Active polysaccharide as well as extraction reagent, extraction method and application thereof

Abstract

The invention discloses an active polysaccharide as well as an extraction reagent, an extraction method and application thereof. The weight-average molecular weight of the active polysaccharide is 7.04 * 10 < 4 > Da; the composition at least comprises the following monosaccharide components: glucose, galactose and fucose in a molar ratio of (28.345): (25.837): (7.464). According to the method, the seaweed gel extraction waste liquid is used as a raw material, ethanol, polyhydric alcohol and quaternary ammonium base are used for preparing an extraction reagent, crude polysaccharide is separated in a short time, and then the purity of the polysaccharide is improved (up to 92.26%) through inorganic matter removal, deproteinization, decoloration and gel column chromatography; the plant pathogen resisting activity of the freeze-dried powder is well preserved through secondary freeze-drying, the minimum inhibition concentration of the freeze-dried powder to alternaria and alternaria is 0.01 mg / mL, and the inhibition rates of the freeze-dried powder to the alternaria and alternaria are 81.98% and 85.47% when the concentration of the freeze-dried powder to the alternaria and alternaria is 0.16 mg / mL. The whole extraction method is simple and environment-friendly, the process conditions are mild, and high-valued utilization of the seaweed gel extraction waste liquid is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of active polysaccharides, and particularly relates to an active polysaccharide and an extraction reagent, an extraction method and an application thereof. Background Art

[0002] Alginate extraction waste liquid is the waste alkali liquid produced in the process of extracting agar from seaweed. The extraction of agar requires four steps, namely alkali treatment, acid treatment, bleaching and boiling. Since the treatment of gelatinous polysaccharides requires a large amount of high-concentration alkali liquid, a large amount of waste alkali liquid will be produced: for every ton of agar produced, about 120 tons of alginate extraction waste alkali liquid will be produced. Alginate extraction waste alkali liquid contains a large amount of acid, alkali, polysaccharide, protein, pigment, inorganic salt and other organic matter. It is directly discharged or discharged after simple treatment, which not only seriously pollutes the environment, but also wastes a large amount of organic matter. Therefore, extracting polysaccharides from alginate extraction waste liquid can realize the high-value utilization of alginate extraction waste liquid.

[0003] However, domestic and foreign research has mainly focused on extracting active polysaccharides directly from seaweed. The extraction methods mainly include enzyme extraction, ultrasound-assisted two-phase aqueous method, microwave superheated water extraction, etc., such as the Chinese invention patent applications with publication numbers CN109608559A and CN117247849A. These methods have the following shortcomings: (1) They basically use water as the solvent, but water has high polarity and low selectivity, which will result in low purity of the extracted polysaccharides; (2) The composition of the waste liquid from seaweed extraction is more complex, and it is much more difficult to extract active polysaccharides from the waste liquid from seaweed extraction than to extract active polysaccharides directly from seaweed; in addition, the special marine environment causes marine polysaccharides to be highly sulfated, with low mass spectrometry and liquid phase responses, and the sulfate structure is easily destroyed during the separation and purification process. The complex structure, high polarity, large molecular weight, and susceptibility to the influence of the separation and purification environment make the research on active polysaccharides from seaweed (including extraction methods, performance activity, etc.) more difficult.

[0004] Therefore, how to effectively extract active polysaccharides from alginate extraction waste liquid with reagents and methods is a problem that urgently needs to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide an active polysaccharide and its extraction reagent, extraction method and application. The extraction method is simple and environmentally friendly. While obtaining active polysaccharides with higher purity, its original biological activity is maintained, which can lay a foundation for the extraction and application of active polysaccharides in alginate extraction waste liquid.

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

[0007] An active polysaccharide, the weight average molecular weight of the active polysaccharide is 7.04×10 4Da; the active polysaccharide comprises 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] Detected by 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 pathogenic bacteria activity, and the minimum inhibition concentration to Alternaria and Alternaria is 0.01 mg / mL. At 0.16 mg / mL, the inhibition rates to Alternaria and Alternaria can reach 81.98% and 85.47% respectively.

[0009] Therefore, the present invention also provides the use of the above-mentioned active polysaccharide in resisting plant pathogens, wherein the plant pathogens at least include Alternaria alternata and Alternaria alternata.

[0010] The active polysaccharide of the present invention is mainly obtained by improved extraction reagent, so the present invention also provides an extraction reagent for extracting the above active polysaccharide. The extraction reagent comprises the following components by mass: 2-4 parts of ethanol, 2-4 parts of polyol and 1 part of quaternary ammonium base.

[0011] In order to reduce the influence of water polarity and improve the purity of polysaccharides, the present invention adopts anhydrous ethanol or a high volume fraction (not less than 75%) ethanol aqueous solution as a solvent in the extraction reagent. In a high concentration ethanol environment, the interaction between polysaccharide molecules is enhanced, which promotes the aggregation of polysaccharide molecules to form precipitation; polyols and quaternary ammonium bases are fully dissolved in anhydrous ethanol or a high volume fraction ethanol aqueous solution. On the one hand, the quaternary ammonium base can separate and precipitate acidic and neutral polysaccharides, and the polyols have a chelating effect, so that the extraction reagent can more stably play the role of precipitating polysaccharides; on the other hand, the two react with each other to form a hydrogen bond network, further strengthening the interaction between polysaccharide molecules. In this way, the active polysaccharide finally obtained not only has a higher purity, but also retains its original biological activity.

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

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

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

[0015] Preferably, in the above extraction reagent, the mass ratio of ethanol to polyol is 1: 1. Under 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 extraction of polysaccharides.

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

[0017] S1: mixing the alginate extraction waste liquid with the above-mentioned extraction reagent, stirring, standing, discarding the supernatant, and obtaining a crude polysaccharide precipitate;

[0018] S2 sequentially removes inorganic matter, removes protein, decolorizes and freeze-dries the crude polysaccharide precipitate to obtain polysaccharide powder;

[0019] S3 dissolves the polysaccharide powder and performs gel column chromatography, collects the fractions, combines, concentrates, and freeze-dries them to obtain the active polysaccharide.

[0020] The present invention uses alginate extraction waste liquid as raw material, and uses ethanol, polyol, and quaternary ammonium base to prepare coupling extraction reagents, firstly separate crude polysaccharides in a relatively short time, and then improve the purity of polysaccharides by removing inorganic substances, removing proteins, decolorizing, and Sephadex LH-20 gel column chromatography; and by secondary freeze-drying, the anti-plant pathogenic bacteria activity is better preserved. The whole extraction method is simple, environmentally friendly, and has mild process conditions, which lays a foundation for the extraction and application of active polysaccharides in alginate extraction waste liquid, and realizes the high-value reuse of alginate extraction waste liquid.

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

[0022] Preferably, 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 a dialysate;

[0023] Add Sevag reagent to the dialysate, stir and allow to stand for stratification, collect the upper layer of liquid, concentrate and obtain a deproteinized solution;

[0024] AB-8 macroporous adsorption resin and deproteinization solution were mixed at a mass ratio of 1:(20-25) and then shaken to depigment to obtain a depigmentation solution;

[0025] The depigmented solution is precooled at -50-55°C and then freeze-dried to a moisture content of 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 having a concentration of 0.6-0.8 M.

[0027] Compared with the prior art, the technical effects of the present invention are embodied 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 pathogenic bacteria activity. The minimum inhibitory concentration for Alternaria and Alternaria is 0.01 mg / mL. At 0.16 mg / mL, the inhibition rate for Alternaria and Alternaria can reach 81.98% and 85.47% respectively.

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

[0030] (3) The present invention uses alginate extraction waste liquid as raw material, and uses ethanol, polyol, and quaternary ammonium base to prepare coupling extraction reagents, firstly separate crude polysaccharides in a relatively short time, and then improve the purity of polysaccharides by removing inorganic substances, removing proteins, decolorizing, and Sephadex LH-20 gel column chromatography; and by secondary freeze-drying, the anti-plant pathogenic bacteria activity is better preserved. The whole extraction method is simple, environmentally friendly, and has mild process conditions, which lays a foundation for the extraction and application of active polysaccharides in alginate extraction waste liquid, and realizes the high-value reuse of alginate extraction waste liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the HPGPC analysis chart of the active polysaccharide of the present invention;

[0032] Wherein, 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 monosaccharide composition analysis diagram of the active polysaccharide of the present invention;

[0034] Figure 3 The results of the antibacterial plate test of the active polysaccharide prepared in Example 3 of the present invention on Alternaria and Alternaria at different concentrations;

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

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

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

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

[0039] Figure 8 The test results of the antibacterial rate of the active polysaccharide prepared in Comparative Example 5 of the present invention against Alternaria at different concentrations;

[0040] Fig. 9 These are the test results of the antibacterial rate of the active polysaccharide prepared in Comparative Example 5 of the present invention against Alternaria at different concentrations. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

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

[0043] Among them, the seaweed gel extraction waste liquid is the waste alkali liquid discharged from the alkali treatment link in the seaweed gel extraction production process obtained from the agar production factory; Alternaria alternata 336535, a plant pathogenic fungus, was purchased from Henan Industrial Microbiological Strain Engineering Technology Research Center; Alternaria spp.123548, a plant pathogenic fungus, was purchased from Henan Industrial Microbiological Strain Engineering Technology Research Center.

[0044] Example 1

[0045] The present embodiment provides a method for extracting active polysaccharides, comprising the following steps:

[0046] S1: mixing the alginate extraction waste liquid with the above-mentioned extraction reagent, stirring, standing, discarding the supernatant, and obtaining a crude polysaccharide precipitate;

[0047] Specifically, it includes:

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

[0049] (2) Separation of crude polysaccharides: first, take anhydrous ethanol, butanediol, and berberine, and mix them in a mass ratio of 3:3:1 to obtain an extraction reagent; then, mix the extraction reagent with the concentrated waste liquid in a volume ratio of 1:2, fully stir at 40°C for 30 minutes, cool and stand for 2 hours, discard the supernatant, and obtain a crude polysaccharide precipitate;

[0050] S2 sequentially removes inorganic matter, removes protein, decolorizes and freeze-dries the crude polysaccharide precipitate to obtain polysaccharide powder;

[0051] Specifically, it includes:

[0052] (3) Removal of inorganic substances: Take a dialysis bag with a molecular weight cutoff of 500 Da, heat it in water for 15 min, then cool it, pour in the viscous crude polysaccharide precipitate after centrifugation in step (2), dialyze it with distilled water for 1 day, and then dialyze it with tap water for 1 day (the dialysis process lasts for 2 days in total), and obtain a dialysate;

[0053] (4) Deproteinization: Sevag reagent (chloroform: n-butanol (v / v) = 2:1) was added to the dialysate, and magnetic stirring was performed. After standing for stratification, the upper layer solution was collected and concentrated by rotary evaporation for 15 min to obtain a deproteinized solution;

[0054] (5) Decolorization: Mix AB-8 macroporous adsorption resin and deproteinized solution at a mass ratio of 1:20, place on a shaker at 50°C and 100 rpm for 2 h, and collect the filtrate after filtration to obtain a depigmented solution;

[0055] (6) Freeze-drying: The depigmented solution was precooled at -50°C for 30 minutes, and then freeze-dried for two days until the water content was 0, thereby obtaining polysaccharide powder;

[0056] S3: dissolving the polysaccharide powder and performing gel column chromatography, collecting the fractions, combining, concentrating, and freeze-drying to obtain the active polysaccharide of this example;

[0057] Specifically, it includes:

[0058] (7) Gel column chromatography: first fill the column with Sephadex LH-20 filler, then weigh 15 mg of the polysaccharide powder obtained in step (6), prepare a polysaccharide solution with a concentration of 15 mg / mL with distilled water, filter through a 0.22 μm water filter membrane, and then load the sample so that the sample liquid level is higher than the filler surface; finally, elute with 0.6 M sodium chloride solution at a flow rate of 0.30 mL / min and collect with an automatic collector;

[0059] (8) Secondary freeze-drying: The eluate obtained in step (7) was tested tube by tube using the phenol-sulfuric acid method to draw an elution curve; the components were combined according to the elution results, and then pre-cooled at -55°C for 30 minutes and freeze-dried for two days to obtain the active polysaccharide of this example; the purity of the polysaccharide was 90.17% as detected by the phenol-sulfuric acid method.

[0060] Example 2

[0061] This embodiment provides a method for extracting active polysaccharides. The extraction method is basically the same as that of 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 provides a method for extracting active polysaccharides. The extraction method is basically the same as that of 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 provides a method for extracting active polysaccharides. The extraction method is basically the same as that of Example 1, except that the extraction reagent in step (2) is composed of anhydrous ethanol, butanediol and berberine in a mass ratio of 2:4:1.

[0066] Example 5

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

[0068] Example 6

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

[0070] Example 7

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

[0072] Example 8

[0073] The present embodiment provides a method for extracting active polysaccharides, comprising the following steps:

[0074] S1: mixing the alginate extraction waste liquid with the above-mentioned extraction reagent, stirring, standing, discarding the supernatant, and obtaining a crude polysaccharide precipitate;

[0075] Specifically, it includes:

[0076] (1) Waste liquid concentration: Take the alginate extraction waste liquid (i.e., the waste alkali liquid obtained from the alginate production plant and discharged from the alkali treatment step in the alginate production process), first filter it with a 150-mesh screen, collect the filtrate, and concentrate the filtrate at 55° C. to obtain a concentrated waste liquid with a volume of 1 / 3 of the original volume;

[0077] (2) Separation of crude polysaccharides: first, take 95% ethanol aqueous solution, sorbitol, and choline chloride, and mix them in a mass ratio of 3:3:1 to obtain an extraction reagent; then, mix the extraction reagent with the concentrated waste liquid in a volume ratio of 1:2, fully stir at 40°C for 30 minutes, cool and let stand for 3 hours, discard the supernatant, and obtain a crude polysaccharide precipitate;

[0078] S2 sequentially removes inorganic matter, removes protein, decolorizes and freeze-dries the crude polysaccharide precipitate to obtain polysaccharide powder;

[0079] Specifically, it includes:

[0080] (3) Removal of inorganic substances: Take a dialysis bag with a molecular weight cutoff of 500 Da, heat it in water for 15 min, then cool it, pour in the viscous crude polysaccharide precipitate after centrifugation in step (2), dialyze it with distilled water for 1 day, and then dialyze it with tap water for 1 day (the dialysis process lasts for 2 days in total), and obtain a dialysate;

[0081] (4) Deproteinization: Sevag reagent (chloroform: n-butanol (v / v) = 2:1) was added to the dialysate, and magnetic stirring was performed. After standing for stratification, the upper layer solution was collected and concentrated by rotary evaporation for 20 min to obtain a deproteinized solution;

[0082] (5) Decolorization: Mix AB-8 macroporous adsorption resin and deproteinized solution at a mass ratio of 1:25, place on a shaker at 50°C and 100 rpm for 2 h, and collect the filtrate after filtration to obtain a depigmented solution;

[0083] (6) Freeze-drying: The depigmented solution was precooled at -55°C for 30 minutes, and then freeze-dried for two days until the water content was 0, thereby obtaining polysaccharide powder;

[0084] S3: dissolving the polysaccharide powder and performing gel column chromatography, collecting the fractions, combining, concentrating, and freeze-drying to obtain the active polysaccharide of this example;

[0085] Specifically, it includes:

[0086] (7) Gel column chromatography: first fill the column with Sephadex LH-20 filler, then weigh 20 mg of the polysaccharide powder obtained in step (6), prepare a polysaccharide solution with a concentration of 20 mg / mL with distilled water, filter through a 0.22 μm water filter membrane, and then load the sample so that the sample liquid level is higher than the filler surface; finally, elute with 0.8 M sodium chloride solution at a flow rate of 0.30 mL / min and collect with an automatic collector;

[0087] (8) Secondary freeze-drying: The eluate obtained in step (7) is tested tube by tube using the phenol-sulfuric acid method to draw an elution curve; the components are combined according to the elution results, and then pre-cooled at -55°C for 30 minutes and freeze-dried for two days to obtain the active polysaccharide of this example.

[0088] Example 9

[0089] The present embodiment provides a method for extracting active polysaccharides, comprising the following steps:

[0090] S1: mixing the alginate extraction waste liquid with the above-mentioned extraction reagent, stirring, standing, discarding the supernatant, and obtaining a crude polysaccharide precipitate;

[0091] Specifically, it includes:

[0092] (1) Waste liquid concentration: Take the alginate extraction waste liquid (i.e., the waste alkali liquid obtained from the alginate production plant and discharged from the alkali treatment step in the alginate production process), first filter it with a 150-mesh screen, collect the filtrate, and concentrate the filtrate at 55° C. to obtain a concentrated waste liquid with a volume of 1 / 2 of the original volume;

[0093] (2) Separation of crude polysaccharides: first, take anhydrous ethanol, glycerol, and betaine, and mix them in a mass ratio of 3:3:1 to obtain an extraction reagent; then, mix the extraction reagent with the concentrated waste liquid in a volume ratio of 1:1, fully stir at 40°C for 30 minutes, cool and stand for 3 hours, discard the supernatant, and obtain a crude polysaccharide precipitate;

[0094] S2 sequentially removes inorganic matter, removes protein, decolorizes and freeze-dries the crude polysaccharide precipitate to obtain polysaccharide powder;

[0095] Specifically, it includes:

[0096] (3) Removal of inorganic substances: Take a dialysis bag with a molecular weight cutoff of 500 Da, heat it in water for 15 min, then cool it, pour in the viscous crude polysaccharide precipitate after centrifugation in step (2), dialyze it with distilled water for 1 day, and then dialyze it with tap water for 1 day (the dialysis process lasts for 2 days in total), and obtain a dialysate;

[0097] (4) Deproteinization: Sevag reagent (chloroform: n-butanol (v / v) = 2:1) was added to the dialysate, and magnetic stirring was performed. After standing for stratification, the upper layer solution was collected and concentrated by rotary evaporation for 20 min to obtain a deproteinized solution;

[0098] (5) Decolorization: Mix AB-8 macroporous adsorption resin and deproteinized solution at a mass ratio of 1:25, place on a shaker at 50°C and 100 rpm for 2 h, and collect the filtrate after filtration to obtain a depigmented solution;

[0099] (6) Freeze-drying: The depigmented solution was precooled at -50°C for 30 minutes, and then freeze-dried for two days until the water content was 0, thereby obtaining polysaccharide powder;

[0100] S3: dissolving the polysaccharide powder and performing gel column chromatography, collecting the fractions, combining, concentrating, and freeze-drying to obtain the active polysaccharide of this example;

[0101] Specifically, it includes:

[0102] (7) Gel column chromatography: first fill the column with Sephadex LH-20 filler, then weigh 20 mg of the polysaccharide powder obtained in step (6), prepare a polysaccharide solution with a concentration of 20 mg / mL with distilled water, filter through a 0.22 μm water filter membrane, and load the sample so that the sample liquid level is higher than the filler surface; finally, elute with 0.7 M sodium chloride solution at a flow rate of 0.30 mL / min and collect the solution with an automatic collector;

[0103] (8) Secondary freeze-drying: The eluate obtained in step (7) was tested tube by tube using the phenol-sulfuric acid method to draw an elution curve; the components were combined according to the elution results, and then pre-cooled at -55°C for 30 minutes and freeze-dried for two days to obtain the active polysaccharide of this example; the purity of the polysaccharide was 91.54% as detected by the phenol-sulfuric acid method.

[0104] Comparative Example 1

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

[0106] Comparative Example 2

[0107] This embodiment provides a method for extracting active polysaccharides. The extraction method is basically the same as that of 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 provides a method for extracting active polysaccharides. The extraction method is basically the same as that in Embodiment 1, except that the extraction reagent in step (2) consists only of anhydrous ethanol.

[0110] Comparative Example 4

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

[0112] Comparative Example 5

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

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

[0115] 1. Purity Analysis

[0116] The phenol-sulfuric acid method was used to detect the purity of each polysaccharide. The results are shown in Table 1.

[0117] Table 1 Purity test results of each polysaccharide

[0118] Polysaccharides purity 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] It can be seen from Table 1 that when the extraction reagent is anhydrous ethanol or a high volume fraction (not less than 75%) ethanol aqueous solution, the purity of the obtained active polysaccharide is about 90%; when the volume fraction of the ethanol aqueous solution is lower than 75%, the purity of the obtained active polysaccharide drops to about 75%.

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

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

[0122] 2. Molecular weight analysis

[0123] The molecular weight of the polysaccharides extracted from Examples 1-3 (with relatively high purity and more accurate test results) was detected by gel permeation chromatography. The detection method was:

[0124] First, prepare the sample into a 0.10 mg / mL solution, filter it through a 0.22 μm water filter membrane, and set aside; then perform chromatographic analysis using the following chromatographic conditions:

[0125] Chromatographic columns: 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] Different molecular weight dextran (Mw1270, 5220, 11600, 48600, 80900, 147600, 273000, 409800Da) was used as standard to draw a standard curve. The molecular weight of each active polysaccharide sample was calculated according to its retention time and compared with the standard curve. The results are shown in Figure 1 and Table 2.

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

[0132] project Mp Mn M PD 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] It can be seen from Table 2 that the weight average molecular weight of the polysaccharides prepared in the present invention is 7.04×10 4 Da level, the detection results of each embodiment are highly consistent.

[0134] 3. Monosaccharide composition analysis

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

[0136] 1) Acid hydrolysis: first weigh an appropriate amount of sample into a 10 mL ampoule, add 3.0 mL of 2 mol / L TFA into the 10 mL ampoule, fill with nitrogen, seal the tube, and acid hydrolyze at 120 °C for 4 h; then remove the sample, add methanol nitrogen to evaporate TFA, and add 30 mL of water to re-dissolve;

[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, 500 μL of 0.4 mol / L PMP-methanol, react at 70°C for 1 hour; cool in cold water for 10 minutes, add 500 μL of 0.3 mol / L HCI to neutralize, then add 1 mL of chloroform and vortex for 1 minute, centrifuge at 3000 r / min for 10 minutes, carefully take the supernatant, extract 3 times, take the supernatant to obtain the sample solution, and set aside;

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

[0139] 4) HPLC chromatographic analysis: The sample solution and the mixed control solution were subjected to HPLC chromatographic analysis respectively, and the chromatographic conditions were as follows:

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

[0141] Column temperature: 30°C;

[0142] Mobile phase: 0.05 M 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) Draw a standard curve: Draw a monosaccharide standard curve based on the peak area and molar concentration. The monosaccharide content in the polysaccharide sample is calculated against the standard curve. The results are as follows: Figure 2 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, and the molar ratio is 28.345:25.837:7.464.

[0147] 4. Minimum Inhibitory Concentration Analysis

[0148] Taking the active polysaccharide prepared in Example 3 as an example, the active polysaccharide freeze-dried powder 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. Next, three parallels were set for each gradient, and 100 mL of PDA culture medium was prepared. After sterilization, it was taken out and cooled to 55-60°C. Four plates can be poured from each bottle of PDA culture medium. After the plate was cooled and solidified, 1 mL of the polysaccharide sample solution was absorbed for coating, and then 0.2 mL of two bacterial solutions (Alternaria alternata and Alternaria alternata) were absorbed and inoculated on the plate. In addition, a PDA plate without polysaccharide sample solution was used as a blank control and cultured at 37°C for 24 hours. The minimum inhibitory concentration of the polysaccharide was determined by observing with the naked eye whether there was colony growth on the plate. The results are as follows: Figure 3 And as shown in Table 3.

[0149] Table 3 Minimum inhibitory concentration analysis results

[0150]

[0151] From Table 3 and Figure 3 It can be seen that the minimum inhibitory concentration of the active polysaccharide of the present invention on Alternaria alternata and Alternaria alternata is 0.01 mg / mL.

[0152] 5. Antibacterial Experiment

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

[0154] The active polysaccharide freeze-dried 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 culture medium was prepared, sterilized and taken out, and cooled to 55-60°C. 10 mL of the sample solution was added to the culture medium to form a 10% concentration system (the final concentration of the polysaccharide in the culture medium was 0.16 mg / mL, 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL, respectively). The same volume of sterile water was used as a blank control, and amphotericin B (final concentration of 0.0025 mg / mL) was used as a positive control. The sample solution was mixed evenly with the culture medium, and after mixing evenly, it was poured into a sterile culture dish, and each gradient was repeated 3 times in parallel. After solidification, take out the bacterial plate that has grown in the previous step, use a 6mm diameter puncher to punch holes on the bacterial plate, and transfer the 6mm bacterial cake to the center of the plate with the mycelium facing down. Place the plate in a 28℃ constant temperature incubator for incubation. When the blank control group is full, use the cross method to measure the diameter of the bacterial circle of each experimental group and the control group, and calculate the antibacterial rate according to this formula:

[0155] (Blank group bacterial circle diameter - test group bacterial circle diameter) / blank group bacterial circle diameter × 100%.

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

[0157] Depend on Figure 4 and Figure 5 It can be seen that when the concentration of the active polysaccharide in Example 3 of the present invention is 0.16 mg / mL, the inhibition rate of Alternaria alternata and Alternaria alternata can reach 81.98% and 85.47% respectively; Figure 6 and Figure 7 As shown in Figure 2, when the active polysaccharide of Example 5 of the present invention has a concentration of 0.16 mg / mL, the inhibition rate of Alternaria alternata and Alternaria alternata can reach 75.83% and 81.62% respectively; Figure 8 and Fig. 9 As shown, when the concentration of the active polysaccharide in Comparative Example 5 was 0.16 mg / mL, the inhibition rates against Alternaria alternata and Alternaria alternata were 64.66% and 73.17%, respectively, indicating that the active polysaccharide prepared by the present invention has good anti-plant pathogenic bacteria activity, and the higher the purity, the better the anti-plant pathogenic bacteria activity.

Claims

1. An active polysaccharide, characterized in that: The weight average molecular weight is 7.04×10 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.

2. An extraction reagent for extracting the active polysaccharide according to claim 1, characterized in that: The composition comprises the following components by weight: 2-4 parts of ethanol, 2-4 parts of polyol and 1 part of quaternary ammonium base.

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

1.

4. The extraction reagent according to claim 2, characterized in that The ethanol is anhydrous ethanol or an ethanol aqueous solution with a volume fraction of not less than 75%.

5. The extraction reagent according to claim 2, characterized in that The polyol includes sorbitol, glycerol or butylene glycol.

6. The extraction reagent according to claim 2, characterized in that The quaternary ammonium base includes choline chloride, betaine or berberine.

7. The method for extracting active polysaccharides according to claim 1, characterized in that: The following steps are involved: S1: mixing, stirring, and standing the alginate extraction waste liquid with the extraction reagent according to any one of claims 2 to 6, discarding the supernatant, and obtaining a crude polysaccharide precipitate; S2 sequentially removes inorganic matter, removes protein, decolorizes and freeze-dries the crude polysaccharide precipitate to obtain polysaccharide powder; S3 dissolves the polysaccharide powder and performs gel column chromatography, collects the fractions, combines, concentrates, and freeze-dries them to obtain the active polysaccharide.

8. The extraction method according to claim 7, characterized in that In step S1, the alginate extraction waste liquid is first filtered and heated at 50-55° C. to be concentrated to 1 / 2-1 / 3 of the original volume to obtain a concentrated waste liquid; then the concentrated waste liquid is mixed with the extraction reagent in a volume ratio of 1:(1-2); and the standing time is 2-3 hours.

9. The extraction method according to claim 7, characterized in that In step S2, a dialysis bag with a molecular weight cutoff of 500 Da is used to remove inorganic substances from the crude polysaccharide precipitate to obtain a dialysate; Add Sevag reagent to the dialysate, stir and allow to stand for stratification, collect the upper layer of liquid, concentrate and obtain a deproteinized solution; AB-8 macroporous adsorption resin and deproteinization solution were mixed at a mass ratio of 1:(20-25) and then shaken to depigment to obtain a depigmentation solution; The depigmented solution is precooled at -50-55°C and then freeze-dried to a moisture content of 0 to obtain the polysaccharide powder.

10. The use of the active polysaccharide in resisting plant pathogens as claimed in claim 1, characterized in that: The plant pathogenic bacteria at least include Alternaria alternata and Alternaria alternata.

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