A G-type alginate oligosaccharide, its preparation method and application

Abstract

The present invention discloses a G-type alginate oligosaccharide, its preparation method and application, which relates to the technical field of G-type alginate oligosaccharide preparation. When preparing the G-type alginate oligosaccharide, the present invention uses kelp as the raw material, organically combines chemical, physical and biological enzymatic hydrolysis methods, and obtains a G-type alginate oligosaccharide with a content of more than 90.12%, which has a high purity. After 6 months of accelerated test, the decrease in the content of G-type alginate oligosaccharide does not exceed 1.3%, improving the stability of G-type alginate oligosaccharide. The extraction rate of G-type alginate oligosaccharide is 3.63%-4.05%. The whole method does not require high-temperature and high-pressure treatment, effectively saving energy consumption and having a low production cost, and has a broad market prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of G-type fuco-oligosaccharides, and specifically provides a G-type fuco-oligosaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] Fuco-oligosaccharides are degradation products of alginate (alginic acid, algal polysaccharide), a component of brown algae cell walls. They are linear oligo-polymers composed of β-D-mannuronic acid (β-1,4-D-mannuronic acid, abbreviated as M) and α-L-guluronic acid (α-1,4-L-guluronic acid, abbreviated as G), with a degree of polymerization between 2 and 10. After depolymerization, their water solubility increases and they are easily absorbed by the body. They are a safe and non-toxic compound. The further decomposition products of fuco-oligosaccharides are guluronic acid oligosaccharides, mannuronic acid oligosaccharides, and fragments of guluronic acid and mannuronic acid mosaics. Among them, guluronic acid oligosaccharides are the G segment of fuco-oligosaccharides, also called G-type fuco-oligosaccharides. Research has found that G-type fuco-oligosaccharides have gel properties that can prevent viral RNA from passing through the cell membrane and prevent virus infection; they can effectively inhibit the expression of excessive inflammatory mediators and inflammatory cytokines, and inhibit the nuclear transcription signaling pathway and the MAPK signaling pathway, thereby inhibiting the occurrence of inflammatory reactions; they can effectively scavenge free radicals such as superoxide anions (O2-), hydroxyl radicals (OH-), and hydrogen peroxide (H2O2), and at the same time have strong activity in chelating ferrous ions, reducing lipid peroxidation, and thus delaying cell aging; and they can enhance immunity and have anti-tumor functions, and can be widely used in the fields of food, medicine, and cosmetics, with great application and development value.

[0003] The existing technology mainly focuses on the extraction and preparation process of fuco-oligosaccharides, and there is less research on further optimizing and purifying G-type fuco-oligosaccharides, and there is also the problem of low purity of fuco-oligosaccharides. Therefore, there is an urgent need to develop a G-type fuco-oligosaccharide, a preparation method thereof, and an application to solve such problems. Summary of the Invention

[0004] The purpose of the present invention is to provide a G-type fuco-oligosaccharide, a preparation method thereof, and an application, aiming at the deficiencies of the existing technology. When preparing G-type fuco-oligosaccharides, the present invention uses kelp as a raw material, combines chemical, physical, and biological enzymatic hydrolysis methods organically, and obtains a G-type fuco-oligosaccharide with a content of more than 90.12%, which has a high purity, improves the stability of G-type fuco-oligosaccharides, and the extraction rate of G-type fuco-oligosaccharides is 3.63%-4.05%. The whole method does not require high-temperature and high-pressure treatment, effectively saves energy consumption, and has a low production cost.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0006] The present invention provides a preparation method of G-type fuco-oligosaccharides, including:

[0007] S1. After removing impurities from kelp, wash it clean, add purified water 10 to 15 times the mass of the kelp raw material, soak for 1 to 2 hours, and then use a pulper to break it into kelp pulp with a mesh size of 100 to 300 meshes;

[0008] S2. Add hydrogen peroxide to the kelp pulp obtained in S1 to a concentration of 0.1% - 0.3%, react at 20°C - 30°C for 2 to 4 hours, and after the reaction, raise the temperature to 65°C - 85°C and treat for 30 to 40 minutes to obtain a preliminary hydrolysis mixture;

[0009] S3. After the temperature of the preliminary hydrolysis mixture drops to room temperature, add a mixed enzyme accounting for 2% - 3% of the total weight of the preliminary hydrolysis mixture, stir evenly, then add a pH regulator until the pH is 6.5 - 7.5, and perform microwave-assisted enzymatic hydrolysis extraction at 30°C - 45°C for 60 to 80 minutes, and boil for 6 to 8 minutes to inactivate the enzyme to obtain a fucoidan oligosaccharide mixture;

[0010] S4. Filter the fucoidan oligosaccharide mixture, concentrate the obtained filtrate under reduced pressure to 1 / 3 - 1 / 2 of the original volume, add glacial acetic acid to adjust the pH to 2.8 - 3.8, and after brown precipitates are produced, centrifuge to remove the supernatant to obtain a crude product of G-type fucoidan oligosaccharide;

[0011] S5. Dissolve the crude product of G-type fucoidan oligosaccharide in purified water 4 to 6 times the mass of the crude product of G-type fucoidan oligosaccharide, heat-treat at 55°C - 65°C for 40 to 60 minutes under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type fucoidan oligosaccharide.

[0012] The present invention is further configured such that in S3, the pH regulator is at least one of tartaric acid, citric acid, and lactic acid;

[0013] The present invention is further configured such that in S3, during the microwave-assisted enzymatic hydrolysis extraction, the microwave frequency is 500 MHz - 800 MHz;

[0014] The present invention is further configured such that in S4, the rotation speed of the centrifugation is 400 rpm - 600 rpm, and the centrifugation time is 8 to 10 minutes;

[0015] The present invention is further configured such that in S5, the filtration is performed with a 200 - 300 mesh gauze;

[0016] The present invention also discloses a G-type fucoidan oligosaccharide prepared by the above method.

[0017] The present invention also discloses a preparation method of a dressing prepared from the above-mentioned G-type alginate oligosaccharide as a raw material, including: Step 1. Raw material preparation: The G-type alginate oligosaccharide extracted from raw kelp by the preparation method of the G-type alginate oligosaccharide is used as a base material, and high-purity G-type alginate oligosaccharide with a content of more than 90.12% is taken. Polyvinyl alcohol is used as a gum base material, and auxiliary materials are prepared, including physiological saline, antibacterial agents, thickeners, moisturizers, and antioxidants;

[0018] In Step 2, the mass ratio of the G-type alginate oligosaccharide to the gum base material is 1:3 - 5. The G-type alginate oligosaccharide is added to polyvinyl alcohol at an addition rate of 15 g / min - 25 g / min, and stirred and mixed evenly at a speed of 50 revolutions per minute - 60 revolutions per minute;

[0019] Then, the temperature is raised. When the temperature reaches 30°C - 40°C, the G-type alginate oligosaccharide and polyvinyl alcohol are stirred and mixed using a stirring device at a rotation speed of 120 rpm - 150 rpm, and the stirring time is 2 hours - 4 hours;

[0020] In Step 3, continuous stirring is carried out, and at the same time, physiological saline with a concentration of 0.9% and accounting for 5% - 10% of the total mixture mass is added to adjust the viscosity of the mixture;

[0021] An antibacterial agent accounting for 0.1% - 0.5% of the total mixture mass is added. The antibacterial agent includes at least one of benzoic acid, sodium benzoate, and parabens;

[0022] A thickener accounting for 1% - 5% of the total mixture mass is added. The thickener includes at least one of hydroxypropyl methylcellulose, polyacrylamide, and sodium carboxymethylcellulose;

[0023] A moisturizer accounting for 1% - 3% of the total mixture mass is added. The moisturizer includes at least one of glycerol, propylene glycol, and hyaluronic acid;

[0024] The antioxidant is selected from vitamin antioxidants or thiol antioxidants, and an antioxidant accounting for 0.1% - 0.3% of the total mixture mass is added;

[0025] In Step 4, mixing and stirring are carried out. At a temperature of 25°C - 35°C, mixing and stirring are continued at a rotation speed of 120 rpm - 150 rpm to fully blend the added materials, and the stirring time lasts for 1 hour - 2 hours to obtain a mixed colloidal solution;

[0026] Step 5. Prepare the mold as needed, control the mold temperature at 20°C - 30°C, pour the mixed colloidal solution into the mold. After complete filling, conduct drying and curing at 50°C - 80°C for 20 min - 35 min. Control the thickness of the dried product at 0.2 - 0.4 cm, perform outer packaging, and irradiate and sterilize with a cobalt-60 dose of 15 - 25 kGy for 4 - 8 h to obtain the G-type fucoidan oligosaccharide dressing.

[0027] The present invention also discloses the application of the above-mentioned G-type fucoidan oligosaccharide in medicine.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention provides a preparation method of G-type fucoidan oligosaccharide. This method uses kelp as the raw material, organically combines chemical, physical, and biological enzymatic hydrolysis methods. After the kelp is soaked and pulped, its tissue structure is destroyed. Then, hydrogen peroxide is added for oxidative hydrolysis to further destroy the cell structure of the kelp, initially decomposing alginate into fucoidan oligosaccharide. After that, hydrogen peroxide is removed through heat treatment, and microwave-assisted mixed enzymatic hydrolysis is continued to further decompose alginate to obtain a fucoidan oligosaccharide mixture. After filtration and concentration, glacial acetic acid is added to adjust the pH to 2.8 - 3.8, so that the G-type fucoidan oligosaccharide precipitates. After centrifugation, the M-type fucoidan oligosaccharide in the supernatant is removed. The crude product of G-type fucoidan oligosaccharide is redissolved in purified water, and heat treatment is carried out under ventilation conditions to remove the glacial acetic acid in the crude product. Then, filtration, concentration, and freeze-drying are carried out to obtain G-type fucoidan oligosaccharide. The entire preparation process does not require high-temperature and high-pressure treatment. The chemical reagents hydrogen peroxide and acetic acid used during this period can be removed by heating, avoiding the introduction of chemical impurities and ensuring the purity of G-type fucoidan oligosaccharide. This preparation method is easy to operate, has low production costs, and has good market promotion prospects.

[0030] 2. In the present invention, after using alginate lyase and kelp hydrolase in combination, control the mass ratio of alginate lyase to kelp hydrolase at 3 - 4:1. Combining with microwave treatment can effectively increase the content of G-type fucoidan oligosaccharide.

[0031] 3. In the present invention, the content of the prepared G-type fucoidan oligosaccharide is above 90.12%, its purity is relatively high, which improves the stability of G-type fucoidan oligosaccharide. After being stored for three months, the content of G-type fucoidan oligosaccharide decreases by no more than 1.3%. The extraction rate of G-type fucoidan oligosaccharide is 3.63% - 4.05%.

[0032] 4. In the present invention, the prepared G-type fucoidan oligosaccharide dressing can promote wound healing and can be used in dressing products for drugs or medical devices. Description of the Drawings

[0033] Figure 1 It is a flow chart of the preparation method of G-type fucoidan oligosaccharide of the present invention. Detailed implementation manners

[0034] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the scope of protection of the present invention.

[0035] In the present invention, unless otherwise specified, all preparation raw materials and equipment and instruments are commercially available products well-known to those skilled in the art.

[0036] The present invention provides a method for preparing G-type alginate oligosaccharide, which comprises the following steps:

[0037] S1. After removing impurities from kelp and cleaning it, add purified water 10 to 15 times the mass of the kelp raw material, soak for 1 to 2 h, and then break it into kelp pulp with a mesh size of 100 to 300 by a pulper;

[0038] S2. Add hydrogen peroxide to the kelp pulp obtained in S1 to a concentration of 0.1% - 0.3%, react at 22 - 30 °C for 2 - 4 h, and after the reaction is completed, raise the temperature to 65 - 85 °C and treat for 30 - 40 min to obtain a preliminary hydrolysis mixture;

[0039] S3. After the temperature of the preliminary hydrolysis mixture drops to room temperature, add a mixed enzyme accounting for 2% - 3% of the total weight of the preliminary hydrolysis mixture, stir evenly, then add a pH regulator to adjust the pH to 6.5 - 7.5, and perform microwave-assisted enzymatic hydrolysis extraction at 30 - 45 °C for 60 - 80 min, and boil for 6 - 8 min to inactivate the enzyme to obtain an alginate oligosaccharide mixture;

[0040] S4. Filter the alginate oligosaccharide mixture, concentrate the obtained filtrate under reduced pressure to 1 / 3 - 1 / 2 of the original volume, add glacial acetic acid to adjust the pH to 2.8 - 3.8, and after brown precipitates are generated, centrifuge to remove the supernatant to obtain a crude product of G-type alginate oligosaccharide;

[0041] S5. Dissolve the crude product of G-type alginate oligosaccharide in purified water 4 to 6 times the mass of the crude product of G-type alginate oligosaccharide, heat-treat at 55 - 65 °C for 40 - 60 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type alginate oligosaccharide.

[0042] Preferably, in S1, after the seaweed is decontaminated, it is cleaned, 12 times of purified water is added, and after soaking for 1.5 h, it is ground into seaweed pulp with a mesh size of 100 by a pulper. The pulping treatment can destroy the tissue structure of the seaweed, facilitating the full contact of hydrogen peroxide, the mixed enzyme and the seaweed during subsequent oxidative decomposition and microwave-assisted enzymatic hydrolysis, and improving the yield of G-type alginate oligosaccharide.

[0043] Preferably, in S2, hydrogen peroxide is added to the seaweed pulp to a concentration of 0.2%, and the reaction is carried out at 28 °C for 3 h. After the reaction is completed, the temperature is raised to 80 °C and treated for 35 min to obtain a preliminary hydrolysis mixture. Hydrogen peroxide is an oxidant. Adding hydrogen peroxide to the seaweed pulp helps to hydrolyze alginate to obtain alginate oligosaccharide. At the same time, hydrogen peroxide decomposes into water and oxygen under high-temperature conditions, avoiding the residue of chemical substances.

[0044] Preferably, in S3, after adding 2.5% of the total weight of the mixed enzyme to the preliminary hydrolysis mixture and stirring evenly, a pH regulator is added dropwise until the pH is 7.2, and then microwave-assisted enzymatic hydrolysis extraction is carried out at 35 °C for 70 min, and the enzyme is inactivated by boiling for 6 min to obtain an alginate oligosaccharide mixture. In the conventional enzymatic hydrolysis method, the reaction conditions are relatively mild, but there is also the problem of a long reaction time, which is not conducive to improving production efficiency. By using microwave-assisted enzymatic hydrolysis extraction, the enzymatic hydrolysis efficiency can be effectively improved, the required time for enzymatic hydrolysis can be shortened, and at the same time, it is beneficial to the formation of the target product G-type alginate oligosaccharide. Among them, when the pH regulator is a solid preparation, it needs to be diluted into an aqueous solution before being added dropwise. If it is a liquid preparation, it can be directly added dropwise.

[0045] Preferably, in S4, the alginate oligosaccharide mixture is filtered, and the obtained filtrate is concentrated under reduced pressure to 1 / 3 of the original volume. Glacial acetic acid is added to adjust the pH to 3.0. After brown precipitates are produced, centrifugation is carried out to remove the supernatant to obtain the crude product of G-type alginate oligosaccharide. The water solubility of alginate oligosaccharide is relatively good. After the filtrate is concentrated, glacial acetic acid is added to adjust the pH to 3.0, so that the G-type alginate oligosaccharide precipitates, and centrifugation is used to remove the M-type alginate oligosaccharide in the supernatant, thus obtaining the crude product of G-type alginate oligosaccharide.

[0046] Preferably, in S5, the crude product of G-type alginate oligosaccharide is redissolved with 5 times of purified water, heated at 60 °C for 50 min under ventilation conditions, filtered, concentrated under reduced pressure, and freeze-dried to obtain G-type alginate oligosaccharide.

[0047] The flow chart of the preparation method of G-type alginate oligosaccharide is shown in Figure 1 。

[0048] The present invention also provides the G-type alginate oligosaccharide prepared by the above preparation method.

[0049] The present invention also provides the application of the above G-type alginate oligosaccharide in medicine. This G-type alginate oligosaccharide can promote wound healing and can be used in dressing products for drugs or medical devices.

[0050] The present invention also discloses a preparation method of a dressing prepared from the above-mentioned G-type alginate oligosaccharide as a raw material, including: Step 1. Raw material preparation, using the G-type alginate oligosaccharide extracted from raw kelp by the preparation method of the G-type alginate oligosaccharide as a base material, taking high-purity G-type alginate oligosaccharide with a content of more than 90.12%, using polyvinyl alcohol as a gum base material, and preparing auxiliary materials, including physiological saline, antibacterial agents, thickeners, moisturizers, and antioxidants;

[0051] Step 2. The mass ratio of the G-type alginate oligosaccharide to the gum base material is 1:3-5. The G-type alginate oligosaccharide is added to polyvinyl alcohol at an addition rate of 15 g / min-25 g / min and stirred and mixed evenly at a speed of 50 revolutions per minute-60 revolutions per minute;

[0052] Then heat up. When the temperature reaches 30°C-40°C, use a stirring device to stir and mix the G-type alginate oligosaccharide and polyvinyl alcohol at a rotation speed of 120 rpm-150 rpm, and the stirring time is 2 hours-4 hours;

[0053] Step 3. Keep stirring, and at the same time add physiological saline with a concentration of 0.9% accounting for 5%-10% of the total mixture mass to adjust the viscosity of the mixture;

[0054] Add antibacterial agents accounting for 0.1%-0.5% of the total mixture mass. The antibacterial agents include at least one of benzoic acid, sodium benzoate, and parabens;

[0055] Add thickeners accounting for 1%-5% of the total mixture mass. The thickeners include at least one of hydroxypropyl methylcellulose, polyacrylamide, and sodium carboxymethylcellulose;

[0056] Add moisturizers accounting for 1%-3% of the total mixture mass. The moisturizers include at least one of glycerol, propylene glycol, and hyaluronic acid;

[0057] Select vitamin antioxidants or thiol antioxidants as antioxidants, and add antioxidants accounting for 0.1%-0.3% of the total mixture mass;

[0058] Step 4. Mix and stir. At a temperature of 25°C-35°C, continue to mix and stir at a rotation speed of 120 rpm-150 rpm to fully blend the added materials, and the stirring time lasts for 1 hour-2 hours to obtain a mixed colloidal solution;

[0059] Step 5. Prepare the mold as needed, control the mold temperature at 20°C - 30°C, pour the mixed colloidal solution into the mold. After complete filling, conduct drying and curing at 50°C - 80°C for 20 min - 35 min. Control the thickness of the dried product at 0.2 - 0.4 cm, perform outer packaging, and irradiate and sterilize with a cobalt-60 dose of 15 - 25 kGy for 4 - 8 h to obtain the G-type fucoidan oligosaccharide dressing.

[0060] The following further describes the present invention in detail with specific examples and drawings:

[0061] Example 1: Preparation of G-type fucoidan oligosaccharide

[0062] S1. Remove impurities from 2 kg of kelp, wash it clean, add 12 times the purified water, soak for 1.5 h, and then use a pulper to break it into 100-mesh kelp pulp;

[0063] S2. Add hydrogen peroxide to the kelp pulp obtained in S1 to a concentration of 0.2%, react at 28°C for 3 h, and after the reaction is completed, raise the temperature to 80°C and treat for 35 min to obtain a preliminary hydrolysis mixture;

[0064] S3. After the temperature of the preliminary hydrolysis mixture drops to room temperature, add 2.0% of alginate lyase and 0.5% of kelp hydrolase based on the total weight of the preliminary hydrolysis mixture, stir evenly, then add tartaric acid until the pH is 7.2, and perform microwave (microwave frequency is 600 MHz) assisted enzymatic extraction at 35°C for 70 min, and boil for 6 min to inactivate the enzyme to obtain a fucoidan oligosaccharide mixture;

[0065] S4. Filter the fucoidan oligosaccharide mixture, concentrate the obtained filtrate under reduced pressure to 1 / 3 of the original volume, add glacial acetic acid to adjust the pH to 3.0, and after brown precipitates are produced, centrifuge to remove the supernatant to obtain the crude product of G-type fucoidan oligosaccharide;

[0066] S5. Add 5 times the purified water to the crude product of G-type fucoidan oligosaccharide for re-dissolution, heat-treat at 60°C for 50 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type fucoidan oligosaccharide.

[0067] Example 2: Preparation of G-type fucoidan oligosaccharide

[0068] S1. Remove impurities from 2 kg of kelp, wash it clean, add 10 times the purified water, soak for 1 h, and then use a pulper to break it into 200-mesh kelp pulp;

[0069] S2. Add hydrogen peroxide to the kelp pulp obtained in S1 to a concentration of 0.1%, react at 22°C for 4 h, and after the reaction is completed, raise the temperature to 85°C and treat for 30 min to obtain a preliminary hydrolysis mixture;

[0070] S3. After the temperature of the preliminarily hydrolyzed mixture drops to room temperature, add alginate lyase accounting for 2.0% of the total weight of the preliminarily hydrolyzed mixture and laminaria japonica hydrolase accounting for 1.0%, stir evenly, then add tartaric acid until the pH reaches 6.8, and perform microwave (microwave frequency is 800 MHz) assisted enzymatic hydrolysis extraction at 45°C for 60 min, and boil for 6 min to inactivate the enzyme to obtain an alginate oligosaccharide mixture;

[0071] S4. Filter the alginate oligosaccharide mixture, concentrate the obtained filtrate under reduced pressure to 1 / 2 of the original volume, add glacial acetic acid to adjust the pH to 2.8, wait until brown precipitates are produced, then centrifuge to remove the supernatant to obtain a crude product of G-type alginate oligosaccharide;

[0072] S5. Add 4 times of purified water to the crude product of G-type alginate oligosaccharide for re-dissolution, heat-treat at 55°C for 60 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type alginate oligosaccharide.

[0073] Example 3: Preparation of G-type alginate oligosaccharide

[0074] S1. Remove impurities from 2 kg of laminaria japonica, wash it clean, add 10 times of purified water, soak for 2 h, and then use a pulper to break it into laminaria japonica pulp with 300 meshes;

[0075] S2. Add hydrogen peroxide to the laminaria japonica pulp obtained in S1 until the concentration reaches 0.3%, react at 30°C for 2 h, and after the reaction ends, raise the temperature to 68°C and treat for 40 min to obtain a preliminarily hydrolyzed mixture;

[0076] S3. After the temperature of the preliminarily hydrolyzed mixture drops to room temperature, add alginate lyase accounting for 2.1% of the total weight of the preliminarily hydrolyzed mixture and laminaria japonica hydrolase accounting for 0.6%, stir evenly, then add tartaric acid until the pH reaches 7.5, and perform microwave (microwave frequency is 500 MHz) assisted enzymatic hydrolysis extraction at 30°C for 80 min, and boil for 8 min to inactivate the enzyme to obtain an alginate oligosaccharide mixture;

[0077] S4. Filter the alginate oligosaccharide mixture, concentrate the obtained filtrate under reduced pressure to 1 / 3 of the original volume, add glacial acetic acid to adjust the pH to 3.3, wait until brown precipitates are produced, then centrifuge to remove the supernatant to obtain a crude product of G-type alginate oligosaccharide;

[0078] S5. Add 6 times of purified water to the crude product of G-type alginate oligosaccharide for re-dissolution, heat-treat at 65°C for 40 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type alginate oligosaccharide.

[0079] Comparative Example 1: On the basis of Example 1, laminaria japonica hydrolase is not used, and the others are the same as Example 1.

[0080] Comparative Example 2:

[0081] S1. After removing impurities from 2 kg of kelp, wash it clean, add 12 times purified water, soak for 1.5 h, and then use a pulper to break it into 100-mesh kelp pulp;

[0082] S2. Add hydrogen peroxide to the kelp pulp obtained in S1 to a concentration of 0.2%, react at 28 °C for 3 h, and after the reaction, raise the temperature to 80 °C and treat for 35 min to obtain a preliminary hydrolysis mixture;

[0083] S3. After the temperature of the preliminary hydrolysis mixture drops to room temperature, add 0.5% alginate lyase and 0.5% kelp hydrolase based on the total weight of the preliminary hydrolysis mixture, stir evenly, then add tartaric acid dropwise until the pH reaches 7.2, and perform microwave (microwave frequency is 600 MHz) assisted enzymatic hydrolysis extraction at 35 °C for 70 min, and boil for 6 min to inactivate the enzyme to obtain a mixture of fucoidan oligosaccharides;

[0084] S4. Filter the mixture of fucoidan oligosaccharides, concentrate the obtained filtrate under reduced pressure to 1 / 3 of the original volume, add glacial acetic acid to adjust the pH to 3.0, and after brown precipitates are produced, centrifuge to remove the supernatant to obtain a crude product of G-type fucoidan oligosaccharides;

[0085] S5. Add 5 times purified water to the crude product of G-type fucoidan oligosaccharides for re-dissolution, heat-treat at 60 °C for 50 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type fucoidan oligosaccharides.

[0086] Comparative Example 3:

[0087] S1. After removing impurities from 2 kg of kelp, wash it clean, add 12 times purified water, soak for 1.5 h, and then use a pulper to break it into 100-mesh kelp pulp;

[0088] S2. Add hydrogen peroxide to the kelp pulp obtained in S1 to a concentration of 0.2%, react at 28 °C for 3 h, and after the reaction, raise the temperature to 80 °C and treat for 35 min to obtain a preliminary hydrolysis mixture;

[0089] S3. After the temperature of the preliminary hydrolysis mixture drops to room temperature, add 2.0% alginate lyase and 0.2% kelp hydrolase based on the total weight of the preliminary hydrolysis mixture, stir evenly, then add tartaric acid dropwise until the pH reaches 7.2, and perform microwave (microwave frequency is 600 MHz) assisted enzymatic hydrolysis extraction at 35 °C for 70 min, and boil for 6 min to inactivate the enzyme to obtain a mixture of fucoidan oligosaccharides;

[0090] S4. Filter the mixture of fucoidan oligosaccharides, concentrate the obtained filtrate under reduced pressure to 1 / 3 of the original volume, add glacial acetic acid to adjust the pH to 3.0, and after brown precipitates are produced, centrifuge to remove the supernatant to obtain a crude product of G-type fucoidan oligosaccharides;

[0091] S5. Add 5 times of purified water to the crude G-type alginate oligosaccharide for re-dissolution, heat-treat at 60 °C for 50 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type alginate oligosaccharide.

[0092] Comparative Example 4:

[0093] S1. Remove impurities from 2 kg of kelp, wash it clean, add 12 times of purified water, soak for 1.5 h, and then use a pulper to break it into 100-mesh kelp pulp;

[0094] S2. Add alginate lyase at 2.0% of the total weight of the kelp pulp and kelp hydrolase at 0.1% to the kelp pulp. After stirring evenly, add tartaric acid until the pH reaches 7.2, and then carry out microwave (microwave frequency is 600 MHz) assisted enzymatic hydrolysis extraction at 35 °C for 70 min, and boil for 6 min to inactivate the enzyme to obtain a mixed solution of alginate oligosaccharides;

[0095] S3. Filter the mixed solution of alginate oligosaccharides, concentrate the obtained filtrate to 1 / 3 of the original volume under reduced pressure, add glacial acetic acid to adjust the pH to 3.0. After brown precipitates are formed, centrifuge to remove the supernatant to obtain the crude G-type alginate oligosaccharide;

[0096] S4. Add 5 times of purified water to the crude G-type alginate oligosaccharide for re-dissolution, heat-treat at 60 °C for 50 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type alginate oligosaccharide.

[0097] Comparative Example 5:

[0098] S1. Remove impurities from 2 kg of kelp, wash it clean, add 12 times of purified water, soak for 1.5 h, and then use a pulper to break it into 100-mesh kelp pulp;

[0099] S2. Add hydrogen peroxide to the kelp pulp obtained in S1 until the concentration is 0.2%, react at 28 °C for 3 h, and after the reaction, raise the temperature to 80 °C and treat for 35 min to obtain a preliminary hydrolysis mixed solution;

[0100] S3. After the temperature of the preliminary hydrolysis mixed solution drops to room temperature, add alginate lyase at 2.0% of the total weight of the preliminary hydrolysis mixed solution and kelp hydrolase at 0.5%. After stirring evenly, add tartaric acid until the pH reaches 7.2, and then carry out enzymatic hydrolysis extraction at 35 °C for 70 min, and boil for 6 min to inactivate the enzyme to obtain a mixed solution of alginate oligosaccharides;

[0101] S4. Filter the mixed solution of alginate oligosaccharides, concentrate the obtained filtrate to 1 / 3 of the original volume under reduced pressure, add glacial acetic acid to adjust the pH to 3.0. After brown precipitates are formed, centrifuge to remove the supernatant to obtain the crude G-type alginate oligosaccharide;

[0102] S5. Add 5 times purified water to the crude product of G-type alginate oligosaccharide for re-dissolution, heat-treat at 60 °C for 50 min under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type alginate oligosaccharide.

[0103] Detect the content of the G-type alginate oligosaccharide prepared in Examples 1-3 and Comparative Examples 1-6. The content detection method of the G-type alginate oligosaccharide refers to the method in Appendix C of our company's record standard Q / YTR 0002S-2022 "G-Type Alginate Oligosaccharide" for calculation of the extraction rate of the G-type alginate oligosaccharide; and place it under the conditions of temperature 40 °C ± 2 °C and relative humidity 75% ± 5% for 6 months to conduct a stability investigation on the content of the prepared G-type alginate oligosaccharide. The results are shown in Table 1.

[0104] Table 1: Content Table of G-Type Alginate Oligosaccharide

[0105]

[0106]

[0107] It can be seen from Table 1 that the content of the G-type alginate oligosaccharide prepared in Examples 1-3 is 90.12% - 91.24%, and the average content is 90.56%, which is higher than that of Comparative Examples 1-5. The extraction rate is between 3.63% - 4.05%. After 6 months of accelerated test, the decrease in the content of the G-type alginate oligosaccharide does not exceed 1.3%. This shows that the G-type alginate oligosaccharide prepared by the method of the present invention has a high purity and good stability, which is convenient for later storage and processing.

[0108] In Comparative Example 1, no kelp hydrolase is used, and the content of the G-type alginate oligosaccharide is only 82.36%. In Comparative Examples 2 and 3, when the weight ratio of alginate lyase to kelp hydrolase is 1:1 and 10:1 respectively, the content of the G-type alginate oligosaccharide is 85.62% and 86.78% respectively. This shows that controlling the mass ratio of alginate lyase to kelp hydrolase to be 3 - 4:1 and combining with microwave treatment can effectively increase the content of the G-type alginate oligosaccharide.

[0109] In Comparative Example 4, without oxidative hydrolysis pretreatment of the kelp pulp, the content of the G-type alginate oligosaccharide is only 75.37%, and after 6 months of accelerated test, the content of the G-type alginate oligosaccharide decreases by 13.28%, and the stability of the G-type alginate oligosaccharide is poor. In Comparative Example 5, without ultrasonic-assisted enzymatic hydrolysis, the content of the G-type alginate oligosaccharide is only 80.55%, and after 6 months of accelerated test, the content of the G-type alginate oligosaccharide decreases by 7.36%.

[0110] Example 4: Preparation of G-Type Alginate Oligosaccharide Dressing

[0111] The G-type alginate oligosaccharide used in this example is prepared in Example 1. The preparation method of the obtained dressing includes:

[0112] Step 1. Preparation of raw materials: 50 g of G-type alginate oligosaccharide obtained from Example 1 was used, 150 g of polyvinyl alcohol was used as the gum base material, and auxiliary materials were prepared, including 18 g of 0.9% normal saline, 1 g of benzoic acid, 5 g of hydroxypropyl methylcellulose, 3 g of propylene glycol, and 0.5 g of tetracosyl mercaptan;

[0113] Step 2. Add the G-type alginate oligosaccharide to polyvinyl alcohol at an addition rate of 25 g / min and stir and mix evenly at a speed of 60 revolutions per minute;

[0114] Then raise the temperature. When the temperature reaches 38 °C, use a stirring device to stir and mix the G-type alginate oligosaccharide and polyvinyl alcohol at a rotation speed of 150 rpm, and the stirring time is 2 hours;

[0115] Step 3. Keep stirring and add normal saline, benzoic acid, hydroxypropyl methylcellulose, propylene glycol, and tetracosyl mercaptan;

[0116] Step 4. Mix and stir. At a temperature of 35 °C, continue to mix and stir at a rotation speed of 150 rpm to fully blend the added materials, and the stirring time lasts for 1 hour to obtain a mixed colloidal solution;

[0117] Step 5. Prepare a mold as needed, control the mold temperature at 30 °C, pour the mixed colloidal solution into the mold, and dry and cure it at 60 °C for 30 min after complete filling. The thickness of the dried product is 0.3 cm, and it is externally packaged and irradiated and sterilized with 18 kGy for 6 h to obtain the G-type alginate oligosaccharide dressing.

[0118] Example 5: Preparation of G-type alginate oligosaccharide dressing

[0119] The G-type alginate oligosaccharide used in this example was prepared in Example 1, and the preparation method of the obtained dressing includes:

[0120] Step 1. Preparation of raw materials: 50 g of G-type alginate oligosaccharide obtained from Example 1 was used, 200 g of polyvinyl alcohol was used as the gum base material, and auxiliary materials were prepared, including 18 g of 0.9% normal saline, 1 g of benzoic acid, 5 g of hydroxypropyl methylcellulose, 3 g of propylene glycol, and 0.5 g of tetracosyl mercaptan;

[0121] Step 2. Add the G-type alginate oligosaccharide to polyvinyl alcohol at an addition rate of 25 g / min and stir and mix evenly at a speed of 60 revolutions per minute;

[0122] Then raise the temperature. When the temperature reaches 38 °C, use a stirring device to stir and mix the G-type alginate oligosaccharide and polyvinyl alcohol at a rotation speed of 150 rpm, and the stirring time is 2 hours;

[0123] Step 3. Keep stirring, and add normal saline, benzoic acid, hydroxypropyl methylcellulose, propylene glycol, and tetracosyl mercaptan;

[0124] Step 4. Mix and stir. At a temperature of 35°C, continue to mix and stir at a speed of 150 rpm to fully blend the added materials. Stir for 1 hour to obtain a mixed colloidal solution;

[0125] Step 5. Prepare a mold as needed, control the mold temperature at 30°C, pour the mixed colloidal solution into the mold. After complete filling, dry and cure at 60°C for 30 min. The thickness of the dried product is 0.3 cm. Package it and sterilize it by irradiation at 18 kGy for 6 h to obtain the G-type fucoidan oligosaccharide dressing.

[0126] Example 6: Preparation of G-type fucoidan oligosaccharide dressing

[0127] The G-type fucoidan oligosaccharide used in this example was prepared in Example 1. The preparation method of the obtained dressing includes:

[0128] Step 1. Prepare raw materials. Use 50 g of the G-type fucoidan oligosaccharide prepared in Example 1, use polyvinyl alcohol as the gum base material at 250 g, and prepare auxiliary materials, including 18 g of 0.9% normal saline, 1 g of benzoic acid, 5 g of hydroxypropyl methylcellulose, 3 g of propylene glycol, and 0.5 g of tetracosyl mercaptan;

[0129] Step 2. Add the G-type fucoidan oligosaccharide to polyvinyl alcohol at a feeding rate of 25 g / min, and stir and mix evenly at a speed of 60 revolutions per minute;

[0130] Then raise the temperature. When the temperature reaches 38°C, use a stirring device to stir and mix the G-type fucoidan oligosaccharide and polyvinyl alcohol at a speed of 150 rpm for 2 hours;

[0131] Step 3. Keep stirring, and add normal saline, benzoic acid, hydroxypropyl methylcellulose, propylene glycol, and tetracosyl mercaptan;

[0132] Step 4. Mix and stir. At a temperature of 35°C, continue to mix and stir at a speed of 150 rpm to fully blend the added materials. Stir for 1 hour to obtain a mixed colloidal solution;

[0133] Step 5. Prepare a mold as needed, control the mold temperature at 30°C, pour the mixed colloidal solution into the mold. After complete filling, dry and cure at 60°C for 30 min. The thickness of the dried product is 0.3 cm. Package it and sterilize it by irradiation at 18 kGy for 6 h to obtain the G-type fucoidan oligosaccharide dressing.

[0134] Experimental Example 1: Wound healing efficacy test

[0135] The wound healing efficacy test was performed on the G-type brown algae oligosaccharide dressing prepared in Examples 4-6.

[0136] Rats were used for animal experiments, and four wounds were produced on the back of each rat. Then, the rats were divided into groups according to the treatment methods of the four wounds of each rat (each rat could complete four groups of experiments at the same time), with 10 rats in each group, including a blank control group and wounds of each group treated with the G-type brown algae oligosaccharide dressing prepared in Examples 4-6.

[0137] The method for making full-thickness skin wounds on the back of rats comprises the following steps: injecting sodium pentobarbital (30 mg / kg) in the abdomen to anesthetize the rats, cutting the hair on the back to expose the skin, and trying not to cut the skin of the rats. After shaving cleanly, wash with warm water, and then wipe with a sterilized absorbent cotton ball dipped in iodine to disinfect. Mark the wounds of 4 full-thickness skins, the wound diameter is 1.8 cm, and then separate the tissues with dissecting scissors and tweezers, and then select 3 wounds of each rat to apply the G-type brown algae oligosaccharide dressing prepared in Examples 4-6, and the remaining wound is not treated as a blank control group. After treating a wound, cover it with sterile gauze and wrap it with a sterile elastic bandage. Change the agent every 2 days, and these treated mice are kept in separate cages. After the wound is made, the wound healing effect is evaluated every two days.

[0138] Wound repair evaluation method: Use a digital camera to observe and record the wound repair and granulation tissue growth every two days.

[0139] The degree of healing is expressed by the formula: wound healing rate = (A0-At) / A0×100%, where A0 and At represent the initial wound area and the wound area at time point t, respectively.

[0140] In order to measure wound healing, the residual wound edge was traced on a transparent film every two days after surgery, the marked tracer film was scanned, and the wound area was calculated using image analysis software. If the wound was covered with a new layer of epidermis, it was considered to have healed. The healing time was the time from the operation to the complete coverage of the wound by the epidermis. The results are shown in Table 2.

[0141] Table 2 Statistical results of wound healing

[0142]

[0143] It can be seen from Table 2 that compared with the blank control group, the G-type brown algae oligosaccharide dressing provided by the present invention can basically achieve wound healing on the 9th to 10th day, and the wound healing effect is better than that of the blank control group.

[0144] In the present invention, kelp is soaked and pulped to destroy its tissue structure, and then hydrogen peroxide is added for oxidative hydrolysis to further destroy the cell structure of kelp, initially decomposing alginate into alginate oligosaccharides. After that, hydrogen peroxide is removed by heat treatment, and then microwave-assisted enzymatic hydrolysis is continued to further decompose alginate to obtain a mixed solution of alginate oligosaccharides. After filtration and concentration, glacial acetic acid is added to adjust the pH to 2.8 - 3.8, so that the G-type alginate oligosaccharides precipitate. After centrifugation, the M-type alginate oligosaccharides in the supernatant are removed. The crude product of G-type alginate oligosaccharides is redissolved in purified water, and then heat treatment is carried out under ventilation conditions to remove the glacial acetic acid in the crude product. After that, filtration, concentration, and freeze-drying are carried out to obtain G-type alginate oligosaccharides. Alginate lyase is an enzyme that can decompose alginate, catalyzing in a β-way, forming an unsaturated double bond between C4 and C5, and producing a degradation process at the reducing end, degrading long-chain polysaccharides into short-chain oligosaccharides. Kelp hydrolase is a special hydrolase developed for kelp (brown algae) by introducing excellent strains and refining through liquid deep fermentation. It can specifically hydrolyze dried kelp or fresh kelp. The alginate lyase and kelp hydrolase in the present invention are both purchased from Shanghai Huashangxiangyang Biotechnology Co., Ltd. The inventor found that after combining the alginate lyase and kelp hydrolase and controlling the mass ratio of alginate lyase to kelp hydrolase to be 3 - 4:1, the yield of G-type alginate oligosaccharides can be effectively improved by combining with microwave treatment.

[0145] When preparing G-type alginate oligosaccharides in the present invention, kelp is used as the raw material, and chemical, physical, and biological enzymatic hydrolysis methods are organically combined to obtain G-type alginate oligosaccharides with a content of more than 90.12%. Its purity is relatively high, improving the stability of G-type alginate oligosaccharides. The extraction rate of G-type alginate oligosaccharides is 3.63% - 4.05%. The whole method does not require high-temperature and high-pressure treatment, effectively saving energy consumption, and effectively avoiding the problem that the traditional single chemical extraction method has an impact on environmental pollution, or the single physical extraction method uses high-temperature and high-pressure treatment with large energy consumption.

[0146] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A method for preparing G-type alginate oligosaccharide, characterized in that, Including: S1. After removing impurities from kelp and cleaning it, add purified water 10 to 12 times the mass of the kelp raw material, soak for 1 to 2 hours, and then use a pulper to break it into kelp pulp with a mesh size of 100 to 300 meshes; S2. Add hydrogen peroxide to the kelp pulp obtained in S1 to a concentration of 0.1% - 0.3%, react at 22°C - 30°C for 2 to 4 hours, and after the reaction, raise the temperature to 68°C - 85°C and treat for 30 to 40 minutes to obtain a preliminary hydrolysis mixture; S3. After the temperature of the preliminary hydrolysis mixture drops to room temperature, add a mixed enzyme accounting for 2% - 3% of the total weight of the preliminary hydrolysis mixture, stir evenly, then add a pH regulator until the pH is 6.8 - 7.5, and perform microwave-assisted enzymatic hydrolysis extraction at 30°C - 45°C for 60 to 80 minutes, and boil for 6 to 8 minutes to inactivate the enzyme to obtain a fucoidan oligosaccharide mixture; The mixed enzyme is alginate lyase and kelp hydrolase, and the mass ratio of alginate lyase to kelp hydrolase is 3 - 4:1; The pH regulator is at least one of tartaric acid, citric acid, and lactic acid; During the microwave-assisted enzymatic hydrolysis extraction, the microwave frequency is 500 MHz - 800 MHz; S4. Filter the fucoidan oligosaccharide mixture, concentrate the obtained filtrate under reduced pressure to 1 / 3 - 1 / 2 of the original volume, add glacial acetic acid to adjust the pH to 2.8 - 3.3, wait for brown precipitates to form, then centrifuge to remove the supernatant to obtain a crude product of G-type fucoidan oligosaccharide; S5. Redissolve the crude product of G-type fucoidan oligosaccharide in purified water 4 to 6 times the mass of the crude product of G-type fucoidan oligosaccharide, heat-treat at 55°C - 65°C for 40 to 60 minutes under ventilation conditions, filter, concentrate under reduced pressure, and freeze-dry to obtain G-type fucoidan oligosaccharide; The content of the obtained G-type fucoidan oligosaccharide is 90.12 - 91.24%, and the extraction rate is 3.63% - 4.05%.

2. The preparation method of a G-type alginate oligosaccharide according to claim 1, wherein In S4, the rotation speed of the centrifugation is 400 rpm - 600 rpm, and the centrifugation time is 8 - 10 minutes.

3. A method for preparing G-type alginate oligosaccharide according to claim 1, characterized in that, In S5, the filtration is performed with a gauze with a mesh size of 200 - 300 meshes.

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