A method for removing endotoxin from alginate oligosaccharide and alginate oligosaccharide with endotoxin removed
By combining Lewis acid synergistic treatment and adsorbent adsorption, the problem of endotoxin residue in alginate oligosaccharides was solved, achieving the preparation of alginate oligosaccharides with low impurity introduction and low endotoxin content, which is suitable for biomedical applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2022-12-11
- Publication Date
- 2026-07-24
AI Technical Summary
In existing technologies, residual endotoxins in the preparation of alginate oligosaccharides affect their biocompatibility and biomedical applications, and traditional removal methods suffer from poor safety, high corrosiveness, high energy consumption for waste liquid treatment, and significant pollution.
The method employs a combination of Lewis acid synergistic treatment and adsorption by an adsorbent. The specific steps include centrifugation to remove insoluble particles, treatment with mixed Lewis acids, acidification to form a precipitate, washing the precipitate, adding an alkaline solution to dissolve the precipitate and filtering, adsorption by an adsorption medium, and precipitation of alginate oligosaccharides with an organic solvent. Finally, drying yields alginate oligosaccharides free of endotoxins.
It effectively reduces the endotoxin content in alginate oligosaccharides, is simple to operate, suitable for industrial production, and does not introduce impurities, thus improving the biocompatibility and biomedical application potential of alginate oligosaccharides.
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Figure CN118165139B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of removing endotoxins from oligomers, and particularly to the technical field of removing endotoxins from oligosaccharide materials. Specifically, it relates to a method for removing endotoxins from alginate oligosaccharides and an alginate oligosaccharide material for removing endotoxins. Background Technology
[0002] Alginate oligosaccharides (AOS), also known as alginate oligosaccharides, are products obtained from the chemical or enzymatic degradation of alginate, with a degree of polymerization typically ranging from 2 to 20. Structurally, alginate oligosaccharide molecules contain two monosaccharide units: α-L-mannuronic acid and its C5 epimer β-D-guluronic acid. These two monosaccharide units can be randomly arranged to form oligosaccharide fragments with various structures. Alginate oligosaccharides possess a variety of bioactivities, such as antioxidant, anti-inflammatory, antitumor, and immunomodulatory effects, and have been widely used in biomedical research. However, existing methods for preparing alginate oligosaccharides often result in residual impurities such as endotoxins, affecting their biocompatibility and biomedical applications. Nevertheless, existing patents and research papers lack reports on technologies and research aimed at removing endotoxins from alginate oligosaccharides. Researchers have proposed techniques to reduce endotoxin levels in oligomeric hyaluronic acid, oligoheparin, and oligopeptides by treating these materials with strong acids, strong alkalis, strong oxidants, and surfactants. However, these techniques suffer from poor safety and problems such as high corrosivity, high energy consumption for wastewater treatment, and significant pollution.
[0003] To address the aforementioned problems, this invention provides a method for removing endotoxins from alginate oligosaccharides. This invention relates to a method for removing endotoxins from alginate oligosaccharides and endotoxin-free alginate oligosaccharides. Using commercially available alginate oligosaccharides as raw materials, insoluble particles are removed by centrifugation. A mixed Lewis acid is added for synergistic treatment. The treatment solution is acidified to form a precipitate, which is then washed with deionized water. An alkaline solution is added to dissolve the precipitate, and the precipitate is filtered. The filtrate is adsorbed onto an adsorption medium, and the alginate oligosaccharides are precipitated using an organic solvent under sterile conditions. The precipitate is then dried to obtain endotoxin-free alginate oligosaccharides. This invention utilizes synergistic treatment with mixed Lewis acids and adsorption with an adsorbent to remove endotoxins from alginate oligosaccharides. No impurities are introduced during the preparation process, resulting in a low endotoxin content in the obtained alginate oligosaccharides. The operation is simple, and the preparation process is suitable for industrial production. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a method for removing endotoxins from alginate oligosaccharides. The present invention solves the problems of endotoxin impurities in traditional alginate oligosaccharide materials affecting the biocompatibility and biomedical applications of alginate oligosaccharides.
[0005] This invention provides a method for removing endotoxins from alginate oligosaccharides and an alginate oligosaccharide material for removing endotoxins:
[0006] Including the following technical solutions:
[0007] Commercially available alginate oligosaccharides were used as raw materials. They were dissolved in water, and insoluble particles were removed by centrifugation. Mixed Lewis acids were added for synergistic treatment. The treated solution was acidified to form a precipitate. The precipitate was then washed with deionized water, and an alkaline solution was added to dissolve the precipitate. The precipitate was then filtered. After the filtrate was adsorbed by an adsorption medium, the alginate oligosaccharides were precipitated with an organic solvent and dried to obtain endotoxin-free alginate oligosaccharides.
[0008] The mixed Lewis acid is preferably a mixture of aluminum chloride and zinc chloride.
[0009] The alginate oligosaccharide is one or more of sodium alginate oligosaccharide and potassium alginate oligosaccharide, and its degree of polymerization is 2-50.
[0010] The specific operation process for removing insoluble particles from alginate oligosaccharides by centrifugation is as follows: 1) Dissolve commercially available alginate oligosaccharides in water at a concentration of 1-500 mg / mL; 2) Centrifuge the alginate oligosaccharide solution at a centrifugal force of 1000-20000g to remove insoluble impurities.
[0011] The specific process of mixed Lewis acid treatment is as follows: add mixed Lewis acid at a weight ratio of 100:1 to 1:1 between alginate oligosaccharide and mixed Lewis acid, and mechanically stir for 0.5 to 48 hours at 4°C to room temperature.
[0012] Furthermore, the mixed Lewis acid is preferably a mixture of aluminum chloride and zinc chloride, with a preferred molar ratio of 0.5:1 to 2:1.
[0013] The specific procedure for acidifying the solution to form a precipitate is to adjust the pH of the solution to below 3 using one or more of hydrochloric acid, sulfuric acid, or nitric acid, so that the alginate oligosaccharide is acidified and precipitates out.
[0014] The specific procedure for adding an alkaline solution to dissolve the precipitate and filtering is as follows: 1) Add an alkaline solution with a concentration of 1-300 mg / mL to the precipitate to dissolve it, and the pH of the solution is 6.5-7.5; filter the alginate oligosaccharide solution through a 0.22 μm microporous membrane to remove insoluble particles.
[0015] Furthermore, the alkaline solution is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate solutions.
[0016] The specific process of adsorption by the adsorption medium is as follows: 1) Add the adsorption medium at a weight ratio of sodium alginate oligosaccharide to adsorption medium of 20:1-1:10, and mechanically stir at 4℃ to room temperature for 0.5-48 hours. Filter the solution containing the adsorption medium through a microporous membrane to remove the adsorption medium and obtain an alginate oligosaccharide solution; 2) Repeat step 1) 0-10 times; 3) Filter the alginate oligosaccharide solution through a 0.22μm filter membrane to obtain a sterile alginate oligosaccharide solution.
[0017] Furthermore, the adsorption medium is activated carbon, kaolin, diatomaceous earth, polymyxin B agarose resin, or a mixture thereof in any proportion.
[0018] Furthermore, the microporous filter membrane can be a cellulose filter membrane, a polypropylene filter membrane, a polyethersulfone filter membrane, a polytetrafluoroethylene filter membrane, a ceramic filter membrane, etc.
[0019] The specific process of precipitating and drying alginate oligosaccharides using an organic solvent is as follows: the organic solvent is slowly added to the adsorbed alginate oligosaccharide solution, causing the alginate oligosaccharides to precipitate. The precipitate is then dried to obtain alginate oligosaccharide material with endotoxins removed.
[0020] Furthermore, the organic solution is one or a mixture of two or more of C1-C3 alcohols, ethers, and ketones in any proportion, and the volume ratio of the alginate oligosaccharide solution to the organic solution is 1:2 to 1:10.
[0021] The drying method is one or more of the following: freeze drying, vacuum drying, and oven drying.
[0022] The beneficial effects of this invention are as follows: This invention utilizes mixed Lewis acids to treat alginate oligosaccharides, causing partial hydrolysis of the endotoxins in them, thereby improving the efficiency of activated carbon adsorption in removing endotoxins from alginate oligosaccharides. It utilizes the synergistic treatment of mixed Lewis acids and the adsorption of adsorbents to remove endotoxins from alginate oligosaccharides. No impurities are introduced during the preparation process, resulting in alginate oligosaccharides with low endotoxin content. The operation is simple, and the preparation process is suitable for industrial production. Attached Figure Description
[0023] Figure 1 The results of bacterial endotoxin detection in sodium alginate oligosaccharides prepared by different methods are shown below; where A is commercially available sodium alginate oligosaccharide, B is sodium alginate oligosaccharide of Comparative Example 1 without treatment with mixed Lewis acids, C is sodium alginate oligosaccharide prepared by replacing mixed Lewis acids with aluminum chloride in Comparative Example 2, D is sodium alginate oligosaccharide prepared by replacing mixed Lewis acids with zinc chloride in Comparative Example 3, and E is sodium alginate oligosaccharide prepared in Example 2 of this invention. Detailed Implementation
[0024] Example 1
[0025] (1) Dissolve 50g of commercially available water-soluble potassium alginate oligosaccharide in 100mL of distilled water and stir magnetically for 0.5h at 400rpm until dissolved;
[0026] (2) Centrifuge the potassium alginate oligosaccharide solution from step (1) at a centrifugal force of 20,000g to remove insoluble particles;
[0027] (3) Add aluminum chloride and zinc chloride mixed Lewis acid with a molar ratio of 0.5:1 to the potassium alginate oligosaccharide solution in step (2) at a weight ratio of 100:1 to potassium alginate oligosaccharide and mixed Lewis acid, and mechanically stir for 24 hours at room temperature.
[0028] (4) Adjust the pH of the solution obtained in step (3) to below 3 with concentrated hydrochloric acid to acidify the alginate oligosaccharide and precipitate it. Then wash the precipitate thoroughly with deionized water.
[0029] (5) Add a potassium carbonate solution with a concentration of 200 mg / mL to the precipitate to dissolve it and adjust the pH of the solution to 6.5-7.5; filter the solution through a 0.22 μm cellulose microporous membrane to remove insoluble particles;
[0030] (6) Add the adsorption medium of activated carbon: diatomaceous earth: polymyxin B agarose resin = 1:1:0.05 (weight ratio) to the solution obtained in step (5) at a weight ratio of 10:1 between potassium alginate oligosaccharide and adsorption medium. Stir mechanically at 4°C for 48 hours. Filter the solution containing activated carbon through a 0.22μm polyethersulfone microporous filter membrane to remove carbon and obtain the alginate oligosaccharide solution.
[0031] (7) Repeat step (6) 5 times;
[0032] (8) Filter the sodium alginate oligosaccharide solution obtained in step (7) through a 0.22 μm polyethersulfone filter membrane to obtain a sterile alginate oligosaccharide solution;
[0033] (9) Under sterile conditions, 100 mL of anhydrous ethanol is slowly added to 50 mL of sodium alginate oligosaccharide solution from step (8) to form a precipitate of sodium alginate oligosaccharide. The precipitate is dried under vacuum to obtain sodium alginate oligosaccharide material with endotoxin removed.
[0034] (10) The residual endotoxin content in the prepared sodium alginate oligosaccharide was determined using a BET32C endotoxin analyzer manufactured by Tianjin University Radio Factory, according to the turbidimetric method specified in Chapter 1143 of the General Section IV of the Pharmacopoeia of the People's Republic of China (2020 Edition). The endotoxin content in commercially available sodium alginate oligosaccharide was 185,000 EU / g. Figure 1 -A), the sodium alginate oligosaccharide prepared by this invention has an endotoxin content of 61 EU / g.
[0035] Example 2
[0036] (1) Dissolve 0.1g of commercially available water-soluble sodium alginate oligosaccharide in 100mL of distilled water and stir magnetically for 0.5h at 400rpm until dissolved;
[0037] (2) Centrifuge the sodium alginate oligosaccharide solution from step (1) at a centrifugal force of 1000g to remove insoluble particles.
[0038] (3) Add aluminum chloride and zinc chloride mixed Lewis acid with a molar ratio of 1:2 to the sodium alginate oligosaccharide solution in step (2) at a weight ratio of 1:1 to 1:1, and mechanically stir at 4°C for 48 hours.
[0039] (4) Adjust the pH of the solution obtained in step (3) to below 3 with concentrated sulfuric acid to acidify the alginate oligosaccharide and precipitate it. Then wash the precipitate thoroughly with deionized water.
[0040] (5) Add a sodium hydroxide and sodium carbonate solution with a concentration of 1 mg / mL to the precipitate to dissolve the precipitate and adjust the pH of the solution to 6.5-7.5; filter the solution through a 0.22 μm polypropylene microporous membrane to remove insoluble particles;
[0041] (6) Add the adsorption medium of activated carbon: diatomaceous earth: polymyxin B agarose resin = 1:0.5:0.1 (weight ratio) to the solution obtained in step (5) at a weight ratio of 1:10 between sodium alginate oligosaccharide and adsorption medium. Stir mechanically for 24 hours at room temperature. Filter the solution containing activated carbon through a 0.22μm polytetrafluoroethylene microporous membrane to remove carbon and obtain the alginate oligosaccharide solution.
[0042] (7) Filter the sodium alginate oligosaccharide solution obtained in step (6) through a 0.22 μm polytetrafluoroethylene filter membrane to obtain a sterile alginate oligosaccharide solution;
[0043] (8) Under sterile conditions, 400 mL of anhydrous ethanol was slowly added to 100 mL of the sodium alginate oligosaccharide solution from step (7), causing the sodium alginate oligosaccharide to form a flocculent precipitate. The precipitate was then vacuum dried to obtain the endotoxin-free sodium alginate oligosaccharide material. The endotoxin content in the prepared sodium alginate oligosaccharide was 9 EU / g. Figure 1 Sample E in the sample).
[0044] Example 3
[0045] (1) Dissolve 30g of commercially available sodium alginate oligosaccharide in 100mL of distilled water and stir magnetically for 24h at 400rpm until dissolved.
[0046] (2) Centrifuge the sodium alginate oligosaccharide solution from step (1) at a centrifugal force of 15000g to remove insoluble particles.
[0047] (3) Add aluminum chloride and zinc chloride mixed Lewis acid with a molar ratio of 2:1 to the sodium alginate oligosaccharide solution in step (2) at a weight ratio of 10:1 to 10:1 and mechanically stir for 2 hours at room temperature.
[0048] (4) Adjust the pH of the solution obtained in step (3) to below 3 with nitric acid to acidify the alginate oligosaccharide and precipitate it. Then wash the precipitate thoroughly with deionized water.
[0049] (5) Add a sodium hydroxide oligosaccharide solution with a concentration of 100 mg / mL to the precipitate to dissolve the precipitate and adjust the pH of the solution to 6.5-7.5; filter the solution through a 0.22 μm ceramic microporous membrane to remove insoluble particles;
[0050] (6) Add activated carbon to the solution obtained in step (5) at a weight ratio of 10:1 for sodium alginate oligosaccharide and activated carbon, stir mechanically for 0.5 hours at room temperature, filter the solution containing activated carbon through a microporous membrane to remove carbon, and obtain an alginate oligosaccharide solution.
[0051] (7) Repeat step (6) twice;
[0052] (8) Under sterile conditions, 1000 mL of anhydrous ethanol was slowly added to 100 mL of the alginate oligosaccharide solution from step (7) to form a flocculent precipitate of alginate oligosaccharide. The precipitate was freeze-dried to obtain the endotoxin-free alginate oligosaccharide material. The prepared sodium alginate endotoxin content was 26 EU / g.
[0053] Example 4
[0054] (1) Dissolve 5g of commercially available water-soluble potassium alginate oligosaccharide in 100mL of distilled water and stir magnetically for 4 hours at 400rpm until dissolved;
[0055] (2) Same as the steps in Example 2;
[0056] (3) Add aluminum chloride and zinc chloride mixed Lewis acid with a molar ratio of 1:1 to the potassium alginate oligosaccharide solution in step (2) at a weight ratio of 5:1 to 5:1 and mechanically stir for 2 hours at room temperature.
[0057] (4) Same as the step in Example 1;
[0058] (5) Add a 50 mg / mL potassium hydroxide solution to the precipitate to dissolve it and adjust the pH of the solution to 6.5-7.5; filter the solution through a 0.22 μm polypropylene microporous membrane to remove insoluble particles; (6) Add activated carbon to the solution obtained in step (5) at a weight ratio of 2:1 for potassium alginate oligosaccharide and activated carbon, stir mechanically for 1 hour at room temperature, and filter the activated carbon solution through a microporous membrane to remove carbon, thereby obtaining an alginate oligosaccharide solution;
[0059] (7) Repeat step (6) 3 times;
[0060] (8) Under sterile conditions, 600 mL of anhydrous ethanol was slowly added to 60 mL of the alginate oligosaccharide solution from step (7) to form a flocculent precipitate of alginate oligosaccharide. The precipitate was freeze-dried to obtain the endotoxin-free alginate oligosaccharide material. The endotoxin content of the prepared potassium alginate oligosaccharide was 11 EU / g.
[0061] Comparative Example 1
[0062] The preparation method of sodium alginate oligosaccharide in this comparative example is basically the same as that in Example 3, except that step (3) is omitted, i.e., mixed Lewis acid treatment is not used. The endotoxin content of the obtained sodium alginate oligosaccharide is 28300 EU / g. Figure 1 Sample B in Comparative Example 1). Compared with the sodium alginate oligosaccharide prepared in Example 3, the sodium alginate oligosaccharide prepared in Comparative Example 1 had a higher endotoxin content.
[0063] Comparative Example 2
[0064] The preparation method of the sodium alginate oligosaccharide in this comparative example is the same as that in Example 3, except that step (3) only uses aluminum chloride treatment (the amount used is the same as the mass of the mixed Lewis acid in Example 3), and does not use mixed Lewis acid treatment. The endotoxin content of the obtained sodium alginate oligosaccharide is 22400 EU / g ( Figure 1 Sample C). Compared with the sodium alginate oligosaccharide prepared in Example 3, the sodium alginate oligosaccharide prepared in Comparative Example 2 had a higher endotoxin content.
[0065] Comparative Example 3
[0066] The preparation method of the potassium alginate oligosaccharide in this comparative example is the same as that in Example 4, except that step (3) only uses zinc chloride treatment (the amount used is the same as the mass of the mixed Lewis acid in Example 4), and does not use mixed Lewis acid treatment. The endotoxin content of the obtained potassium alginate oligosaccharide is 21800 EU / g. Figure 1 Sample D). Compared with the potassium alginate oligosaccharide prepared in Example 4, the potassium alginate oligosaccharide prepared in Comparative Example 3 had a higher endotoxin content.
[0067] The above comparative examples show that treatment with mixed Lewis amino acids can significantly improve the endotoxin removal efficiency in alginate oligosaccharides, resulting in purer alginate oligosaccharides.
Claims
1. A method for removing endotoxins from alginate oligosaccharides, characterized in that: Commercially available alginate oligosaccharides were used as raw materials, dissolved in water, and insoluble particles were removed by centrifugation. A mixed Lewis acid was added for co-treatment. The treated solution was acidified to form a precipitate, which was then washed with deionized water. An alkaline solution was added to dissolve the precipitate, and the solution was filtered. The filtrate was adsorbed onto an adsorption medium, and the alginate oligosaccharides were precipitated with an organic solvent and dried to obtain endotoxin-free alginate oligosaccharides. The specific process of the mixed Lewis acid treatment was as follows: mixed Lewis acids were added at a weight ratio of 100:1 to 1:
1. o Mechanically stir at room temperature for 0.5-48 hours; the mixed Lewis acid is a mixture of aluminum chloride and zinc chloride in a molar ratio of 0.5:1-2:
1.
2. The method according to claim 1, characterized in that: The alginate oligosaccharide is one or more of sodium alginate oligosaccharide and potassium alginate oligosaccharide, and its degree of polymerization is 2-50.
3. The method according to claim 1, characterized in that: The specific procedure for removing insoluble particles from alginate oligosaccharides by centrifugation is as follows: 1) Dissolve commercially available alginate oligosaccharides in water to a w / v concentration of 1-500 mg / mL; 2) Centrifuge the alginate oligosaccharide solution at a centrifugal force of 1000-20000g to remove insoluble impurities.
4. The method according to claim 1, characterized in that: The specific procedure for acidifying the solution to form a precipitate is to adjust the pH of the solution to below 3 using one or more of hydrochloric acid, sulfuric acid, or nitric acid, so that the alginate oligosaccharide is acidified and precipitates out.
5. The method according to claim 1, characterized in that: The specific procedure for adding an alkaline solution to dissolve the precipitate and filtering is as follows: 1) Add an alkaline solution with a concentration of 1-300 mg / mL to the precipitate to dissolve it, and the pH of the solution is 6.5-7.5; filter the alginate oligosaccharide solution through a 0.22 μm microporous membrane to remove insoluble particles. The alkaline solution is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate solutions.
6. The method according to claim 1, characterized in that: The specific adsorption process of the adsorption medium is as follows: 1) Add the adsorption medium at a weight ratio of sodium alginate oligosaccharide to adsorption medium of 10:1-1:10; 4 o Mechanically stir the solution at room temperature for 0.5-48 hours, then filter the solution containing the adsorption medium through a microporous membrane to remove the adsorption medium, thus obtaining an alginate oligosaccharide solution. 2) Repeat step 1) 0-10 times; 3) Filter the alginate solution through a 0.22μm filter membrane to obtain a sterile alginate oligosaccharide solution; The adsorption medium is one of activated carbon, kaolin, diatomaceous earth, polymyxin B agarose resin, or a mixture of two or more of them in any proportion.
7. The method according to claim 5, characterized in that: The microporous filter membrane is one or more of the following: cellulose filter membrane, polypropylene filter membrane, polyethersulfone filter membrane, polytetrafluoroethylene filter membrane, and ceramic filter membrane.
8. The method according to claim 1, characterized in that: The specific process of precipitating and drying alginate oligosaccharides with organic solvent is as follows: Under sterile conditions, organic solvent is slowly added to the alginate oligosaccharide solution after adsorption by adsorbent, so that the alginate oligosaccharides form a precipitate. The precipitate is dried to obtain alginate oligosaccharide material with endotoxin removed. The organic solution is one or more of the following: alcohols, ethers, and ketones of C1-C3, or a mixture of two or more of them in any proportion. The volume ratio of the alginate oligosaccharide solution to the organic solution is 1:2 to 1:
10.
9. The method according to claim 8, characterized in that: The drying method is one or more of the following: freeze drying, vacuum drying, and oven drying.