Active polysaccharide, extraction reagent, extraction method and application thereof

Abstract

The application discloses active polysaccharide, an extraction reagent and method thereof and application of the active polysaccharide. 4 The active polysaccharide has a weight average molecular weight of 7.04*10 4 The application uses seaweed gel extraction waste liquid as raw material, and uses ethanol, polyhydric alcohol and quaternary ammonium base to prepare the extraction reagent, separates crude polysaccharide in a short time, removes inorganic substances, proteins and color, and carries out gel column chromatography, so that the purity of the polysaccharide is improved (up to 92.26 %); and the activity of resisting plant pathogenic fungi is well preserved through secondary freeze-drying; the minimum inhibitory concentration of the polysaccharide to alternaria alternata and alternaria alternata is 0.01 mg / mL, and the inhibition rate is 81.98 % and 85.47 % respectively at 0.16 mg / mL. The whole extraction method is simple and environment-friendly, the process condition is mild, and the high-value utilization of the seaweed gel extraction waste liquid is realized.

Description

Technical Field

[0001] This invention belongs to the field of active polysaccharide technology, and specifically relates to an active polysaccharide, its extraction reagent, extraction method and application. Background Technology

[0002] Alginate extraction wastewater is the alkaline waste generated during the agar extraction process from seaweed. Agar extraction involves four steps: alkali treatment, acid treatment, bleaching, and boiling. Because the processing of agar polysaccharides requires a large amount of high-concentration alkaline solution, a significant amount of waste alkaline solution is generated: approximately 120 tons of agar extraction waste alkaline solution are produced for every ton of agar produced. This waste alkaline solution contains large amounts of acids, alkalis, polysaccharides, proteins, pigments, inorganic salts, and other organic matter. Direct discharge or discharge after only simple treatment not only severely pollutes the environment but also wastes a large amount of organic matter. Therefore, extracting polysaccharides from agar extraction wastewater can achieve high-value utilization of this wastewater.

[0003] However, domestic and international research mainly focuses on the direct extraction of active polysaccharides from seaweed. The extraction methods mainly include enzymatic extraction, ultrasonic-assisted aqueous two-phase extraction, and microwave superheated water extraction, such as Chinese invention patent applications with publication numbers CN109608559A and CN117247849A. These methods have the following shortcomings: (1) They are basically water-based solvents, but water has high polarity and low selectivity, which leads to low purity of the extracted polysaccharides; (2) The composition of seaweed gum extraction waste liquid is more complex, and it is much more difficult to extract active polysaccharides from seaweed gum extraction waste liquid than to extract active polysaccharides directly from seaweed; In addition, the special marine environment leads to high sulfation of marine polysaccharides, resulting in low mass spectrometry and liquid phase response, and the sulfuric acid structure is easily destroyed during the separation and purification process. The complex structure, high polarity, large molecular weight, and susceptibility to the separation and purification environment make the study of active polysaccharides from seaweed (including extraction methods, performance activities, etc.) more difficult.

[0004] Therefore, finding effective reagents and methods for extracting active polysaccharides from alginate extraction waste liquid is a problem that urgently needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide an active polysaccharide, its extraction reagent, extraction method, and application. The extraction method is simple and environmentally friendly, and while obtaining a high-purity active polysaccharide, it maintains its original biological activity, which can lay the foundation for the extraction and application of active polysaccharides from alginate extraction waste liquid.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0007] An active polysaccharide with a weight-average molecular weight of 7.04 × 10⁻⁶. 4Da; The active polysaccharide includes at least the following monosaccharide components: glucose, galactose and fucose, and the molar ratio of glucose, galactose and fucose is 28.345:25.837:7.464.

[0008] According to the phenol-sulfuric acid method, the active polysaccharide of the present invention not only has a purity of up to 92.26%, but also has good anti-plant pathogen activity. The minimum inhibitory concentration against Alternaria alternata and Alternaria solani is 0.01 mg / mL. At 0.16 mg / mL, the inhibition rate against Alternaria alternata and Alternaria solani can reach 81.98% and 85.47%, respectively.

[0009] Therefore, the present invention also provides the application of the above-mentioned active polysaccharides in the resistance to plant pathogens, wherein the plant pathogens include at least Alternaria and Alternaria.

[0010] The acquisition of the active polysaccharides of this invention mainly relies on a modified extraction reagent. Therefore, this invention also provides an extraction reagent for extracting the aforementioned active polysaccharides. By mass, the extraction reagent comprises the following components: 2-4 parts ethanol, 2-4 parts polyol, and 1 part quaternary ammonium base.

[0011] To reduce the influence of water polarity and improve polysaccharide purity, this invention uses anhydrous ethanol or a high-volume-fraction (not less than 75%) aqueous ethanol solution as the solvent in the extraction reagent. In a high-concentration ethanol environment, the intermolecular forces of polysaccharide molecules are enhanced, promoting their aggregation and precipitation. The polyol and quaternary ammonium base are fully dissolved in the anhydrous ethanol or high-volume-fraction aqueous ethanol solution. On one hand, the quaternary ammonium base can separate and precipitate acidic and neutral polysaccharides, while the polyol has a chelating effect, allowing the extraction reagent to more stably precipitate polysaccharides. On the other hand, the two react with each other to form a hydrogen bond network, further strengthening the intermolecular forces of polysaccharide molecules. This results in the final active polysaccharide not only having high purity but also retaining its original biological activity.

[0012] Preferably, in the above-mentioned extraction reagent, the ethanol is anhydrous ethanol or an aqueous solution of ethanol with a volume fraction of not less than 75%.

[0013] Preferably, the polyol in the above-mentioned extraction reagent includes sorbitol, glycerol, or butanediol.

[0014] Preferably, the quaternary ammonium base in the above-mentioned extraction reagent includes choline chloride, betaine, or berberine.

[0015] Preferably, in the above-mentioned extraction reagent, the mass ratio of ethanol to polyol is 1:1. At this ratio, polysaccharides can be precipitated as much as possible; a decrease in the content of ethanol or polyol (especially ethanol) will lead to insufficient polysaccharide extraction.

[0016] The present invention also provides a method for extracting the above-mentioned active polysaccharides, the method comprising the following steps:

[0017] S1. The waste liquid from seaweed extraction is mixed with the above-mentioned extraction reagent, stirred, and allowed to stand. The supernatant is discarded to obtain crude polysaccharide precipitate.

[0018] S2 sequentially performs inorganic removal, protein removal, decolorization, and freeze-drying on the crude polysaccharide precipitate to obtain polysaccharide powder;

[0019] S3 dissolves the polysaccharide powder and performs gel column chromatography. The fractions are collected, combined, concentrated, and freeze-dried to obtain the active polysaccharide.

[0020] This invention uses alginate extraction waste liquid as raw material and prepares a coupling extraction reagent with ethanol, polyol, and quaternary ammonium base. First, crude polysaccharides are separated in a short time. Then, the purity of the polysaccharides is improved by removing inorganic matter, proteins, and colors, followed by Sephadex LH-20 gel column chromatography. Furthermore, secondary freeze-drying effectively preserves its anti-plant pathogen activity. The entire extraction method is simple, environmentally friendly, and uses mild process conditions, laying the foundation for the extraction and application of active polysaccharides from alginate extraction waste liquid and realizing the high-value reuse of alginate extraction waste liquid.

[0021] Preferably, in step S1 of the above extraction method, the seaweed extract waste liquid is first filtered and heated at 50-55℃ to concentrate it to 1 / 2-1 / 3 of the original volume to obtain concentrated waste liquid; then the concentrated waste liquid is mixed with the extraction reagent at a volume ratio of 1:(1-2); and the standing time is 2-3 hours.

[0022] As a preferred embodiment, in step S2 of the above extraction method, a dialysis bag with a molecular weight cutoff of 500 Da is used to remove inorganic substances from the crude polysaccharide precipitate to obtain dialysis solution.

[0023] Add Sevag reagent to the dialysate, stir, let stand to separate the layers, collect the upper liquid, concentrate, and obtain the deproteinized solution;

[0024] The AB-8 macroporous adsorption resin and the deproteinizing solution were mixed at a mass ratio of 1:(20-25) and shaken to remove pigments, thus obtaining a depigmented solution.

[0025] The depigmentation solution was pre-cooled at -50 to -55°C and then freeze-dried until the water content was 0 to obtain the polysaccharide powder.

[0026] Preferably, in step S3 of the above extraction method, Sephadex LH-20 is used for gel column chromatography, and isocratic elution is performed with a sodium chloride solution with a concentration of 0.6-0.8M.

[0027] Compared with the prior art, the technical effects of the present invention are reflected in:

[0028] (1) The active polysaccharide of the present invention not only has a purity of up to 92.26%, but also has good anti-plant pathogen activity. The minimum inhibitory concentration against Alternaria and Alternaria is 0.01 mg / mL. At 0.16 mg / mL, the inhibition rate against Alternaria and Alternaria can reach 81.98% and 85.47%, respectively.

[0029] (2) To reduce the influence of water polarity and improve polysaccharide purity, the extraction reagent of this invention uses anhydrous ethanol or a high-volume-fraction (not less than 75%) aqueous ethanol solution as the solvent. In a high-concentration ethanol environment, the interaction forces between polysaccharide molecules are enhanced, promoting the aggregation of polysaccharide molecules to form a precipitate. The polyol and quaternary ammonium base are fully dissolved in anhydrous ethanol or a high-volume-fraction aqueous ethanol solution. On the one hand, the quaternary ammonium base can separate and precipitate acidic and neutral polysaccharides, while the polyol has a chelating effect, enabling the extraction reagent to more stably precipitate polysaccharides. On the other hand, the two react with each other to form a hydrogen bond network, further strengthening the interaction forces between polysaccharide molecules. Thus, the final obtained active polysaccharide not only has high purity but also retains its original biological activity.

[0030] (3) This invention uses alginate extraction waste liquid as raw material and prepares coupling extraction reagents with ethanol, polyols, and quaternary ammonium bases. First, crude polysaccharides are separated in a short time. Then, the purity of the polysaccharides is improved by removing inorganic matter, proteins, and colors, followed by Sephadex LH-20 gel column chromatography. Furthermore, secondary freeze-drying effectively preserves its anti-plant pathogen activity. The entire extraction method is simple, environmentally friendly, and uses mild process conditions, laying the foundation for the extraction and application of active polysaccharides from alginate extraction waste liquid and realizing the high-value reuse of alginate extraction waste liquid. Attached Figure Description

[0031] Figure 1 This is the HPGPC analysis chromatogram of the active polysaccharide of this invention;

[0032] Where Distribution Plots represents the distribution points; dw / dlogM represents the logarithmic differential weight distribution of molecular weight; MW represents the weight-average molecular weight; and %Ht represents the weight fraction.

[0033] Figure 2 This is a diagram showing the monosaccharide composition analysis of the active polysaccharide of this invention;

[0034] Figure 3 The results of antibacterial plate tests on Alternaria alternata and Alternaria spp. at different concentrations of the active polysaccharide prepared in Example 3 of this invention are shown.

[0035] Figure 4The results of the antibacterial rate test of the active polysaccharide prepared in Example 3 of the present invention against Alternaria alterniflora at different concentrations are shown.

[0036] Figure 5 The results of the antibacterial rate test of the active polysaccharide prepared in Example 3 of the present invention against Alternaria at different concentrations are shown.

[0037] Figure 6 The results of the antibacterial rate test of the active polysaccharide prepared in Example 5 of the present invention against Alternaria alterniflora at different concentrations are shown.

[0038] Figure 7 The results of the antibacterial rate test of the active polysaccharide prepared in Example 5 of the present invention against Alternaria at different concentrations are shown.

[0039] Figure 8 The results of the antibacterial rate test of the active polysaccharide prepared in Comparative Example 5 of this invention against Alternaria alterniflora at different concentrations are shown.

[0040] Figure 9 The results show the antibacterial rate of the active polysaccharide prepared in Comparative Example 5 of this invention against Alternaria at different concentrations. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0043] Among them, the seaweed gum extraction waste liquid is the waste alkaline liquid discharged from the alkali treatment stage of the seaweed gum extraction process obtained from the agar production plant; Alternaria alternata 336535, a plant pathogenic fungus, was purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains; Alternaria spp.123548, a plant pathogenic fungus, was purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains.

[0044] Example 1

[0045] This embodiment describes a method for extracting active polysaccharides, comprising the following steps:

[0046] S1. The waste liquid from seaweed extraction is mixed with the above-mentioned extraction reagent, stirred, and allowed to stand. The supernatant is discarded to obtain crude polysaccharide precipitate.

[0047] Specifically, including:

[0048] (1) Waste liquid concentration: Take the seaweed extract waste liquid (i.e. the waste alkaline liquid obtained from the seaweed production plant and discharged from the alkaline treatment stage in the seaweed production process), filter it first with a 150-mesh sieve, collect the filtrate, and concentrate the filtrate at 50°C to obtain a concentrated waste liquid with 1 / 3 of the original volume.

[0049] (2) Crude polysaccharide separation: First, take anhydrous ethanol, butanediol and berberine, mix them in a mass ratio of 3:3:1 to obtain the extraction reagent; then mix the extraction reagent with the concentrated waste liquid in a volume ratio of 1:2, stir thoroughly at 40℃ for 30 min, cool and let stand for 2 h, discard the supernatant to obtain crude polysaccharide precipitate;

[0050] S2 sequentially performs inorganic removal, protein removal, decolorization, and freeze-drying on the crude polysaccharide precipitate to obtain polysaccharide powder;

[0051] Specifically, including:

[0052] (3) Remove inorganic substances: Take a dialysis bag with a molecular weight cutoff of 500 Da, heat it in water for 15 minutes and then cool it. Pour in the crude polysaccharide precipitate that was centrifuged in step (2) and it is viscous. Dialyze it with distilled water for 1 day and then with tap water for 1 day (the dialysis process lasts for 2 days in total) to obtain the dialysate.

[0053] (4) Deproteinization: Add Sevag reagent (chloroform: n-butanol (v / v) = 2:1) to the dialysate, stir magnetically, let stand for separation, collect the upper layer solution, and concentrate by rotary evaporation for 15 min to obtain the deproteinized solution.

[0054] (5) Decolorization: Mix AB-8 macroporous adsorption resin and deproteinizing solution at a mass ratio of 1:20, place on a shaker at 50℃ and 100r / min for 2 hours for adsorption, filter and collect the filtrate to obtain the decolorized solution.

[0055] (6) Freeze-drying: Place the depigmentation solution at -50℃ for 30 minutes, and then freeze-dry for two days until the water content is 0, which yields polysaccharide powder;

[0056] S3 dissolves the polysaccharide powder and performs gel column chromatography. The fractions are collected, combined, concentrated, and freeze-dried to obtain the active polysaccharide of this embodiment.

[0057] Specifically, including:

[0058] (7) Gel column chromatography: First, the column was packed with Sephadex LH-20 packing material. Then, 15 mg of polysaccharide powder obtained in step (6) was weighed and prepared into a polysaccharide solution with a concentration of 15 mg / mL using distilled water. The solution was filtered through a 0.22 μm aqueous filter membrane and loaded onto the column, ensuring that the liquid level was higher than the surface of the packing material. Finally, the solution was eluted with 0.6 M sodium chloride solution at a flow rate of 0.30 mL / min and collected using an automatic collector.

[0059] (8) Secondary freeze-drying: The eluent obtained in step (7) was tested tube by tube using the phenol-sulfuric acid method, and the elution curve was plotted. The components were combined according to the elution results, and then pre-cooled at -55℃ for 30 min and freeze-dried for two days to obtain the active polysaccharide of this embodiment. Its purity was 90.17% as determined by the phenol-sulfuric acid method.

[0060] Example 2

[0061] This embodiment describes a method for extracting active polysaccharides. This method is basically the same as that in Example 1, except that the extraction reagent in step (2) is composed of anhydrous ethanol, butanediol and berberine in a mass ratio of 2:2:1.

[0062] Example 3

[0063] This embodiment describes a method for extracting active polysaccharides. This method is basically the same as that in Example 1, except that the extraction reagent in step (2) is composed of anhydrous ethanol, butanediol and berberine in a mass ratio of 4:4:1.

[0064] Example 4

[0065] This embodiment describes an extraction method for active polysaccharides, which is basically the same as that in Example 1, except that the extraction reagent in step (2) consists of anhydrous ethanol, butanediol and berberine in a mass ratio of 2:4:1.

[0066] Example 5

[0067] This embodiment describes an extraction method for active polysaccharides, which is basically the same as that in Example 1, except that the extraction reagent in step (2) consists of anhydrous ethanol, butanediol and berberine in a mass ratio of 4:2:1.

[0068] Example 6

[0069] This embodiment describes a method for extracting active polysaccharides. The extraction method is basically the same as that in Example 1, except that the extraction reagent in step (2) consists of 95% ethanol, butanediol and berberine in a mass ratio of 3:3:1.

[0070] Example 7

[0071] This embodiment describes a method for extracting active polysaccharides. The extraction method is basically the same as that in Example 1, except that the extraction reagent in step (2) consists of 75% ethanol, butanediol and berberine in a mass ratio of 3:3:1.

[0072] Example 8

[0073] This embodiment describes a method for extracting active polysaccharides, comprising the following steps:

[0074] S1. The waste liquid from seaweed extraction is mixed with the above-mentioned extraction reagent, stirred, and allowed to stand. The supernatant is discarded to obtain crude polysaccharide precipitate.

[0075] Specifically, including:

[0076] (1) Waste liquid concentration: Take the seaweed extract waste liquid (i.e. the waste alkaline liquid obtained from the seaweed production plant and discharged from the alkaline treatment stage in the seaweed production process), filter it first with a 150-mesh sieve, collect the filtrate, and concentrate the filtrate at 55℃ to obtain a concentrated waste liquid with 1 / 3 of the original volume.

[0077] (2) Crude polysaccharide separation: First, take 95% ethanol aqueous solution, sorbitol and choline chloride and mix them in a mass ratio of 3:3:1 to obtain the extraction reagent; then mix the extraction reagent with the concentrated waste liquid in a volume ratio of 1:2, stir thoroughly at 40℃ for 30 min, cool and let stand for 3 h, discard the supernatant to obtain crude polysaccharide precipitate;

[0078] S2 sequentially performs inorganic removal, protein removal, decolorization, and freeze-drying on the crude polysaccharide precipitate to obtain polysaccharide powder;

[0079] Specifically, including:

[0080] (3) Remove inorganic substances: Take a dialysis bag with a molecular weight cutoff of 500 Da, heat it in water for 15 minutes and then cool it. Pour in the crude polysaccharide precipitate that was centrifuged in step (2) and it is viscous. Dialyze it with distilled water for 1 day and then with tap water for 1 day (the dialysis process lasts for 2 days in total) to obtain the dialysate.

[0081] (4) Deproteinization: Add Sevag reagent (chloroform: n-butanol (v / v) = 2:1) to the dialysate, stir magnetically, let stand for separation, collect the upper layer solution, and concentrate by rotary evaporation for 20 min to obtain the deproteinized solution.

[0082] (5) Decolorization: Mix AB-8 macroporous adsorption resin and deproteinizing solution at a mass ratio of 1:25, place on a shaker at 50℃ and 100r / min for 2 hours for adsorption, filter and collect the filtrate to obtain the decolorized solution.

[0083] (6) Freeze-drying: Place the depigmentation solution at -55℃ for 30 minutes, and then freeze-dry for two days until the water content is 0, which yields polysaccharide powder.

[0084] S3 dissolves the polysaccharide powder and performs gel column chromatography. The fractions are collected, combined, concentrated, and freeze-dried to obtain the active polysaccharide of this embodiment.

[0085] Specifically, including:

[0086] (7) Gel column chromatography: First, the column was packed with Sephadex LH-20 packing material. Then, 20 mg of polysaccharide powder obtained in step (6) was weighed and prepared into a polysaccharide solution with a concentration of 20 mg / mL using distilled water. The solution was filtered through a 0.22 μm aqueous filter membrane and loaded onto the column, ensuring that the liquid level was higher than the surface of the packing material. Finally, the solution was eluted with 0.8 M sodium chloride solution at a flow rate of 0.30 mL / min and collected using an automatic collector.

[0087] (8) Secondary freeze-drying: The eluent obtained in step (7) was tested tube by tube using the phenol-sulfuric acid method, and the elution curve was plotted. The components were combined according to the elution results, and then pre-cooled at -55℃ for 30 min and freeze-dried for two days to obtain the active polysaccharide of this embodiment.

[0088] Example 9

[0089] This embodiment describes a method for extracting active polysaccharides, comprising the following steps:

[0090] S1. The waste liquid from seaweed extraction is mixed with the above-mentioned extraction reagent, stirred, and allowed to stand. The supernatant is discarded to obtain crude polysaccharide precipitate.

[0091] Specifically, including:

[0092] (1) Waste liquid concentration: Take the seaweed extract waste liquid (i.e. the waste alkaline liquid obtained from the seaweed production plant and discharged from the alkaline treatment stage in the seaweed production process), filter it first with a 150-mesh sieve, collect the filtrate, and concentrate the filtrate at 55℃ to obtain a concentrated waste liquid with 1 / 2 of the original volume.

[0093] (2) Crude polysaccharide separation: First, take anhydrous ethanol, glycerol and betaine, mix them in a mass ratio of 3:3:1 to obtain the extraction reagent; then mix the extraction reagent with the concentrated waste liquid in a volume ratio of 1:1, stir thoroughly at 40℃ for 30 min, cool and let stand for 3 h, discard the supernatant to obtain crude polysaccharide precipitate;

[0094] S2 sequentially performs inorganic removal, protein removal, decolorization, and freeze-drying on the crude polysaccharide precipitate to obtain polysaccharide powder;

[0095] Specifically, including:

[0096] (3) Remove inorganic substances: Take a dialysis bag with a molecular weight cutoff of 500 Da, heat it in water for 15 minutes and then cool it. Pour in the crude polysaccharide precipitate that was centrifuged in step (2) and it is viscous. Dialyze it with distilled water for 1 day and then with tap water for 1 day (the dialysis process lasts for 2 days in total) to obtain the dialysate.

[0097] (4) Deproteinization: Add Sevag reagent (chloroform: n-butanol (v / v) = 2:1) to the dialysate, stir magnetically, let stand for separation, collect the upper layer solution, and concentrate by rotary evaporation for 20 min to obtain the deproteinized solution.

[0098] (5) Decolorization: Mix AB-8 macroporous adsorption resin and deproteinizing solution at a mass ratio of 1:25, place on a shaker at 50℃ and 100r / min for 2 hours for adsorption, filter and collect the filtrate to obtain the decolorized solution.

[0099] (6) Freeze-drying: Place the depigmentation solution at -50℃ for 30 minutes, and then freeze-dry for two days until the water content is 0, which yields polysaccharide powder;

[0100] S3 dissolves the polysaccharide powder and performs gel column chromatography. The fractions are collected, combined, concentrated, and freeze-dried to obtain the active polysaccharide of this embodiment.

[0101] Specifically, including:

[0102] (7) Gel column chromatography: First, the column was packed with Sephadex LH-20 packing material. Then, 20 mg of polysaccharide powder obtained in step (6) was weighed and prepared into a polysaccharide solution with a concentration of 20 mg / mL using distilled water. The solution was filtered through a 0.22 μm aqueous filter membrane and loaded onto the column, ensuring that the liquid level was higher than the surface of the packing material. Finally, the solution was eluted with 0.7 M sodium chloride solution at a flow rate of 0.30 mL / min and collected using an automatic collector.

[0103] (8) Secondary freeze-drying: The eluent obtained in step (7) was tested tube by tube using the phenol-sulfuric acid method, and the elution curve was plotted. The components were combined according to the elution results, and then pre-cooled at -55℃ for 30 min and freeze-dried for two days to obtain the active polysaccharide of this embodiment. Its purity was 91.54% as determined by the phenol-sulfuric acid method.

[0104] Comparative Example 1

[0105] This embodiment describes a method for extracting active polysaccharides. This method is basically the same as that in Example 1, except that the extraction reagent in step (2) consists of a 95% aqueous ethanol solution and butanediol in a mass ratio of 1:1.

[0106] Comparative Example 2

[0107] This embodiment describes a method for extracting active polysaccharides. This method is basically the same as that in Example 1, except that the extraction reagent in step (2) consists of a 95% ethanol aqueous solution and berberine in a mass ratio of 3:1.

[0108] Comparative Example 3

[0109] This embodiment describes a method for extracting active polysaccharides. This method is basically the same as that in Example 1, except that the extraction reagent in step (2) consists only of anhydrous ethanol.

[0110] Comparative Example 4

[0111] This embodiment describes a method for extracting active polysaccharides. This method is basically the same as that in Example 1, except that the extraction reagent in step (2) consists only of a 95% aqueous ethanol solution.

[0112] Comparative Example 5

[0113] This embodiment describes a method for extracting active polysaccharides. The extraction method is basically the same as that in Example 1, except that the extraction reagent in step (2) consists of a 50% aqueous ethanol solution, butanediol, and berberine in a mass ratio of 3:3:1.

[0114] The active polysaccharides obtained in Examples 1-9 and Comparative Examples 1-5 were analyzed for their purity, molecular weight, monosaccharide composition, and anti-plant pathogen activity.

[0115] I. Purity Analysis

[0116] The purity of each polysaccharide was determined by the phenol-sulfuric acid method, and the results are shown in Table 1.

[0117] Table 1. Purity test results of various polysaccharides

[0118] Example 1 92.17％ Example 2 89.13％ Example 3 92.26％ Example 4 86.58％ Example 5 88.75％ Example 6 90.91％ Example 7 87.65％ Example 8 91.06％ Example 9 91.54％ Comparative Example 1 90.02％ Comparative Example 2 87.38％ Comparative Example 3 85.66％ Comparative Example 4 83.12％ Comparative Example 5 75.32％

[0119] As shown in Table 1, when anhydrous ethanol or a high volume fraction (not less than 75%) of ethanol aqueous solution is used as the extraction reagent, the purity of the obtained active polysaccharides is about 90%; while when the volume fraction of the ethanol aqueous solution is less than 75%, the purity of the obtained active polysaccharides drops to about 75%.

[0120] As can be seen from Examples 3-5, the purity of the extracted active polysaccharide is higher when the mass ratio of ethanol solution to polyol is 1:1; and as can be seen from Examples 1-3, the higher the content of ethanol solution and polyol in the extraction reagent, the higher the purity of the extracted active polysaccharide.

[0121] In summary, the importance of ethanol (or ethanol solution), polyols, and quaternary ammonium bases in extraction reagents is as follows: ethanol (or ethanol solution) > polyols > quaternary ammonium bases.

[0122] II. Molecular weight analysis

[0123] The molecular weight of the polysaccharides extracted in Examples 1-3 (which have relatively high purity and provide more accurate results) was determined by gel permeation chromatography. The detection method was as follows:

[0124] First, prepare a 0.10 mg / mL solution of the sample, filter it through a 0.22 μm aqueous filter membrane, and set it aside for later use; then perform chromatographic analysis under the following chromatographic conditions:

[0125] Chromatographic column: 2x PLgel 8um aquagel-OH Mixed-M 7.5*300mm;

[0126] Detector: Differential refractive index detector;

[0127] Mobile phase: ultrapure water;

[0128] Flow rate: 0.5 mL / min;

[0129] Injection volume: 100 μL.

[0130] Standard curves were plotted using dextran of different molecular weights (Mw 1270, 5220, 11600, 48600, 80900, 147600, 273000, 409800 Da) as standards. The molecular weight of each active polysaccharide sample was calculated based on its retention time relative to the standard curve. The results are shown in [Figure number missing]. Figure 1 And Table 2.

[0131] Table 2. GPC molecular weight determination results of active polysaccharides

[0132] Example 1 47169 36465 70389 1.93.032 Example 2 47172 36489 70433 1.93.025 Example 3 47165 36475 70428 1.93.085

[0133] As can be seen from Table 2, the weight-average molecular weight of the polysaccharides obtained by this invention is all above 7.04 × 10⁻⁶. 4 At the Da level, the detection results of each embodiment showed high consistency.

[0134] III. Monosaccharide Composition Analysis

[0135] Taking the active polysaccharide from Example 3 (highest purity) as an example, the monosaccharide composition of this active polysaccharide was detected by HPLC (Shimadzu LC-20AD); the analytical method was as follows:

[0136] 1) Acid hydrolysis: Weigh an appropriate amount of sample into a 10mL ampoule, add 3.0mL of 2mol / L TFA to the 10mL ampoule, fill with nitrogen, seal the ampoule, and hydrolyze at 120℃ for 4h; then remove the sample, add methanol and nitrogen to evaporate the TFA, and add 30mL of water to reconstitute.

[0137] 2) Sample solution derivatization: Accurately pipette 250 μL of the hydrolyzed sample solution into a 5 mL EP tube, add 250 μL of 0.6 mol / L NaOH and 500 μL of 0.4 mol / L PMP-methanol, and react at 70 °C for 1 h; cool in cold water for 10 min, add 500 μL of 0.3 mol / L HCl to neutralize, then add 1 mL of chloroform and vortex for 1 min, centrifuge at 3000 r / min for 10 min, carefully collect the supernatant, extract 3 times, and collect the supernatant to obtain the sample solution for later use;

[0138] 3) Preparation of mixed control solution: Accurately weigh the monosaccharide standards (mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, glucose, N-acetylglucosamine, galactose, xylose, arabinose, fucose), dissolve and dilute with water to a concentration of 50 μg of each in a mixed control solution per 1 mL. Pass the solution through a 0.22 μm membrane to obtain the mixed control solution for later use.

[0139] 4) HPLC chromatographic analysis: The sample solution and the mixed control solution were analyzed by HPLC under the following chromatographic conditions:

[0140] Chromatographic column: Xtimate C18 4.6*200mm 5μm;

[0141] Column temperature: 30℃;

[0142] Mobile phase: 0.05M potassium dihydrogen phosphate solution (adjusted to pH 6.70 with sodium hydroxide solution) - acetonitrile = 83-17;

[0143] Flow rate: 1.0 mL / min;

[0144] Injection volume: 20 μL.

[0145] 5) Plotting the standard curve: Plot the monosaccharide standard curve based on peak area and molar concentration. The monosaccharide content in the polysaccharide sample is calculated by referring to the standard curve. The results are as follows: Figure 2 As shown.

[0146] Depend on Figure 2 It can be seen that the monosaccharides of the active polysaccharide of the present invention mainly include glucose, galactose and fucose, with a molar ratio of 28.345:25.837:7.464.

[0147] IV. Minimum Inhibitory Concentration Analysis

[0148] Taking the active polysaccharide prepared in Example 3 as an example, the lyophilized powder of the active polysaccharide was dissolved in distilled water and diluted to 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, 0.01 mg / mL, and 0.005 mg / mL to obtain polysaccharide sample solutions of different concentrations. Then, three replicates were set up for each gradient. 100 mL of PDA medium was prepared, sterilized, and cooled to 55-60℃. Each bottle of PDA medium could be used to pour four plates. After the plates cooled and solidified, 1 mL of the polysaccharide sample solution was spread on the plate, and then 0.2 mL of each of the two bacterial suspensions (Alternaria alternata and Alternaria solani) was inoculated onto the plate. A PDA plate without polysaccharide sample solution was used as a blank control. The plates were incubated at 37℃ for 24 h, and the presence or absence of bacterial colony growth was observed visually to determine the minimum inhibitory concentration (MIC) of the polysaccharide. The results are as follows: Figure 3 As shown in Table 3.

[0149] Table 3. Results of Minimum Inhibitory Concentration Analysis

[0150]

[0151] From Table 3 and Figure 3 It is evident that the minimum inhibitory concentration of the active polysaccharide of the present invention against Alternaria alternata and Alternaria solani is 0.01 mg / mL.

[0152] V. Antibacterial Test

[0153] Taking the active polysaccharides prepared in Examples 3, 5, and Comparative Example 5 as examples, their antibacterial activity against Alternaria and Alternaria was detected using the drug-containing plate method. The detection method was as follows:

[0154] The lyophilized active polysaccharide powder was dissolved in distilled water and diluted to 1.6 mg / mL, 0.80 mg / mL, 0.40 mg / mL, 0.20 mg / mL, and 0.10 mg / mL to obtain polysaccharide sample solutions of different concentrations. Next, 90 mL of PDA medium was prepared, sterilized, and cooled to 55-60°C. 10 mL of the sample solution was added to the medium to form a 10% concentration system (the final polysaccharide concentrations in the medium were 0.16 mg / mL, 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL, respectively). An equal volume of sterile water was used as a blank control, and amphotericin B (final concentration 0.0025 mg / mL) was used as a positive control. The sample solutions and medium were mixed thoroughly, and then poured into sterile petri dishes, with each gradient repeated in triplicate. After solidification, remove the fully colonized mycelium plate from the previous step. Use a 6mm diameter punch to make holes in the plate, transferring 6mm mycelial discs to the center of the plate with the mycelial side facing down. Place the plates in a 28℃ incubator. Once the control group has fully colonized, measure the diameter of the mycelial rings in each experimental group and the control group using the cross-sectional method. Calculate the inhibition rate using this formula:

[0155] (Diameter of bacterial rings in the blank group - diameter of bacterial rings in the experimental group) / diameter of bacterial rings in the blank group × 100%.

[0156] The results are as follows Figure 4 and Figure 5 , Figure 6 and Figure 7 , Figure 8 and Figure 9 As shown.

[0157] Depend on Figure 4 and Figure 5 As can be seen, the active polysaccharide of Example 3 of the present invention, at a concentration of 0.16 mg / mL, achieved inhibition rates of 81.98% and 85.47% against Alternaria and Alternaria, respectively; Figure 6 and Figure 7 As shown, the active polysaccharide of Example 5 of the present invention, at a concentration of 0.16 mg / mL, achieved inhibition rates of 75.83% and 81.62% against Alternaria and Alternaria, respectively; while... Figure 8 and Figure 9 As shown, the active polysaccharide of Comparative Example 5, at a concentration of 0.16 mg / mL, exhibited inhibition rates of 64.66% and 73.17% against Alternaria and Alternaria, respectively. This indicates that the active polysaccharide prepared in this invention has good anti-plant pathogen activity, and the higher the purity, the better its anti-plant pathogen activity.

Claims

1. An active polysaccharide, characterized in that, The weight-average molecular weight is 7.04 × 10⁻⁶. 4 Da; includes at least the following monosaccharide components: glucose, galactose and fucose, and the molar ratio of glucose, galactose and fucose is 28.345:25.837:7.464; The extraction method for the active polysaccharides includes the following steps: S1 mixes the alginate extraction waste liquid with the extraction reagent, stirs, and lets stand. The supernatant is discarded to obtain crude polysaccharide precipitate. The extraction reagent comprises the following components by mass: 2-4 parts ethanol, 2-4 parts polyol and 1 part quaternary ammonium base; wherein the ethanol is anhydrous ethanol or an aqueous solution of ethanol with a volume fraction of not less than 75%. S2 sequentially performs inorganic removal, protein removal, decolorization, and freeze-drying on the crude polysaccharide precipitate to obtain polysaccharide powder; S3 dissolves the polysaccharide powder and performs gel column chromatography. The fractions are collected, combined, concentrated, and freeze-dried to obtain the active polysaccharide.

2. An extraction reagent for extracting the active polysaccharide as described in claim 1, characterized in that, By mass, it includes the following components: 2-4 parts ethanol, 2-4 parts polyol and 1 part quaternary ammonium base; The ethanol is anhydrous ethanol or an aqueous solution of ethanol with a volume fraction of not less than 75%. The polyol is selected from sorbitol, glycerol, or butanediol; The quaternary ammonium base is selected from choline chloride, betaine, or berberine.

3. The extraction reagent as described in claim 2, characterized in that, The mass ratio of ethanol to polyol is 1:

1.

4. The method for extracting active polysaccharides as described in claim 1, characterized in that, Includes the following steps: S1. The waste liquid from seaweed gum extraction is mixed with the extraction reagent described in any one of claims 2-3, stirred, and allowed to stand. The supernatant is discarded to obtain crude polysaccharide precipitate. S2 sequentially performs inorganic removal, protein removal, decolorization, and freeze-drying on the crude polysaccharide precipitate to obtain polysaccharide powder; S3 dissolves the polysaccharide powder and performs gel column chromatography. The fractions are collected, combined, concentrated, and freeze-dried to obtain the active polysaccharide.

5. The extraction method as described in claim 4, characterized in that, In step S1, the seaweed extract waste liquid is first filtered and heated at 50-55℃ to concentrate it to 1 / 2-1 / 3 of the original volume to obtain concentrated waste liquid; then the concentrated waste liquid is mixed with the extraction reagent at a volume ratio of 1:(1-2); the standing time is 2-3h.

6. The extraction method as described in claim 4, characterized in that, In step S2, the crude polysaccharide precipitate is treated with a dialysis bag with a molecular weight cutoff of 500 Da to remove inorganic substances and obtain dialysate. Add Sevag reagent to the dialysate, stir, let stand to separate the layers, collect the upper liquid, concentrate, and obtain the deproteinized solution; The AB-8 macroporous adsorption resin and the deproteinizing solution were mixed at a mass ratio of 1:(20-25) and shaken to remove pigments, thus obtaining a depigmented solution. The depigmentation solution was pre-cooled at -50 to -55°C and then freeze-dried until the water content was 0 to obtain the polysaccharide powder.

7. The application of the active polysaccharide as described in claim 1 in the preparation of products resistant to plant pathogens, characterized in that, The plant pathogens mentioned include at least Alternaria and Alternaria.

Images