reaction. Then, 0.5 mol / L ammonia solution is added to the hydrogen-form strong acid ion exchange resin (which adsorbs fully deacetylated chitosan oligosaccharide) at a volume ratio of (0.5-1) mL:1 mL for desorption.

[0013] Preferably, desorption is carried out at a temperature of 15℃-25℃. When the temperature is too high, the Maillard reaction between chitosan oligosaccharide and ammonia is likely to occur; while when the temperature is too low, additional refrigeration is required, which increases production costs.

[0014] The present invention also provides a fully deacetylated chitosan oligosaccharide prepared by the above method and its application. The fully deacetylated chitosan oligosaccharide has a high yield and can be widely used in the preparation of antitumor or immunomodulatory drugs, antibacterial, antiseptic and antioxidant applications.

[0015] The beneficial effects of this invention are as follows: This invention uses a hydrogen-form strong acid ion exchange resin catalysis method to prepare fully deacetylated chitosan oligosaccharides. This preparation method is simple to operate, uses low-cost raw materials, and does not require the use of high-concentration inorganic acids as catalysts, reducing experimental risks and equipment wear. It can efficiently undergo ion exchange reactions with chitosan oligosaccharides, making it a safe, efficient, green, and environmentally friendly preparation method. The fully deacetylated chitosan oligosaccharides obtained using this preparation method have a high yield and can be widely used in the preparation of antitumor or immunomodulatory drugs, as well as in antibacterial, antiseptic, and antioxidant applications. Detailed Implementation

[0016] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, solution and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0017] Example 1

[0018] A fully deacetylated chitosan oligosaccharide, the preparation method of which includes the following steps:

[0019] S1. Weigh 1g of chitosan oligosaccharide (degree of deacetylation 93.1%), add 500mL of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to prepare a chitosan oligosaccharide solution with a mass percentage concentration of 0.2%.

[0020] S2. Add 500 mL of 150 mesh hydrogen-type D001 macroporous strong acid ion exchange resin (manufactured by Jiangyin Suqing Resin Co., Ltd.) to the chitosan oligosaccharide solution obtained in step S1, and stir to adsorb for 10 h.

[0021] S3. Filter out the hydrogen form D001 macroporous strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in step S2, and wash thoroughly with 10L of deionized water to remove impurities.

[0022] S4. Add 500 mL of 0.001 mol / L dilute hydrochloric acid solution to the hydrogen form D001 macroporous strong acid ion exchange resin obtained in step S3 after removing impurities, and keep it at 72℃ for 10 h to complete the deacetylation of chitosan oligosaccharide.

[0023] S5. Filter out the hydrogen-type D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharide obtained in step S4, and wash thoroughly with 10L of deionized water to remove dilute hydrochloric acid and acetate generated by the deacetylation reaction.

[0024] S6. Add 500 mL of 0.5 mol / L ammonia solution to the hydrogen-form D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharides obtained in step S5, and stir at 20°C for 5 h to desorb.

[0025] S7. Freeze-dry the desorption solution obtained in step S6 to obtain 0.51 g of fully deacetylated chitosan oligosaccharide (degree of deacetylation is 99.7%).

[0026] Example 2

[0027] A fully deacetylated chitosan oligosaccharide, the preparation method of which includes the following steps (compared to Example 1 which uses a hydrogen-form D001 macroporous strong acid ion exchange resin with a particle size of 100 mesh):

[0028] S1. Weigh 1.0g of chitosan oligosaccharide (degree of deacetylation 91.4%), add 500mL of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to prepare a chitosan oligosaccharide solution with a mass percentage concentration of 0.2%.

[0029] S2. Add 500 mL of 100-mesh hydrogen-type D001 macroporous strong acid ion exchange resin to the chitosan oligosaccharide solution obtained in step S1, and stir to adsorb for 10 h.

[0030] S3. Filter out the hydrogen form D001 macroporous strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in step S2, and wash thoroughly with 10L of deionized water to remove impurities.

[0031] S4. Add 500 mL of 0.001 mol / L dilute hydrochloric acid solution to the hydrogen form D001 macroporous strong acid ion exchange resin obtained in step S3 after removing impurities, and keep it at 75℃ for 10 h to complete the deacetylation of chitosan oligosaccharide.

[0032] S5. Filter out the hydrogen-type D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharide obtained in step S4, and wash thoroughly with 10L of deionized water to remove dilute hydrochloric acid and acetate generated by the deacetylation reaction.

[0033] S6. Add 500 mL of 0.5 mol / L ammonia solution to the hydrogen-form D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharides obtained in step S5, and stir at 20°C for 5 h to desorb.

[0034] S7. Freeze-dry the desorption solution obtained in step S6 to obtain 0.47 g of fully deacetylated chitosan oligosaccharide (degree of deacetylation is 99.8%).

[0035] Example 3

[0036] A fully deacetylated chitosan oligosaccharide, the preparation method of which includes the following steps (compared to Example 1 which uses a hydrogen-form D001 macroporous strong acid ion exchange resin with a particle size of 200 mesh):

[0037] S1. Weigh 1.0g of chitosan oligosaccharide (degree of deacetylation 96.2%), add 500mL of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to prepare a chitosan oligosaccharide solution with a mass percentage concentration of 0.2%.

[0038] S2. Add 500 mL of 200 mesh hydrogen-type D001 macroporous strong acid ion exchange resin to the chitosan oligosaccharide solution obtained in step S1, and stir to adsorb for 10 h.

[0039] S3. Filter out the hydrogen form D001 macroporous strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in step S2, and wash thoroughly with 10L of deionized water to remove impurities.

[0040] S4. Add 500 mL of 0.001 mol / L dilute hydrochloric acid solution to the hydrogen form D001 macroporous strong acid ion exchange resin obtained in step S3 after removing impurities, and keep it at 70℃ for 8 h to complete the deacetylation of chitosan oligosaccharide.

[0041] S5. Filter out the hydrogen-type D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharide obtained in step S4, and wash thoroughly with 10L of deionized water to remove dilute hydrochloric acid and acetate generated by the deacetylation reaction.

[0042] S6. Add 500 mL of 0.5 mol / L ammonia solution to the hydrogen-form D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharides obtained in step S5, and stir at 20°C for 5 h to desorb.

[0043] S7. Freeze-dry the desorption solution obtained in step S6 to obtain 0.54 g of fully deacetylated chitosan oligosaccharide (degree of deacetylation is 99.6%).

[0044] Example 4

[0045] A fully deacetylated chitosan oligosaccharide, the preparation method of which includes the following steps (the mass percentage concentration of the chitosan oligosaccharide solution is adjusted to 0.1% compared to Example 1):

[0046] S1. Weigh 1.0g of chitosan oligosaccharide (degree of deacetylation 91.4%), add 1L of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to prepare a chitosan oligosaccharide solution with a mass percentage concentration of 0.1%.

[0047] S2. Add 500 mL of 150 mesh hydrogen-type D001 macroporous strong acid ion exchange resin to the chitosan oligosaccharide solution obtained in step S1, and stir to adsorb for 10 h.

[0048] S3. Filter out the hydrogen form D001 macroporous strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in step S2, and wash thoroughly with 10L of deionized water to remove impurities.

[0049] S4. Add 500 mL of 0.001 mol / L dilute hydrochloric acid solution to the hydrogen form D001 macroporous strong acid ion exchange resin obtained in step S3 after removing impurities, and keep it at 72℃ for 10 h to complete the deacetylation of chitosan oligosaccharide.

[0050] S5. Filter out the hydrogen-type D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharide obtained in step S4, and wash thoroughly with 10L of deionized water to remove dilute hydrochloric acid and acetate generated by the deacetylation reaction.

[0051] S6. Add 500 mL of 0.5 mol / L ammonia solution to the hydrogen-form D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharides obtained in step S5, and stir at 20°C for 5 h to desorb.

[0052] S7. Freeze-dry the desorption solution obtained in step S6 to obtain 0.52 g of fully deacetylated chitosan oligosaccharide (degree of deacetylation is 99.7%).

[0053] Example 5

[0054] A fully deacetylated chitosan oligosaccharide, the preparation method of which includes the following steps (the mass percentage concentration of the chitosan oligosaccharide solution is adjusted to 0.1% compared to Example 2):

[0055] S1. Weigh 1.0g of chitosan oligosaccharide (degree of deacetylation 93.1%), add 1L of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to prepare a chitosan oligosaccharide solution with a mass percentage concentration of 0.1%.

[0056] S2. Add 500 mL of 100-mesh hydrogen-type D001 macroporous strong acid ion exchange resin to the chitosan oligosaccharide solution obtained in step S1, and stir to adsorb for 10 h.

[0057] S3. Filter out the hydrogen form D001 macroporous strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in step S2, and wash thoroughly with 10L of deionized water to remove impurities.

[0058] S4. Add 500 mL of 0.001 mol / L dilute hydrochloric acid solution to the hydrogen form D001 macroporous strong acid ion exchange resin obtained in step S3 after removing impurities, and keep it at 75℃ for 10 h to complete the deacetylation of chitosan oligosaccharide.

[0059] S5. Filter out the hydrogen-type D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharide obtained in step S4, and wash thoroughly with 10L of deionized water to remove dilute hydrochloric acid and acetate generated by the deacetylation reaction.

[0060] S6. Add 500 mL of 0.5 mol / L ammonia solution to the hydrogen-form D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharides obtained in step S5, and stir at 20°C for 5 h to desorb.

[0061] S7. Freeze-dry the desorption solution obtained in step S6 to obtain 0.49 g of fully deacetylated chitosan oligosaccharide (degree of deacetylation is 99.8%).

[0062] Example 6

[0063] A fully deacetylated chitosan oligosaccharide, the preparation method of which includes the following steps (the mass percentage concentration of the chitosan oligosaccharide solution is adjusted to 0.1% compared to Example 3):

[0064] S1. Weigh 1.0g of chitosan oligosaccharide (degree of deacetylation 96.2%), add 1L of deionized water, and stir until the chitosan oligosaccharide is completely dissolved to prepare a chitosan oligosaccharide solution with a mass percentage concentration of 0.1%.

[0065] S2. Add 500 mL of 200 mesh hydrogen-type D001 macroporous strong acid ion exchange resin to the chitosan oligosaccharide solution obtained in step S1, and stir to adsorb for 10 h.

[0066] S3. Filter out the hydrogen form D001 macroporous strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in step S2, and wash thoroughly with 10L of deionized water to remove impurities.

[0067] S4. Add 500 mL of 0.001 mol / L dilute hydrochloric acid solution to the hydrogen form D001 macroporous strong acid ion exchange resin obtained in step S3 after removing impurities, and keep it at 70℃ for 8 h to complete the deacetylation of chitosan oligosaccharide.

[0068] S5. Filter out the hydrogen-type D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharide obtained in step S4, and wash thoroughly with 10L of deionized water to remove dilute hydrochloric acid and acetate generated by the deacetylation reaction.

[0069] S6. Add 500 mL of 0.5 mol / L ammonia solution to the hydrogen-form D001 macroporous strong acid ion exchange resin that adsorbs fully deacetylated chitosan oligosaccharides obtained in step S5, and stir at 20°C for 5 h to desorb.

[0070] S7. Freeze-dry the desorption solution obtained in step S6 to obtain 0.56 g of fully deacetylated chitosan oligosaccharide (degree of deacetylation is 99.8%).

[0071] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.

Claims

1. A method for preparing fully deacetylated chitosan oligosaccharide, characterized in that, Includes the following steps: S1. Add the hydrogen-form strong acid ion exchange resin to a chitosan oligosaccharide solution with a concentration of 0.1%-0.2%, and the volume ratio of the chitosan oligosaccharide solution to the hydrogen-form strong acid ion exchange resin is 1:(0.5-1). Stir and adsorb for 5-10 hours. S2. Take out the hydrogen-form strong acid ion exchange resin that adsorbs chitosan oligosaccharides obtained in S1 and wash it to remove impurities. Then add dilute hydrochloric acid solution and heat it at 60℃-80℃ for 5h-10h. S3. Filter out the hydrogen-form strong acid ion exchange resin that has adsorbed fully deacetylated chitosan oligosaccharide obtained in S2, wash it, add ammonia water, stir and desorb for 2-5 hours, and finally freeze-dry the obtained eluent to obtain fully deacetylated chitosan oligosaccharide with a degree of deacetylation >99.5%.

2. The preparation method according to claim 1, characterized in that, The particle size of the hydrogen-form strong acid ion exchange resin is 40-400 mesh.

3. The preparation method according to claim 2, characterized in that, Hydrogen-form strong acid ion exchange resin is a macroporous strong acid ion exchange resin.

4. The preparation method according to claim 3, characterized in that, The particle size of macroporous strong acid ion exchange resin is 100-200 mesh.

5. The preparation method according to claim 1, characterized in that, In S1, the degree of deacetylation of chitosan in the chitosan oligosaccharide solution is 55%-99%.

6. The preparation method according to claim 5, characterized in that, In S1, the degree of deacetylation of chitosan in the chitosan oligosaccharide solution is 90%-99%.

7. The preparation method according to claim 1, characterized in that, The concentration of the dilute hydrochloric acid solution is 0.001 mol / L-0.01 mol / L, and the concentration of the ammonia solution is 0.5 mol / L.

8. The preparation method according to claim 1, characterized in that, Desorption was carried out at a temperature of 15℃-25℃.