Method for preparing modified carrageenan by slurry method and application thereof

Abstract

The application discloses a method for preparing modified carrageenan by using a slurry method and application thereof. The method comprises the following steps: uniformly mixing potassium salt, deionized water, an oxidant and carrageenan to obtain a slurry, preparing modified carrageenan by using the slurry oxidation method, and improving the stability of the modified carrageenan in the oxidation degradation reaction through the electrostatic action between potassium ions and sulfate radicals in the carrageenan, so that the content of the sulfate radicals in the modified carrageenan is improved. The method uses a single hydrogen peroxide oxidant, has simple and efficient preparation, low solvent consumption, is green and environment-friendly, and has the dual advantages of low molecular weight and high sulfate radical content of the prepared modified carrageenan, and has a wide application prospect in the field of anticoagulation.

Description

Technical Field

[0001] This invention relates to the technical field of polysaccharide processing, specifically to a method for preparing modified carrageenan using a slurry method and its application. Background Technology

[0002] Carrageenan is a linear sulfated polysaccharide extracted from red algae. Carrageenan is composed of alternating units of 3-β-D-galactose and 4-linked α-D-galactopyran or 4-linked 3,6-endoether-α-D-galactopyran. Based on the constituent units and the number and position of sulfate groups in the disaccharide units, carrageenan can be broadly classified into kappa (κ), iota (ι), lambda (λ), mu (μ), nu (ν), gamma (γ), alpha (α), delta (δ), theta (θ), and beta (β) carrageenan. Among these, the three most common commercially available carrageenan types are κ, ι, and λ carrageenan, with 1, 2, and 3 sulfate groups in each repeating disaccharide unit, respectively.

[0003] Natural carrageenan has a molecular weight of approximately 450,000 to 650,000 Daltons and is commonly used as a thickener, gelling agent, suspending agent, emulsifier, and stabilizer. However, its high molecular weight limits its applications to some extent. Degrading natural carrageenan yields low molecular weight carrageenan. Compared to raw carrageenan, low molecular weight carrageenan exhibits better solubility, absorption efficiency, and bioavailability. Furthermore, low molecular weight carrageenan displays favorable biological activities, including antibacterial, anticoagulant, antioxidant, and antiviral properties, opening up possibilities for its application in the pharmaceutical field. On the other hand, carrageenan contains a large number of negatively charged sulfate ions, which can enhance the affinity of antithrombin III (AT-III) for activated coagulation factors such as thrombin, thereby producing in vitro and in vivo anticoagulant effects. Therefore, carrageenan can potentially inhibit platelet aggregation. Literature studies have shown that the introduction of sulfate ions can enhance the biological activity of polysaccharides, including antiviral, anticoagulant, antioxidant, and antitumor effects. Therefore, increasing the sulfate concentration could potentially enhance the bioactivity and function of carrageenan, thereby expanding its applications in antiviral, anticoagulant, antioxidant, and antitumor activities. Thus, obtaining a carrageenan with low molecular weight and high sulfate content is of great significance.

[0004] Currently, the main methods for obtaining low molecular weight polysaccharides include physical methods, enzymatic methods, and chemical methods. Physical methods mainly refer to ultrasonic and high-temperature degradation methods, but their disadvantage is poor selectivity. Enzymatic methods use biologically active enzymes to break down carrageenan macromolecules, but enzyme preparations are often expensive and their activity is easily lost. Chemical methods are low-cost, simple in process, and have good scalability.

[0005] Chemical methods for preparing low-molecular-weight sulfated polysaccharides mainly include acid hydrolysis and free radical degradation. Among these, the free radical degradation method commonly used is the transition metal ion combined with H₂O₂ degradation. Compared with acid hydrolysis, the degradation of sulfated polysaccharides by transition metal ions combined with an appropriate concentration of H₂O₂ not only reduces the molecular weight but also effectively avoids the removal of sulfate ions. 2+ The -H2O2 system is one of the most widely used degradation methods. For example, homogeneous Cu... 2+ The -H2O2 system can effectively degrade fucoidan and increase its sulfate content; however, the concentration of fucoidan in this reaction system can only reach 6.67% (w / v), resulting in low reaction efficiency (Research on the chemical composition and activity of kelp fucoidan sulfate, Publication Date: 20040610; Research progress on the preparation of low molecular weight fucoidan and fucoidan oligosaccharides by chemical degradation of fucoidan, Publication Date: 20240719). Furthermore, Cu 2+ It is toxic and poses certain safety risks.

[0006] While reducing the molecular weight of sulfated polysaccharides plays a crucial role in enhancing their bioactivity, studies have reported that degraded fucoidan sulfate with a molecular weight around 2 kDa exhibits significantly lower anticoagulant activity than degraded fucoidan sulfate with molecular weights of 8 kDa and 200 kDa (Study on the Chemical Composition and Activity of Laminaria Fucoidan Sulfate, Publication Date: 20040610). For carrageenan, research also indicates that while partially oxidized carrageenan has a higher molecular weight than fully oxidized carrageenan, partially oxidized carrageenan demonstrates better anticoagulant effects (PMID: 30925999). Therefore, sulfated polysaccharides or carrageenan with moderate molecular weights generally exhibit better anticoagulant effects.

[0007] Therefore, it is of great significance to develop a method that can prepare carrageenan with low molecular weight and high sulfate content while ensuring its anticoagulant activity. Summary of the Invention

[0008] To overcome the aforementioned defects and shortcomings in the existing technology, the present invention provides a method for preparing modified carrageenan using the slurry method and its application.

[0009] The first objective of this invention is to provide a method for preparing high concentrations of modified carrageenan using a slurry method.

[0010] A second objective of this invention is to provide modified carrageenan prepared by the above-described method.

[0011] A third objective of this invention is to provide the application of the above-described modified carrageenan in the preparation of anticoagulants.

[0012] Therefore, this invention claims protection for the following:

[0013] A method for preparing modified carrageenan using a slurry method involves thoroughly mixing potassium salt, water, oxidant, and carrageenan to obtain a slurry, followed by reaction and purification.

[0014] Preferably, the mass-to-volume ratio of carrageenan, water, and potassium salt is 4g:(0-16)mL:(4-400)mg.

[0015] More preferably, the water is deionized water.

[0016] Preferably, the potassium salt is any one of potassium sulfate, potassium nitrate, or potassium dihydrogen phosphate.

[0017] Preferably, the thorough mixing to obtain the slurry is carried out at a temperature of 15–40°C.

[0018] More preferably, the thorough mixing to obtain the slurry is carried out at 30°C.

[0019] Preferably, the reaction is carried out at 64–66°C for 35–45 min, or at 24–26°C for 890–910 min.

[0020] More preferably, the reaction is carried out at 65°C for 40 min, or at 25°C for 900 min.

[0021] Preferably, the carrageenan is iota-carrageenan, Kappa-carrageenan, or Lambda-carrageenan.

[0022] Preferably, the oxidant is a hydrogen peroxide solution with a mass fraction of 28-32%.

[0023] More preferably, the oxidant is a hydrogen peroxide solution with a mass fraction of 30%.

[0024] Preferably, the mass-to-volume ratio of carrageenan to hydrogen peroxide solution is 4 g:(8-26.7) mL.

[0025] More preferably, the carrageenan is iota-carrageenan, and the mass-volume ratio of the iota-carrageenan, water, potassium salt and hydrogen peroxide solution is 4g:(0-16)mL:4mg:(8-26.7)mL, the potassium salt is any one of potassium sulfate, potassium nitrate or potassium dihydrogen phosphate, and the reaction is carried out at 65°C for 40min.

[0026] Alternatively, the carrageenan may be iota-carrageenan, and the mass-volume ratio of iota-carrageenan, water, potassium salt, and hydrogen peroxide solution may be 4g:(0-16)mL:40mg:(8-26.7)mL, the potassium salt may be potassium nitrate, and the reaction may be carried out at 65°C for 40min.

[0027] Under this preferred scheme, compared with the unmodified iota-carrageenan, the prepared modified iota-carrageenan has a sulfate content increased by 29.7-47.1% and a viscosity-average molecular weight decreased by 83.2-95.3%.

[0028] More preferably, the carrageenan is Kappa-carrageenan, and the mass-to-volume ratio of Kappa-carrageenan, water, potassium salt, and hydrogen peroxide solution is 4g:8mL:4mg:16mL, the potassium salt is potassium sulfate, and the reaction is carried out at 65°C for 40 min. Under this preferred embodiment, compared with unmodified Kappa-carrageenan, the prepared modified Kappa-carrageenan has a 27.3% higher sulfate content and a 91.2% lower average molecular weight.

[0029] More preferably, the carrageenan is Lambda-carrageenan, and the mass-to-volume ratio of Lambda-carrageenan, water, potassium salt, and hydrogen peroxide solution is 4g:8mL:4mg:16mL, the potassium salt is potassium sulfate, and the reaction is carried out at 65°C for 40 min. Under this preferred embodiment, compared with unmodified Lambda-carrageenan, the prepared modified Lambda-carrageenan has a 63.0% higher sulfate content and a 46.2% lower average molecular weight.

[0030] Preferably, the purification includes removing potassium ions using a chelating agent, dialysis, filtration, and drying.

[0031] As one feasible approach, the chelating agent is sodium tetraphenylborate.

[0032] Preferably, the ratio of sodium tetraphenylborate to potassium salt is 0.2 mol: (4-400) g.

[0033] Compared to the commonly used homogeneous aqueous solution degradation method in existing technologies (where carrageenan typically needs to be dissolved in a high-temperature aqueous solution above 90°C), this invention employs a heterogeneous slurry method. In the method of this invention:

[0034] (1) Carrageenan powder is directly mixed with a solution of potassium salt, deionized water and 30% hydrogen peroxide solution. High temperature is not used to dissolve the carrageenan. As the oxidation reaction proceeds, the molecular weight of the carrageenan decreases significantly. It will spontaneously dissolve during the dialysis purification process, which greatly saves heat energy.

[0035] (2) In the homogeneous aqueous solution degradation method, Kappa-carrageenan and iota-carrageenan spontaneously react with the introduced potassium ions to form cross-linked macrohydrogels, making it impossible to effectively remove residual potassium ions and perform dialysis purification of modified carrageenan. The slurry method of the present invention effectively solves the technical problem of the spontaneous formation of cross-linked hydrogels by the introduced potassium ions with Kappa-carrageenan and iota-carrageenan.

[0036] (3) The concentration of reactants is high and the amount of solvent is small in the slurry method, that is, the solid-liquid ratio is high, and the reaction efficiency is significantly improved.

[0037] Modified carrageenan prepared by any of the methods described above.

[0038] The above-mentioned modified carrageenan is used in the preparation of anticoagulants.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention discloses a method for preparing modified carrageenan using a slurry method and its applications. The method involves mixing potassium salt, deionized water, and an oxidant with carrageenan to obtain a slurry. Modified carrageenan is then prepared via a slurry oxidation method. The stability of the modified carrageenan in the oxidative degradation reaction is improved through the electrostatic interaction between potassium ions and sulfate ions in the carrageenan, thereby increasing the sulfate content. This invention uses a single hydrogen peroxide oxidant, resulting in a simple, efficient, and environmentally friendly preparation method with low solvent consumption. The modified carrageenan prepared possesses both low molecular weight and high sulfate content, making it promising for broad applications in the anticoagulant field. Attached Figure Description

[0041] Figure 1 The modified iota-carrageenan prepared in Comparative Example 7 is shown in its cooled state.

[0042] Figure 2 The modified iota-carrageenan prepared in Comparative Example 8 is shown in its cooled state.

[0043] Figure 3 The modified iota-carrageenan prepared in Comparative Example 9 is shown in its cooled state.

[0044] Figure 4 The modified iota-carrageenan prepared in Comparative Example 10 is shown in its cooled state.

[0045] Figure 5 The modified iota-carrageenan prepared in Comparative Example 11 is shown in its cooled state.

[0046] Figure 6 The modified Kappa-carrageenan prepared in Comparative Example 12 is shown in its cooled state.

[0047] Figure 7 The modified Kappa-carrageenan prepared in Comparative Example 13 is shown in its cooled state.

[0048] Figure 8 The modified iota-carrageenan prepared in Comparative Example 14 is shown in its reaction state.

[0049] Figure 9 The modified iota-carrageenan prepared in Comparative Example 14 is shown in its cooled state.

[0050] Figure 10 The modified Kappa-carrageenan prepared in Comparative Example 15 is shown in its cooled state.

[0051] Figure 11 The modified Lambda-carrageenan prepared in Comparative Example 16 is shown in its reaction state.

[0052] Figure 12 The modified Lambda-carrageenan prepared in Comparative Example 16 is shown in its cooled state.

[0053] Figure 13 The states of 8%, 9%, and 10% Lambda-carrageenan solutions (w / v) when cooled to 75°C. Detailed Implementation

[0054] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0055] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0056] Example 1: Preparation of modified iota-carrageenan

[0057] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0058] Example 2: Preparation of modified iota-carrageenan

[0059] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 40mg of potassium sulfate. Stir rapidly at 40℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.025mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0060] Example 3: Preparation of modified iota-carrageenan

[0061] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 400mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.025mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0062] Example 4: Preparation of modified iota-carrageenan

[0063] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 25℃ water bath and react for 900min. After the reaction, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove solid particles of potassium tetraphenylborate, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0064] Example 5: Preparation of modified iota-carrageenan

[0065] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium nitrate. Stir rapidly at 15℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0066] Example 6: Preparation of Modified iota-carrageenan

[0067] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 40mg of potassium nitrate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.025mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0068] Example 7: Preparation of Modified iota-carrageenan

[0069] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 400mg of potassium nitrate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.025mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0070] Example 8: Preparation of Modified iota-carrageenan

[0071] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium nitrate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 25℃ water bath and react for 900min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0072] Example 9: Preparation of Modified iota-carrageenan

[0073] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium dihydrogen phosphate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0074] Example 10: Preparation of Modified iota-carrageenan

[0075] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 40mg of potassium dihydrogen phosphate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.025mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0076] Example 11 Preparation of modified iota-carrageenan

[0077] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 400mg of potassium dihydrogen phosphate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.025mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0078] Example 12 Preparation of modified iota-carrageenan

[0079] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium dihydrogen phosphate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 25℃ water bath and react for 900min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0080] Example 13 Preparation of modified Kappa-carrageenan

[0081] Weigh 4g of Kappa-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified Kappa-carrageenan.

[0082] Example 14 Preparation of modified Kappa-carrageenan

[0083] Weigh 4g of Kappa-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 25℃ water bath and react for 900min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified Kappa-carrageenan.

[0084] Example 15 Preparation of modified Lambda-carrageenan

[0085] Weigh 4g of Lambda-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified Lambda-carrageenan.

[0086] Example 16 Preparation of Modified Lambda-Carrageenan

[0087] Weigh 4g of Lambda-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 25℃ water bath and react for 900min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove solid particles of potassium tetraphenylborate, and then freeze-dry for two days to obtain modified Lambda-carrageenan.

[0088] Example 17 Preparation of modified iota-carrageenan

[0089] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 16mL deionized water, 8mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0090] Example 18 Preparation of modified iota-carrageenan

[0091] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution of 24mL of 30% hydrogen peroxide solution (w / v) and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0092] Example 19 Preparation of modified iota-carrageenan

[0093] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 6.7mL deionized water, 13.3mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0094] Example 20 Preparation of modified iota-carrageenan

[0095] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 13.3mL deionized water, 26.7mL of 30% hydrogen peroxide solution (w / v), and 4mg of potassium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction is complete, add 40mL of 0.005mol / L sodium tetraphenylborate solution and stir for 30min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove potassium tetraphenylborate solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0096] Comparative Example 1

[0097] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of copper acetate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction, add 40mL of 0.05mol / L NaOH solution and stir for 30min. Adjust the pH to neutral with acetic acid to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove copper hydroxide solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0098] Comparative Example 2

[0099] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 40mg of copper acetate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40min. After the reaction, add 40mL of 0.1mol / L NaOH solution and stir for 30min. Adjust the pH to neutral with acetic acid to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove copper hydroxide solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0100] Comparative Example 3

[0101] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of copper acetate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 25℃ water bath and react for 900min. After the reaction, add 40mL of 0.05mol / L NaOH solution and stir for 30min. Adjust the pH to neutral with acetic acid to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. After dialysis, filter to remove copper hydroxide solid particles, and then freeze-dry for two days to obtain modified iota-carrageenan.

[0102] Comparative Example 4

[0103] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 4mg of sodium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40 minutes to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. Then, freeze-dry for two days to obtain modified iota-carrageenan.

[0104] Comparative Example 5

[0105] Weigh 4g of iota-carrageenan powder into a beaker, then add a solution consisting of 8mL deionized water, 16mL of 30% hydrogen peroxide solution (w / v), and 40mg of sodium sulfate. Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a water bath at 25℃ and react for 900min to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. Then, freeze-dry for two days to obtain modified iota-carrageenan.

[0106] Comparative Example 6

[0107] Weigh 4g of iota-carrageenan powder into a beaker, then add 8mL of deionized water and 16mL of 30% hydrogen peroxide solution (w / v). Stir rapidly at 30℃ to obtain a slurry mixture. Place the beaker in a 65℃ water bath and react for 40 minutes to obtain a mixed slurry. Dialyze the obtained mixed slurry with deionized water for three days, changing the external deionized water three times a day. Then, freeze-dry for two days to obtain modified iota-carrageenan.

[0108] Performance Test 1

[0109] I. Experimental Methods

[0110] Standard solutions: Prepare Na2SO4 standard solutions with concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL;

[0111] Agarose-BaCl2 reagent: Prepare a 0.01% agarose solution (w / v) by adding 0.5g BaCl2 to 100mL of agarose solution. After preparation, store in a refrigerator at 4℃ overnight.

[0112] Prepare a 1 mg / mL sample solution (using 0.1 M HCl solution as solvent) and hydrolyze it at 90℃ for 6 h. Take 1.1 mL of the hydrolyzed sample solution, add 1.2 mL of 8% trichloroacetic acid solution (w / v) and 0.6 mL of agarose-BaCl2 reagent, mix well, and let stand for 35 min. Use water as a control and measure the absorbance at a wavelength of 500 nm. The sample solution needs to be shaken before the test.

[0113] The prepared standard solution was tested according to the above detection method, the absorbance was recorded, a standard curve was plotted, and the regression equation was obtained. The sulfate content was obtained by substituting the absorbance value of the sample into the regression equation.

[0114] Each sample was measured three times, and the average value was taken as the final result. The blank samples were unmodified iota-carrageenan, Kappa-carrageenan, and Lambda-carrageenan.

[0115] II. Experimental Results

[0116] The results are shown in Table 1. As can be seen from Table 1, the sulfate content of the modified carrageenan prepared in Examples 1-20 was higher than that of the unmodified carrageenan. Different types of potassium salts had varying degrees of influence on the increase in sulfate content. Compared to unmodified iota-carrageenan, the modification with potassium sulfate and hydrogen peroxide resulted in the most significant increase in sulfate content, with a maximum increase of 47.1%. However, the modification with copper ions and hydrogen peroxide did not significantly alter the sulfate content of the unmodified iota-carrageenan.

[0117] Besides potassium sulfate, potassium nitrate and potassium dihydrogen phosphate can also increase the sulfate content in modified iota-carrageenan. The best improvement effect is achieved when the mass-to-volume ratio of carrageenan powder, deionized water, potassium salt, and hydrogen peroxide solution is 1 g: 2 mL: 1 mg: 4 mL.

[0118] Potassium ions also have a positive effect on increasing the sulfate content in modified Kappa-carrageenan and modified Lambda-carrageenan. When Kappa-carrageenan and Lambda-carrageenan were modified with potassium sulfate and hydrogen peroxide, respectively, and the mass-volume ratio of carrageenan powder, deionized water, potassium salt, and hydrogen peroxide solution was 1 g:2 mL:1 mg:4 mL, the sulfate content of modified Kappa-carrageenan and modified Lambda-carrageenan increased by 27.3% and 63.0% respectively compared to unmodified Kappa-carrageenan and Lambda-carrageenan.

[0119] The above results indicate that modifying carrageenan with potassium salts and hydrogen peroxide can significantly increase the sulfate content in the modified carrageenan.

[0120] Table 1. Sulfate content of modified carrageenan prepared in Examples 1-20 and Comparative Examples 1-6

[0121] Group Sulfate content / w% Group Sulfate content / w% Example 1 38.84±0.02 Example 16 33.14±0.04 Example 2 27.93±0.10 Example 17 36.59±0.14 Example 3 27.69±0.04 Example 18 34.23±0.11 Example 4 30.12±0.05 Example 19 36.74±0.09 Example 5 37.16±0.11 Example 20 36.42±0.06 Example 6 36.79±0.08 Comparative Example 1 24.95±0.13 Example 7 28.63±0.10 Comparative Example 2 22.49±0.29 Example 8 30.41±0.08 Comparative Example 3 19.97±0.16 Example 9 38.30±0.16 Comparative Example 4 24.22±0.17 Example 10 30.62±0.19 Comparative Example 5 25.39±0.22 Example 11 27.43±0.21 Comparative Example 6 25.01±0.09 Example 12 28.32±0.18 Unmodified iota-carrageenan 26.40±0.05 Example 13 26.45±0.14 Unmodified Kappa-carrageenan 20.78±0.07 Example 14 22.78±0.02 Unmodified Lambda-carrageenan 24.62±0.05 Example 15 40.12±0.05

[0122] Performance Test 2

[0123] I. Experimental Methods

[0124] The viscosity-average molecular weight (Mη) of modified carrageenan was determined by viscometry at 35 ± 0.1 °C in a 0.7% Na₂SO₄ aqueous solution (w / v). Under certain temperature and solvent conditions, the relationship between intrinsic viscosity [η] and polymer molar mass M is the Mark-Houwink empirical equation:

[0125]

[0126] The viscosity-average molecular weight of the modified carrageenan was measured using an Ubbelohde viscometer. The carrageenan was dissolved in a 0.7% Na2SO4 aqueous solution (w / v) at a concentration between 0.1% and 0.2% (w / v) and the temperature was maintained at 35±0.1℃.

[0127] Based on experiments, the following empirical formula applies to sufficiently dilute polymer solutions:

[0128]

[0129] In the formula, κ and β are called Huggins and Kramer constants, respectively.

[0130] Furthermore, since the determination is usually carried out in dilute solutions, where the density of the solution and the density of the solvent are approximately equal, η can be... r Written as:

[0131]

[0132] t is the time it takes for the liquid surface to flow from graduation a to graduation b when measuring the solution viscosity; t0 is the time it takes for the pure solvent to flow through; therefore, η can be obtained from the above formula by measuring the outflow time of the solvent and solution in the capillary. r Then, [η] is obtained by extrapolation.

[0133] The blank samples were unmodified iota-carrageenan, Kappa-carrageenan, and Lambda-carrageenan.

[0134] II. Experimental Results

[0135] The results are shown in Table 2. The original molecular weights (Mη) of unmodified iota-carrageenan, kappa-carrageenan, and lambda-carrageenan were 558.271±0.970 kDa, 610.194±0.617 kDa, and 1133.247±6.098 kDa, respectively. Compared with unmodified carrageenan, the molecular weight of the modified carrageenan prepared by modifying carrageenan with potassium salt and hydrogen peroxide was significantly reduced.

[0136] Although modifying iota-carrageenan with copper ions and hydrogen peroxide can reduce the molecular weight of the prepared modified iota-carrageenan to below 10 kDa, modified carrageenan within this molecular weight range has poor stability due to its small molecular weight. It is prone to moisture absorption and browning after prolonged storage, and browning may be a deterioration phenomenon of carrageenan.

[0137] Furthermore, based on existing technological disclosures (Research on the chemical composition and activity of kelp fucoidan sulfate, Publication Date: 20040610; PMID: 30925999, Publication Date: 20190319), a small molecular weight of carrageenan can also affect its anticoagulant activity. Therefore, the low molecular weight modified iota-carrageenan prepared using copper ions and hydrogen peroxide not only suffers from deterioration but may also have its anticoagulant properties affected. In contrast, the modified carrageenan prepared in Examples 1-20 of this invention has a high sulfate content and a suitable molecular weight, avoiding the aforementioned drawbacks, and has good application prospects in the preparation of anticoagulants.

[0138] Table 2 shows the molecular weights of the modified carrageenan prepared in Examples 1-20 and Comparative Examples 1-6.

[0139] Group Mη(KDa) Group Mη(KDa) Example 1 73.593±0.204 Example 16 594.170±3.063 Example 2 17.293±0.028 Example 17 48.461±0.938 Example 3 21.490±0.026 Example 18 53.668±0.250 Example 4 77.839±0.060 Example 19 48.355±0.138 Example 5 65.024±0.018 Example 20 26.257±0.189 Example 6 93.523±0.488 Comparative Example 1 2.148±0.144 Example 7 110.292±0.292 Comparative Example 2 2.321±0.132 Example 8 69.362±0.101 Comparative Example 3 3.602±0.030 Example 9 50.683±0.336 Comparative Example 4 80.049±0.504 Example 10 155.715±0.377 Comparative Example 5 95.704±1.298 Example 11 60.141±0.166 Comparative Example 6 156.746±0.158 Example 12 75.867±0.059 Unmodified iota-carrageenan 558.271±0.970 Example 13 53.438±0.509 Unmodified Kappa-carrageenan 610.194±0.617 Example 14 153.892±0.560 Unmodified Lambda-carrageenan 1133.247±6.098 Example 15 609.608±2.045

[0140] Comparative Example 7

[0141] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified iota-carrageenan:

[0142] 1.00 g of iota-carrageenan powder was dissolved in 50.00 mL of deionized water to obtain a 2% carrageenan solution (w / v). Then, a solution consisting of 2 mL of deionized water, 4 mL of 30% hydrogen peroxide solution (w / v), and 1 mg of potassium sulfate was added. The beaker was placed in a water bath at 65°C and reacted for 40 min. After the reaction was complete and cooled to room temperature, the system was found to be in a soft gel state with strong fluidity. Figure 1 ).

[0143] Comparative Example 8

[0144] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified iota-carrageenan:

[0145] Weigh 1.00 g of iota-carrageenan powder and dissolve it in 50.00 mL of deionized water to obtain a 2% carrageenan solution (w / v). Then, add a solution consisting of 2 mL of deionized water, 4 mL of 30% hydrogen peroxide solution (w / v), and 10 mg of potassium sulfate. Place the beaker in a water bath at 65°C and react for 40 min. After the reaction is complete and cooled to room temperature, the system is found to be in a soft gel state with moderate fluidity. Figure 2 ).

[0146] Comparative Example 9

[0147] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified iota-carrageenan:

[0148] 1.00 g of iota-carrageenan powder was dissolved in 50.00 mL of deionized water to obtain a 2% carrageenan solution (w / v). Then, a solution consisting of 2 mL of deionized water, 4 mL of 30% hydrogen peroxide solution (w / v), and 100 mg of potassium sulfate was added. The beaker was placed in a water bath at 65°C and reacted for 40 min. After the reaction was completed and cooled to room temperature, the system was found to be in a soft gel state with weak fluidity. Figure 3 ).

[0149] Comparative Example 10

[0150] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified iota-carrageenan:

[0151] Weigh 2.50 g of iota-carrageenan powder and dissolve it in 50.00 mL of deionized water to obtain a 5% carrageenan solution (w / v). Then, add a solution consisting of 5 mL of deionized water, 10 mL of 30% hydrogen peroxide solution (w / v), and 25 mg of potassium sulfate. Place the beaker in a water bath at 65°C and react for 40 min. After the reaction is complete and cooled to room temperature, the system is found to be a soft, blocky gel, but without flowability. Figure 4 ).

[0152] Comparative Example 11

[0153] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified iota-carrageenan:

[0154] 5.00 g of iota-carrageenan powder was dissolved in 50.00 mL of deionized water to obtain a 10% carrageenan solution (w / v). Then, a solution consisting of 10 mL of deionized water, 20 mL of 30% hydrogen peroxide solution (w / v), and 50 mg of potassium sulfate was added. The beaker was placed in a 65°C water bath and reacted for 40 min. After the reaction was completed and cooled to room temperature, the system was found to be a soft, blocky gel with no flowability. Because the homogeneous dissolution of carrageenan has a high concentration (reaching 10%, w / v), complete dissolution is only possible under high temperature conditions, and partial gelation of the solution occurs rapidly after heating is stopped. The addition of hydrogen peroxide-potassium sulfate solution exacerbates the gelation phenomenon, making it difficult to achieve a completely homogeneous reaction. Figure 5 ).

[0155] Comparative Example 12

[0156] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified Kappa-carrageenan:

[0157] Weigh 1.00 g of Kappa-carrageenan powder and dissolve it in 50.00 mL of deionized water to obtain a 2% carrageenan solution (w / v). Then, add a solution consisting of 2 mL of deionized water, 4 mL of 30% hydrogen peroxide solution (w / v), and 1 mg of potassium sulfate. Place the beaker in a water bath at 65°C and react for 40 min. After the reaction is complete and cooled to room temperature, the system forms a solid, blocky gel with no flowability. Figure 6 ).

[0158] Comparative Example 13

[0159] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified Kappa-carrageenan:

[0160] Weigh 1.00 g of Kappa-carrageenan powder and dissolve it in 50.00 mL of deionized water to obtain a 2% carrageenan solution (w / v). Then, add a solution consisting of 2 mL of deionized water, 4 mL of 30% hydrogen peroxide solution (w / v), and 10 mg of potassium sulfate. Place the beaker in a water bath at 65°C and react for 40 min. After the reaction is complete and cooled to room temperature, the system forms a solid, blocky gel with no flowability. Figure 7 ).

[0161] Comparative Example 14

[0162] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified iota-carrageenan:

[0163] Weigh 2.50 g of iota-carrageenan powder and dissolve it in 50.00 mL of deionized water to obtain a 5% carrageenan solution (w / v). Then, add a solution consisting of 5 mL of deionized water, 10 mL of 30% hydrogen peroxide solution (w / v), and 25 mg of potassium sulfate. Place the beaker in a 70°C water bath and react for 40 min. After the reaction is complete and cooled to room temperature, the system forms a blocky, flexible gel with no flowability. Figure 8 and Figure 9 ).

[0164] Comparative Example 15

[0165] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified Kappa-carrageenan:

[0166] Weigh 2.50 g of Kappa-carrageenan powder and dissolve it in 50.00 mL of deionized water to obtain a 5% carrageenan solution (w / v). Then, add a solution consisting of 2 mL of deionized water, 4 mL of 30% hydrogen peroxide solution (w / v), and 25 mg of potassium sulfate. Place the beaker in a water bath at 75°C and react for 40 min. After the reaction is complete and cooled to room temperature, the system forms a solid, blocky gel with no flowability. Figure 10 ).

[0167] Comparative Example 16

[0168] Unlike the slurry method used in Examples 1-20, this comparative example uses a homogeneous aqueous solution degradation method to prepare modified Lambda-carrageenan:

[0169] Weigh 2.50 g of Lambda-carrageenan powder and dissolve it in 50.00 mL of deionized water to obtain a 5% carrageenan solution (w / v). Then, add a solution consisting of 5 mL of deionized water, 10 mL of 30% hydrogen peroxide solution (w / v), and 25 mg of potassium sulfate. Place the beaker in a water bath at 75°C and react for 40 min. After the reaction is complete, cool to room temperature; the system will be in solution form. Figure 11 and Figure 12 ).

[0170] The homogeneous aqueous solution degradation methods in Comparative Examples 7–16 require prolonged high temperatures of 90°C to completely dissolve iota-carrageenan, kappa-carrageenan, and lambda-carrageenan, a process that consumes a large amount of energy.

[0171] Comparative Examples 7–15 show that when the concentration of iota-carrageenan and kappa-carrageenan solutions increases to 5% (w / v) and the temperature decreases to 65–75°C, both iota-carrageenan and kappa-carrageenan form large macroscopic gels that cannot be effectively mixed with the hydrogen peroxide-potassium salt solution. Furthermore, the large volume of the resulting gels makes direct dialysis purification impossible; they must be broken into smaller pieces for purification, which is unfavorable for large-scale preparation. When the concentration of iota-carrageenan and kappa-carrageenan solutions is further increased to 10% (w / v), the gelation process intensifies further. Even at 90°C, complete dissolution takes a long time, and partial gelation occurs rapidly after heating is stopped, resulting in an inhomogeneous system. Adding hydrogen peroxide-potassium sulfate solution exacerbates the gelation, and upon cooling, a non-flowing gel is formed, making complete homogeneous reaction and subsequent purification difficult.

[0172] Comparative Example 16 shows that although 5% Lambda-carrageenan (w / v) is in a solution state after the reaction, its high viscosity results in poor flowability during the reaction. Furthermore, the 8%, 9%, and 10% Lambda-carrageenan solutions (w / v) lose their flowability at 75°C. Figure 13 It cannot be mixed evenly, and cannot be effectively mixed and reacted.

[0173] However, under the same conditions, in the slurry method, the concentration of iota-carrageenan, Kappa-carrageenan, and Lambda-carrageenan in the hydrogen peroxide-potassium salt solution is usually between 10% and 20% (w / v).

[0174] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing modified carrageenan using a slurry method, characterized in that, Potassium salt, water, oxidant and carrageenan were thoroughly mixed to obtain a slurry, which was then reacted and purified. The potassium salt is any one of potassium sulfate, potassium nitrate, or potassium dihydrogen phosphate.

2. The method according to claim 1, characterized in that, The oxidant is a hydrogen peroxide solution with a mass fraction of 28-32%.

3. The method according to claim 1, characterized in that, The mass-to-volume ratio of carrageenan, water, and potassium salt is 4g:(0-16)mL:(4-400)mg.

4. The method according to claim 2, characterized in that, The mass-to-volume ratio of carrageenan to hydrogen peroxide solution is 4 g: (8–26.7) mL.

5. The method according to claim 1, characterized in that, The process of thoroughly mixing the slurry to obtain the slurry is carried out at a temperature of 15–40°C.

6. The method according to claim 1, characterized in that, The reaction is carried out at 64–66°C for 35–45 min, or at 24–26°C for 890–910 min.

7. The method according to claim 1, characterized in that, The carrageenan is iota-carrageenan, Kappa-carrageenan, or Lambda-carrageenan.

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