A chitosan solution resistant to radiation degradation and its irradiation sterilization method

Abstract

The present invention relates to chitosan, and specifically relates to a chitosan solution resistant to radiation degradation and its radiation sterilization method, belonging to the technical field of biomaterial processing. The present invention discloses a chitosan solution resistant to radiation degradation, which is characterized in that, by mass percentage, the chitosan solution contains the following components: chitosan 0.5% - 2%; glacial acetic acid 0.25% - 1%; glycerol 4% - 6%; n-butanol 3% - 4%; propylene glycol 0.6% - 1%; the balance is water; by adding glycerol, n-butanol and propylene glycol to the chitosan solution, the present invention can reduce the degradation of chitosan during the irradiation of the chitosan solution, improve the stability of the chitosan solution, and at the same time improve the antibacterial effect of the chitosan solution film-forming.

Description

Technical Field

[0001] The present invention relates to a chitosan solution resistant to radiation degradation and its radiation sterilization method, belonging to the technical field of biomaterial processing. Background Art

[0002] Chitosan is a product of chitin deacetylation and widely exists in the exoskeletons of shrimps, crabs and insects. As a natural polymer material, chitosan is widely used in the field of medical materials and can be used to manufacture chitosan membranes and chitosan scaffolds. Chitosan can also be dissolved in acidic solutions to make chitosan antibacterial film-forming sprays. At present, the sterilization of medical materials by wet heat steam sterilization and radiation sterilization will cause a decrease in the molecular weight of chitosan. After chitosan is sterilized by cobalt-60, the cleavage of β-(1→4) ether bonds on the main chain of chitosan is caused, resulting in a significant decrease in its viscosity, which affects the film-forming property and antibacterial property of the chitosan solution.

[0003] Chinese Patent CN1563106A discloses a method for preparing small molecular weight or water-soluble sugars by radiation, and Chinese Patent ZL201710883206.7 discloses a method for preparing low molecular weight chitosan. The prior art only discloses how to prepare low molecular weight chitosan by radiation. At present, there is no technology for reducing the radiation degradation of liquid chitosan. Therefore, there is an urgent need for a radiation sterilization method that can effectively sterilize and slow down the degradation of chitosan solution. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a chitosan solution resistant to radiation degradation, thereby slowing down the degradation of the chitosan solution and improving the radiation sterilization effect.

[0005] The degradation of chitosan is caused by irradiating the chitosan solution in the air. The inventor unexpectedly found that the degradation of chitosan can be reduced by adding glycerol, n-butanol and propylene glycol to the chitosan solution.

[0006] The present invention provides a chitosan solution resistant to radiation degradation, which is characterized in that, by mass percentage, the chitosan solution contains the following components:

[0007] Chitosan 0.5%-2%;

[0008] Glacial acetic acid 0.25%-1%;

[0009] Glycerol 4%-6%;

[0010] n-butanol 3%-4%;

[0011] Propylene glycol 0.6%-1%;

[0012] The balance is water;

[0013] Preferably, the chitosan solution contains the following components:

[0014] Chitosan 0.9% - 1.6%;

[0015] Glacial acetic acid 0.45% - 0.8%;

[0016] Glycerol 4.5% - 5.3%;

[0017] n-Butanol 3.2% - 3.7%;

[0018] Propylene glycol 0.73% - 0.92%;

[0019] The balance is water;

[0020] Preferably, the chitosan solution contains the following components:

[0021] Chitosan 1%;

[0022] Glacial acetic acid 0.5%;

[0023] Glycerol 5%;

[0024] n-Butanol 3.5%;

[0025] Propylene glycol 0.8%;

[0026] The balance is water;

[0027] Preferably, the viscosity-average molecular weight of the chitosan is 110 - 423 KDa.

[0028] Preferably, the viscosity-average molecular weight of the chitosan is 231 KDa.

[0029] Preferably, the deacetylation degree of the chitosan ≥ 85%;

[0030] Preferably, the mass ratio of glycerol, chitosan, and propylene glycol is (40 - 60):(30 - 40):(6 - 10).

[0031] Preferably, the mass ratio of glycerol, chitosan, and propylene glycol is (45 - 53):(32 - 37):(7.3 - 9.2).

[0032] Preferably, the mass ratio of glycerol, chitosan, and propylene glycol is 50:35:8.

[0033] The present invention also provides a method for irradiation sterilization of a radiation-resistant degradation chitosan solution, which is prepared according to the following steps:

[0034] 1) Package the above-mentioned radiation-resistant degradation chitosan solution;

[0035] 2) Perform irradiation sterilization.

[0036] Preferably, the irradiation in step 2) is 60Co-γ ray.

[0037] Preferably, the irradiation dose in step 2) is 3 KGy - 30 KGy.

[0038] Beneficial effects

[0039] By adding glycerol, n-butanol and propylene glycol to the chitosan solution resistant to irradiation degradation, the present invention can reduce the degradation of chitosan during the irradiation process of the chitosan solution, improve the stability of the chitosan solution, and at the same time improve the antibacterial effect of the chitosan solution film-forming. Specific embodiments

[0040] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

[0041] The reagents used in the following examples were purchased through commercial channels;

[0042] Chitosan: Aladdin Chemical Reagent Co., Ltd., the degree of deacetylation is ≥85%, and the viscosity-average molecular weights are 110KDa, 231KDa, and 423KDa respectively.

[0043] Example 1:

[0044] Preparation of chitosan solution resistant to irradiation degradation:

[0045] Add 5 g of chitosan with a viscosity-average molecular weight of 110KDa and a degree of deacetylation of ≥90% to 500 g of purified water, add 2.5 g of glacial acetic acid, stir to fully dissolve the chitosan, continue to stir and add 40 g of glycerol, 30 g of n-butanol, and 6 g of propylene glycol, make up the volume to 1 L, and stir for 10 minutes until the solution is evenly mixed;

[0046] Sterilization of chitosan solution resistant to irradiation degradation:

[0047] 1) Fill the mixed solution into chitosan solution;

[0048] 2) Irradiate the chitosan solution with 60Co γ-ray at room temperature (Guangzhou Huada Biotechnology Co., Ltd.), the dose is 3 - 6 kGy, and the viscosity-average molecular weight of chitosan after irradiation is 80.3 KDa.

[0049] Example 2:

[0050] Preparation of chitosan solution resistant to irradiation degradation:

[0051] Add 10 g of chitosan with a viscosity-average molecular weight of 231 kDa and a degree of deacetylation of ≥90% to 500 g of purified water, add 5 g of glacial acetic acid, stir to fully dissolve the chitosan, continue stirring and add 50 g of glycerol, 35 g of n-butanol, and 8 g of propylene glycol, make up the volume to 1 L, and stir for 10 minutes until the solution is well mixed;

[0052] Sterilization of the radiation-resistant degraded chitosan solution:

[0053] 1) Fill the mixed solution into a chitosan solution;

[0054] 2) Irradiate the chitosan solution, use 60Co γ-ray irradiation for sterilization (Guangzhou Huada Biotechnology Co., Ltd.), the dose is 8 - 16 kGy, and the viscosity-average molecular weight of chitosan after irradiation is 194 kDa.

[0055] Example 3:

[0056] Preparation of the radiation-resistant degraded chitosan solution:

[0057] Add 20 g of chitosan with a viscosity-average molecular weight of 423 kDa and a degree of deacetylation of ≥90% to 500 g of purified water, add 10 g of glacial acetic acid, stir to fully dissolve the chitosan, continue stirring and add 60 g of glycerol, 40 g of n-butanol, and 10 g of propylene glycol, make up the volume to 1 L, and stir for 10 minutes until the solution is well mixed;

[0058] Sterilization of the radiation-resistant degraded chitosan solution:

[0059] 1) Fill the mixed solution into a chitosan solution;

[0060] 2) Irradiate the chitosan solution, use 60Co γ-ray irradiation for sterilization (Guangzhou Huada Biotechnology Co., Ltd.), the dose is 14 - 28 kGy, and the viscosity-average molecular weight of chitosan after irradiation is 308.79 kDa.

[0061] Example 4:

[0062] Preparation of the radiation-resistant degraded chitosan solution:

[0063] Add 9 g of chitosan with a viscosity-average molecular weight of 231 kDa and a degree of deacetylation of ≥90% to 500 g of purified water, add 4.5 g of glacial acetic acid, stir to fully dissolve the chitosan, continue stirring and add 45 g of glycerol, 32 g of n-butanol, and 7.3 g of propylene glycol, make up the volume to 1 L, and stir for 10 minutes until the solution is well mixed;

[0064] Sterilization of the radiation-resistant degraded chitosan solution:

[0065] 1) Fill the mixed solution into a chitosan solution;

[0066] 2) Irradiate the chitosan solution, using 60Co γ-ray irradiation for sterilization (Guangzhou Huada Biotechnology Co., Ltd.), with a dose of 8 - 16 kGy. The viscosity-average molecular weight of chitosan after irradiation is 179.5 KDa.

[0067] Example 5:

[0068] Preparation of radiation-resistant degraded chitosan solution:

[0069] Add 16 g of chitosan with a viscosity-average molecular weight of 231 KDa and a deacetylation degree of ≥90% to 500 g of purified water, add 8 g of glacial acetic acid, stir to fully dissolve the chitosan, continue stirring and add 53 g of glycerol, 37 g of n-butanol, and 9.2 g of propylene glycol, make up the volume to 1 L, and stir for 10 minutes until the solution is evenly mixed;

[0070] Sterilization of radiation-resistant degraded chitosan solution:

[0071] 1) Fill the mixed solution into chitosan solution;

[0072] 2) Irradiate the chitosan solution, using 60Co γ-ray irradiation for sterilization (Guangzhou Huada Biotechnology Co., Ltd.), with a dose of 8 - 16 kGy. The viscosity-average molecular weight of chitosan after irradiation is 179.5 KDa.

[0073] Comparative Example 1:

[0074] Compared with Example 2, the difference is only that Comparative Example 1 does not contain n-butanol and only contains 43 g of propylene glycol;

[0075] Comparative Example 2:

[0076] Compared with Example 2, the difference is only that Comparative Example 2 does not contain propylene glycol and only contains 43 g of n-butanol;

[0077] Comparative Example 3:

[0078] Compared with Example 2, the difference is only that Comparative Example 3 contains 10 g of n-butanol and 33 g of propylene glycol;

[0079] Comparative Example 4:

[0080] Compared with Example 2, the difference is only that Comparative Example 4 does not contain n-butanol and propylene glycol and contains 43 g of isopropanol;

[0081] Control group:

[0082] Compared with Example 2, the chitosan solution was not sterilized;

[0083] Experimental Example 1. Molecular weight determination

[0084] Experimental materials: Chitosan solutions prepared in Examples 1 - 5 and Comparative Examples 1 - 4

[0085] Test method: The viscosity-average molecular weight of chitosan was determined using an Ubbelohde viscometer. The method described in Pan Zuren's "Polymer Chemistry", 5th Edition, Chemical Industry Press, 2014.1 was adopted. The viscosity-average molecular weight was measured using an Ubbelohde viscometer in a constant temperature water bath at 25 ± 0.5 °C, as shown in Table 1:

[0086] Sample Initial viscosity-average molecular weight / * KDa Viscosity-average molecular weight after irradiation / * KDa Proportion of molecular weight decrease Example 1 110 80.3 27.30% Example 2 231 194 16% Example 3 423 308.79 27% Example 4 231 179.5 22.3% Example 5 231 183.4 20.6% Comparative Example 1 231 109 52.8% Comparative Example 2 231 86 62.7% Comparative Example 3 231 62 73.20% Comparative Example 4 231 137 40.70% Control group 231 224 3%

[0087] As can be seen from Table 1, the addition of the molecular weight of chitosan, propylene glycol, glycerol, and n-butanol in the chitosan solution will affect the degradation of the chitosan solution after irradiation. The smaller the molecular weight, the higher the degradation rate. Comparing Comparative Examples 1-4 with Example 2, the difference lies in the content of propylene glycol and n-butanol. Comparative Example 1 does not contain n-butanol, and the viscosity-average molecular weight of chitosan decreased by 52.8%. Comparative Example 2 does not contain propylene glycol, and the viscosity-average molecular weight of chitosan decreased by 62.7%. Comparative Example 3 contains 10 g of n-butanol and 33 g of propylene glycol, and the viscosity-average molecular weight of chitosan decreased by 73.2%. Comparative Example 4 does not contain n-butanol, and isopropanol is used to replace n-butanol, and the viscosity-average molecular weight of chitosan decreased by 40.7%.

[0088] Test Example 2: Film-forming property of chitosan solution

[0089] The chitosan solutions prepared in Examples 1-3, Comparative Examples 1-4, and the control group were sprayed on a dry flat glass and spread out evenly at a temperature of 20 °C - 25 °C and a relative humidity of 70% to test the film-forming time.

[0090] Table 2 Film-forming time

[0091] Sample Film-forming time (min) Example 1 9 Example 2 8 Example 3 7 Comparative Example 1 23 Comparative Example 2 25 Comparative Example 3 22 Comparative Example 4 12 Control group 9

[0092] As can be seen from Table 2, the simultaneous addition of glycerol, n-butanol, and propylene glycol in the chitosan solution can shorten the film-forming time. The film-forming time is determined by the molecular weight of chitosan. Since the molecular weight of the chitosan solution decreases after irradiation, the addition of glycerol, n-butanol, and glycerol in Examples 1-3 can reduce the irradiation degradation of chitosan. The larger the molecular weight, the better the film-forming property. Therefore, the film-forming time of Examples 1-3 is lower than that of the comparative examples.

[0093] Test Example 3: Bacteriostatic performance test:

[0094] Materials:

[0095] ① Strains: Standard strain of Staphylococcus aureus, which shows typical colonies after enrichment in the culture medium and can be passaged normally.

[0096] ② Samples: Chitosan solutions of Examples 1-3 and Comparative Examples 1-4.

[0097] Experiment:

[0098] In an agar culture medium petri dish, the chitosan solutions of Examples 1-3 were sprayed onto the surface of the culture medium at a dosage of 1 mL per square centimeter respectively. Control Examples 1 to 4 were coated onto the surface of the culture medium at a dosage of 1 mL per square centimeter. After coating the chitosan film, the petri dish was placed in a sterile environment with the lid open to form a 40 mm × 40 mm square chitosan solution area and waited for it to form a film. After 60 min, Serratia marcescens aerosol was sprayed onto the surface of the culture medium, and then the lid of the petri dish was covered. It was cultured at 37 °C for 24 h, and the colony growth conditions at the covered and uncovered areas of the chitosan film were observed.

[0099] Then, the chitosan film was removed, and the culture medium with the chitosan film removed was continued to be cultured at 37 °C for 24 h, and the colony growth conditions at the covered and uncovered areas were observed. Each group was operated continuously 3 times to observe the reproducibility. The results are shown in Table 3 below.

[0100] Table 3 Bacteriostatic Effect Test

[0101] Sample Example 1 Example 2 Example 3 Comparative Example 1 Control Example 2 Control Example 3 Control Example 4 Colony growth situation when the chitosan film is not removed There is no colony growth at the covered area of the original chitosan film, and the rest is covered with bacteria There is no colony growth at the covered area of the original chitosan film, and the rest is covered with bacteria There is no colony growth at the covered area of the original chitosan film, and the rest is covered with bacteria Bacteria grow both at the covered area of the original chitosan film and the rest Bacteria grow both at the covered area of the original chitosan film and the rest Bacteria grow both at the covered area of the original chitosan film and the rest Bacteria grow both at the covered area of the original chitosan film and the rest Colony growth situation after removing the chitosan film There is no colony growth at the covered area of the original chitosan film, and the rest is covered with bacteria There is no colony growth at the covered area of the original chitosan film, and the rest is covered with bacteria There is no colony growth at the covered area of the original chitosan film, and the rest is covered with bacteria Bacteria grow both at the covered area of the original chitosan film and the rest Bacteria grow both at the covered area of the original chitosan film and the rest Bacteria grow both at the covered area of the original chitosan film and the rest Bacteria grow both at the covered area of the original chitosan film and the rest Colony growth rate at the covered area of the original chitosan film when the chitosan film is not removed / (%) / / / 25.6±7.8 15.8±5.9 27.6±8.2 23.9±6.9 Colony growth rate at the covered area of the original chitosan film after removing the chitosan film / (%) / / / 32.5±6.7 29.6±9.6 38.9±7.6 34.8±8.4

[0102] As can be seen from Table 3, there was no colony growth at the areas covered by the original chitosan film of the culture media in Examples 1-3, while the rest were covered with bacteria. The state before removing the chitosan film was exactly the same as the state after removing the chitosan film and continuing to culture for 24 h, indicating that adding glycerol, n-butanol, and propylene glycol to the chitosan solution simultaneously could form a chitosan film with bacteriostatic effect. However, in Comparative Examples 1-4, there was colony growth both before and after removing the chitosan film and continuing to culture for 24 h, indicating that n-butanol and glycerol in the chitosan solution could promote the formation of the chitosan film.

[0103] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A chitosan solution resistant to radiation degradation, characterized in that, The chitosan solution contains the following components by mass percentage: Chitosan 0.5% - 2%; Glacial acetic acid 0.25% - 1%; Glycerol 4% - 6%; n-Butanol 3% - 4%; Propylene glycol 0.6% - 1%; The balance is water.

2. The radiation-resistant and degradable chitosan solution according to claim 1, wherein The chitosan solution contains the following components: Chitosan 0.9% - 1.6%; Glacial acetic acid 0.45% - 0.8%; Glycerol 4.5% - 5.3%; n-Butanol 3.2% - 3.7%; Propylene glycol 0.73% - 0.92%; The balance is water.

3. The chitosan solution resistant to radiation degradation according to claim 2, wherein The chitosan solution contains the following components: Chitosan 1%; Glacial acetic acid 0.5%; Glycerol 5%; n-Butanol 3.5%; Propylene glycol 0.8%; The balance is water.

4. The chitosan solution resistant to radiation degradation according to claim 1, characterized in that, The viscosity-average molecular weight of the chitosan is 110 - 423 KDa.

5. The chitosan solution resistant to radiation degradation according to claim 1, wherein The viscosity-average molecular weight of the chitosan is 231 KDa.

6. The radiation-resistant and degradable chitosan solution according to claim 1, wherein The degree of deacetylation of the chitosan is ≥ 85%.

7. The chitosan solution with radiation-resistant degradation according to claim 1, wherein The mass ratio of the glycerol, chitosan, and propylene glycol is (40 - 60):(30 - 40):(6 - 10).

8. The radiation-resistant and degradable chitosan solution according to claim 7, wherein The mass ratio of the glycerol, chitosan, and propylene glycol is (45 - 53):(32 - 37):(7.3 - 9.2).

9. The radiation-resistant and degradable chitosan solution according to claim 7 or 8, characterized in that The mass ratio of the glycerol, chitosan, and propylene glycol is 50:35:

8.

10. A method for irradiation sterilization of a chitosan solution resistant to radiation degradation, characterized in that, It is prepared according to the following steps: 1) Package the radiation-resistant and degradable chitosan solution according to any one of claims 1 - 8; 2) Perform radiation sterilization.

11. The irradiation sterilization method according to claim 10, wherein The radiation in step 2) is 60Co-γ rays.

12. The irradiation sterilization method according to claim 11, characterized in that, The radiation dose in step 2) is 3 KGy - 28 KGy.