Preparation method and application of rape pollen polysaccharide

Abstract

The invention relates to the technical field of pollen polysaccharides, and discloses a preparation method and application of rape pollen polysaccharides, and the preparation method comprises the following steps: treating degreased rape pollen with a mixed enzyme solution of cellulase and pectinase, and centrifuging to obtain a polysaccharide-containing supernatant; mixing the supernate with absolute ethyl alcohol, centrifuging, and dissolving the precipitate with distilled water to obtain a polysaccharide solution; the polysaccharide solution is sulfonated and modified by a chlorosulfonic acid and pyridine mixed solution, and then carboxymethylated with a monochloroacetic acid and NaOH mixed solution to obtain a modified polysaccharide solution. And finally, dialyzing to remove impurities, and freeze-drying to obtain the rape pollen polysaccharide. Through the extraction and modification technology, the application range of polysaccharide products in agriculture is expanded, crop growth can be more effectively promoted, the yield and quality are improved, and meanwhile negative influences on the environment are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of pollen polysaccharides, and in particular to a preparation method of rapeseed pollen polysaccharides and an application thereof. Background Art

[0002] Polysaccharides, as a natural active substance, are widely distributed in plants, animals and microorganisms. They have attracted much attention in the field of modern agriculture due to their multiple biological functions such as immunomodulation, antioxidant and antibacterial. In recent years, researchers have continued to explore the possibility of polysaccharides as plant growth promoters and eco-agricultural adjuvants. In particular, those polysaccharides derived from plant pollen have shown extraordinary application prospects due to their complex structure and high activity. Despite this, the extraction and application of rapeseed pollen polysaccharides still face several challenges:

[0003] Limitations of traditional extraction methods: The traditional water extraction and alcohol precipitation method has low extraction efficiency, resulting in low polysaccharide purity, and high temperature treatment may destroy the structure of polysaccharide molecules, thereby affecting their biological activity. In addition, chemical extraction technologies such as acid-base method will produce a large amount of waste liquid, causing environmental pollution.

[0004] Insufficient functional development: The polysaccharides extracted from rapeseed pollen have poor water solubility, low bioavailability, and relatively single functions, which makes it difficult to meet the needs of crop seed treatment and foliar spraying for high activity and multifunctionality of polysaccharides.

[0005] Application potential of modification technology: Modification of polysaccharides, such as sulfonation and carboxymethylation, has been proven to significantly improve the water solubility, biological activity and absorption and utilization efficiency of polysaccharides in plants. However, the current research on the modification of rapeseed pollen polysaccharides is still insufficient, and its functional development has not yet been fully realized, especially in the application of crop growth regulation, which needs to be explored.

[0006] In view of the above problems, developing an efficient and environmentally friendly method for extracting rapeseed pollen polysaccharides and further improving the performance of polysaccharides through modification technology will provide a new type of efficient and safe multifunctional adjuvant for the field of agricultural growth regulation. At the same time, this will also provide strong technical support for the high-value utilization of pollen resources. Summary of the invention

[0007] In view of this, the present invention proposes a rape pollen polysaccharide and a preparation method thereof, aiming to solve the problems in the current technology.

[0008] The present invention provides a method for preparing rapeseed pollen polysaccharide, comprising the following steps:

[0009] After defatting rapeseed pollen, a mixed enzyme solution of cellulase and pectinase is added for enzymolysis, and then the enzymolysis supernatant containing polysaccharides is obtained by centrifugation;

[0010] The enzymatic supernatant is mixed with anhydrous ethanol, allowed to stand, and then centrifuged, and the precipitate is collected and dissolved with distilled water to obtain a polysaccharide solution;

[0011] Adding a mixed solution of chlorosulfonic acid and pyridine to the polysaccharide solution for sulfonation modification reaction, and subjecting the polysaccharide solution after the sulfonation modification reaction to a mixed solution of monochloroacetic acid and NaOH for carboxymethyl modification reaction to obtain a modified polysaccharide solution;

[0012] The modified polysaccharide solution is dialyzed to remove low molecular weight impurities, and the rapeseed pollen polysaccharide is obtained after freeze-drying.

[0013] Preferably, the mass ratio of the cellulase to the pectinase is 1:1-2, and the total enzyme addition amount is 1%-2% of the mass of the rapeseed pollen.

[0014] Preferably, the solid-liquid ratio of the enzyme solution during the enzymatic hydrolysis process is 1:10-15 (g / mL).

[0015] Preferably, the mass ratio of chlorosulfonic acid to pyridine in the sulfonation reaction is 1:4-5.

[0016] Preferably, the mass ratio of monochloroacetic acid to NaOH in the carboxymethylation reaction is 1:2-2.5.

[0017] Preferably, the molecular weight cut-off range of the dialysis is 1000-3000 Daltons.

[0018] The present invention also provides a rapeseed pollen polysaccharide, which is prepared according to the above-mentioned method for preparing rapeseed pollen polysaccharide.

[0019] The present invention also proposes an application of the rapeseed pollen polysaccharide in promoting the growth of crops.

[0020] The present invention also proposes an application of the rapeseed pollen polysaccharide in anti-oxidation.

[0021] Preferably, the rapeseed pollen polysaccharide is applied by seed treatment and / or foliar spraying.

[0022] More preferably, when the application method is seed treatment, it comprises: dissolving the rapeseed pollen polysaccharide in distilled water at a concentration of 0.2-0.5 mg / mL, and soaking the seeds for 6-12 hours.

[0023] More preferably, when the application method is foliar spraying, the method comprises: dissolving the rapeseed pollen polysaccharide in distilled water at a concentration of 0.1-0.3 mg / mL, and spraying once every 7-10 days.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. Extraction efficiency is significantly improved

[0026] Compared with the traditional hot water extraction method, enzymatic hydrolysis technology significantly improves the extraction efficiency of rapeseed pollen polysaccharides, with an increase of 20%-30%, while reducing energy consumption and environmental pollution during the extraction process.

[0027] By optimizing the solid-liquid ratio and ethanol concentration during the alcohol precipitation process, the purity and recovery rate of polysaccharides were further improved.

[0028] 2. Significant improvement in polysaccharide performance

[0029] After modification, the water solubility of polysaccharides is significantly improved, with an increase of 50%-70%, making it more suitable for plant absorption and utilization.

[0030] Through sulfonation and carboxymethylation treatment, the antioxidant capacity of polysaccharides is enhanced (the clearance rate is increased by 20%-30%), giving polysaccharides more agricultural functions.

[0031] 3. The plant growth stimulation effect is significantly enhanced

[0032] The modified polysaccharides showed significant growth-promoting effects in both seed treatment and foliar spraying, such as a 10%-15% increase in seed germination rate and a 15%-20% increase in plant biomass. This is closely related to the stronger biological activity and plant absorption and utilization rate of the modified polysaccharides.

[0033] 4. Improve plant resistance to stress

[0034] The introduction of modified polysaccharides not only optimizes the physiological state of plants, but also improves their ability to survive and develop in extreme environments. Specifically, through sulfonation and carboxymethylation modification, rapeseed pollen polysaccharides show obvious advantages in enhancing water solubility, antioxidant properties and water retention capacity, which can effectively improve the growth activity and stress resistance of plants under adverse conditions such as high temperature and plateau climate.

[0035] 5. Green environmental protection and economic benefits

[0036] The use of enzymatic extraction and modification technology to replace traditional methods such as high temperature, strong acid and strong alkali can greatly reduce pollution to the environment, while reducing corrosion and loss of equipment and reducing production costs.

[0037] Modified polysaccharide products can be used to regulate the growth of a variety of crops and have broad market prospects and economic benefits.

[0038] In summary, the present invention not only improves the extraction efficiency and purity of rapeseed pollen polysaccharides, but also significantly enhances its functionality and economic value in agricultural applications. Through the extraction and modification process of the present invention, the application scope of polysaccharide products in agriculture has been expanded, which can more effectively promote crop growth, improve yield and quality, and reduce negative impacts on the environment. In addition, the green and environmentally friendly characteristics of the present invention provide new solutions for agricultural production and environmental protection. DETAILED DESCRIPTION

[0039] The exemplary embodiments of the present disclosure will be described in more detail below. Although the exemplary embodiments of the present disclosure are shown below, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0040] Example 1 Preparation of rapeseed pollen polysaccharide

[0041] 1. Raw material preparation

[0042] 1. Raw materials

[0043] Rapeseed pollen, ensure the dryness is below 5%.

[0044] 2. Preprocessing

[0045] Pollen was screened to remove impurities, crushed to 80 mesh to increase the surface area, and soaked in 0.1% NaHCO3 solution (solid-liquid ratio 1:10, w / v) for 12 hours to remove the wax layer and some proteins on the pollen surface.

[0046] 2. Polysaccharide Extraction

[0047] 1. Enzymatic extraction

[0048] Reagents: cellulase and pectinase (mass ratio 2:1), the enzyme dosage is 1.5% of the pollen weight.

[0049] Conditions: temperature 45°C, pH 5.5, enzymatic hydrolysis time 3 hours.

[0050] Process: Extract with continuous stirring, and inactivate the enzyme in a 95℃ water bath for 10 minutes after enzymatic hydrolysis.

[0051] 2. Water extraction and alcohol precipitation

[0052] Water extraction: Heat extraction at 90℃ for 2 times, 2 hours each time, with a solid-liquid ratio of 1:12 (w / v).

[0053] The extracts were combined and filtered to remove impurities.

[0054] Alcohol precipitation: add anhydrous ethanol to a final concentration of 70%, let stand for 8 hours, centrifuge (8000 rpm, 10 minutes), and collect the precipitate.

[0055] 3. Deproteinization

[0056] Method: Sevag method (chloroform: n-butanol = 4:1, v / v), repeated 3 times.

[0057] 3. Polysaccharide modification

[0058] 1. Sulfonation modification

[0059] Reagents: Chlorosulfonic acid-pyridine method.

[0060] Chlorosulfonic acid and pyridine were mixed at a ratio of 1:4 and added dropwise to the polysaccharide solution (polysaccharide concentration 5%, w / v).

[0061] Conditions: react at 50°C for 5 hours, terminate the reaction, wash and neutralize with ethanol, and dry in vacuum.

[0062] Purpose: To improve water solubility and antioxidant activity.

[0063] 2. Carboxymethylation

[0064] Reagents: Monochloroacetic acid and NaOH.

[0065] The extracted polysaccharide was dissolved in an isopropanol solution, and then a sodium hydroxide solution was added and monochloroacetic acid was added dropwise (polysaccharide: monochloroacetic acid = 1:1.8, mass ratio).

[0066] Conditions: Stir the reaction at 55°C for 3 hours, adjust the pH to neutral, precipitate the modified polysaccharide with ethanol, centrifuge (8000 rpm, 10 minutes) and dry.

[0067] Purpose: To impart stronger water solubility and complexing ability.

[0068] Example 2 Preparation of rapeseed pollen polysaccharide

[0069] 1. Raw material preparation

[0070] 1. Raw materials

[0071] Rapeseed pollen, ensure the dryness is below 5%.

[0072] 2. Preprocessing

[0073] Pollen was screened to remove impurities, crushed to 80 mesh to increase the surface area, and soaked in 0.1% NaHCO3 solution (solid-liquid ratio 1:10, w / v) for 12 hours to remove the wax layer and some proteins on the pollen surface.

[0074] 2. Polysaccharide Extraction

[0075] 1. Enzymatic extraction

[0076] Reagents: cellulase and pectinase (mass ratio 1:1), the enzyme dosage is 2.5% of the pollen weight.

[0077] Conditions: temperature 50°C, pH 5.0, enzymatic hydrolysis time 4 hours.

[0078] Process: Extract with continuous stirring, and inactivate the enzyme in a 95℃ water bath for 10 minutes after enzymatic hydrolysis.

[0079] 2. Water extraction and alcohol precipitation

[0080] Water extraction: Heat extraction at 80℃ for 2 times, 2 hours each time, with a solid-liquid ratio of 1:15 (w / v).

[0081] The extracts were combined and filtered to remove impurities.

[0082] Alcohol precipitation: add anhydrous ethanol to a final concentration of 80%, let stand for 12 hours, centrifuge (8000 rpm, 10 minutes), and collect the precipitate.

[0083] 3. Deproteinization

[0084] Method: Sevag method (chloroform: n-butanol = 4:1, v / v), repeated 3 times.

[0085] 3. Polysaccharide modification

[0086] 1. Sulfonation modification

[0087] Reagents: Chlorosulfonic acid-pyridine method.

[0088] Chlorosulfonic acid and pyridine were mixed at a ratio of 1:5 and added dropwise to the polysaccharide solution (polysaccharide concentration 5%, w / v).

[0089] Conditions: react at 40°C for 6 hours, terminate the reaction, wash and neutralize with ethanol, and dry in vacuum.

[0090] Purpose: To improve water solubility and antioxidant activity.

[0091] 2. Carboxymethylation

[0092] Reagents: Monochloroacetic acid and NaOH.

[0093] The extracted polysaccharide was dissolved in an isopropanol solution, and then sodium hydroxide solution was added and monochloroacetic acid was added dropwise (polysaccharide: monochloroacetic acid = 1:2.2, mass ratio).

[0094] Conditions: Stir the reaction at 60°C for 4 hours, adjust the pH to neutral, precipitate the modified polysaccharide with ethanol, centrifuge (8000 rpm, 10 minutes) and dry.

[0095] Purpose: To impart stronger water solubility and complexing ability.

[0096] Example 3 Preparation of rapeseed pollen polysaccharide

[0097] 1. Raw material preparation

[0098] 1. Raw materials

[0099] Rapeseed pollen, ensure the dryness is below 5%.

[0100] 2. Preprocessing

[0101] Pollen was screened to remove impurities, crushed to 80 mesh to increase the surface area, and soaked in 0.1% NaHCO3 solution (solid-liquid ratio 1:10, w / v) for 12 hours to remove the wax layer and some proteins on the pollen surface.

[0102] 2. Polysaccharide Extraction

[0103] 1. Enzymatic extraction

[0104] Reagents: cellulase and pectinase (mass ratio 2:1), the enzyme dosage is 2% of the pollen weight.

[0105] Conditions: temperature 55°C, pH 4.5, enzymatic hydrolysis time 5 hours.

[0106] Process: Extract with continuous stirring, and inactivate the enzyme in a 95℃ water bath for 10 minutes after enzymatic hydrolysis.

[0107] 2. Water extraction and alcohol precipitation

[0108] Water extraction: Heat extraction at 85℃ for 2 times, 1.5 hours each time, with a solid-liquid ratio of 1:10 (w / v).

[0109] The extracts were combined and filtered to remove impurities.

[0110] Alcohol precipitation: add anhydrous ethanol to a final concentration of 75%, let stand for 12 hours, centrifuge (8000 rpm, 10 minutes), and collect the precipitate.

[0111] 3. Deproteinization

[0112] Method: Sevag method (chloroform: n-butanol = 4:1, v / v), repeated 3 times.

[0113] 3. Polysaccharide modification

[0114] 1. Sulfonation modification

[0115] Reagents: Chlorosulfonic acid-pyridine method.

[0116] Chlorosulfonic acid and pyridine were mixed at a ratio of 1:3 and added dropwise to the polysaccharide solution (polysaccharide concentration 5%, w / v).

[0117] Conditions: react at 45°C for 4 hours, terminate the reaction, wash and neutralize with ethanol, and dry in vacuum.

[0118] Purpose: To improve water solubility and antioxidant activity.

[0119] 2. Carboxymethylation

[0120] Reagents: Monochloroacetic acid and NaOH.

[0121] The extracted polysaccharide was dissolved in an isopropanol solution, and then a sodium hydroxide solution was added and monochloroacetic acid was added dropwise (polysaccharide: monochloroacetic acid = 1:2, mass ratio).

[0122] Conditions: Stir the reaction at 58°C for 3.5 hours, adjust the pH to neutral, precipitate the modified polysaccharide with ethanol, centrifuge (8000 rpm, 10 minutes) and dry.

[0123] Purpose: To impart stronger water solubility and complexing ability.

[0124] Comparative Example 1

[0125] This comparative example is a conventional polysaccharide extraction method.

[0126] 1. Extraction stage

[0127] No enzymatic treatment was performed, only hot water extraction was used.

[0128] Water extraction conditions: extraction at 100°C for 3 times, 1 hour each time, with a solid-liquid ratio of 1:10 (w / v).

[0129] Alcohol precipitation: Add ethanol to a final concentration of 80% and let stand for 12 hours.

[0130] 2. Modification

[0131] No modification.

[0132] Comparative Example 2

[0133] This comparative example is a method for extracting polysaccharides without modification. The extraction process is the same as that of Example 1, except that no modification is performed. Specifically:

[0134] 1. Raw material preparation

[0135] 1. Raw materials

[0136] Rapeseed pollen, ensure the dryness is below 5%.

[0137] 2. Preprocessing

[0138] Pollen was screened to remove impurities, crushed to 80 mesh to increase the surface area, and soaked in 0.1% NaHCO3 solution (solid-liquid ratio 1:10, w / v) for 12 hours to remove the wax layer and some proteins on the pollen surface.

[0139] 2. Polysaccharide Extraction

[0140] 1. Enzymatic extraction

[0141] Reagents: cellulase and pectinase (mass ratio 2:1), the enzyme dosage is 1.5% of the pollen weight.

[0142] Conditions: temperature 45°C, pH 5.5, enzymatic hydrolysis time 3 hours.

[0143] Process: Extract with continuous stirring, and inactivate the enzyme in a 95℃ water bath for 10 minutes after enzymatic hydrolysis.

[0144] 2. Water extraction and alcohol precipitation

[0145] Water extraction: Heat extraction at 90℃ for 2 times, 2 hours each time, with a solid-liquid ratio of 1:12 (w / v).

[0146] The extracts were combined and filtered to remove impurities.

[0147] Alcohol precipitation: add anhydrous ethanol to a final concentration of 70%, let stand for 8 hours, centrifuge (8000 rpm, 10 minutes), and collect the precipitate.

[0148] 3. Deproteinization

[0149] Method: Sevag method (chloroform: n-butanol = 4:1, v / v), repeated 3 times.

[0150] Comparative Example 3

[0151] This comparative example is a polysaccharide extraction method that only performs sulfonation modification. The extraction process is the same as that of Example 1, the only difference being that only sulfonation modification is performed without carboxymethyl modification. Specifically:

[0152] 1. Raw material preparation

[0153] 1. Raw materials

[0154] Rapeseed pollen, ensure the dryness is below 5%.

[0155] 2. Preprocessing

[0156] Pollen was screened to remove impurities, crushed to 80 mesh to increase the surface area, and soaked in 0.1% NaHCO3 solution (solid-liquid ratio 1:10, w / v) for 12 hours to remove the wax layer and some proteins on the pollen surface.

[0157] 2. Polysaccharide Extraction

[0158] 1. Enzymatic extraction

[0159] Reagents: cellulase and pectinase (mass ratio 2:1), the enzyme dosage is 1.5% of the pollen weight.

[0160] Conditions: temperature 45°C, pH 5.5, enzymatic hydrolysis time 3 hours.

[0161] Process: Extract with continuous stirring, and inactivate the enzyme in a 95℃ water bath for 10 minutes after enzymatic hydrolysis.

[0162] 2. Water extraction and alcohol precipitation

[0163] Water extraction: Heat extraction at 90℃ for 2 times, 2 hours each time, with a solid-liquid ratio of 1:12 (w / v).

[0164] The extracts were combined and filtered to remove impurities.

[0165] Alcohol precipitation: add anhydrous ethanol to a final concentration of 70%, let stand for 8 hours, centrifuge (8000 rpm, 10 minutes), and collect the precipitate.

[0166] 3. Deproteinization

[0167] Method: Sevag method (chloroform: n-butanol = 4:1, v / v), repeated 3 times.

[0168] 3. Polysaccharide modification

[0169] Reagents: Chlorosulfonic acid-pyridine method.

[0170] Chlorosulfonic acid and pyridine were mixed at a ratio of 1:4 and added dropwise to the polysaccharide solution (polysaccharide concentration 5%, w / v).

[0171] Conditions: react at 50°C for 5 hours, terminate the reaction, wash and neutralize with ethanol, and dry in vacuum.

[0172] Comparative Example 4

[0173] This comparative example is a polysaccharide extraction method that only performs carboxylmethyl modification. The extraction process is the same as that of Example 1, the only difference being that only carboxylmethyl modification is performed, and sulfonation modification is not performed. Specifically:

[0174] 1. Raw material preparation

[0175] 1. Raw materials

[0176] Rapeseed pollen, ensure the dryness is below 5%.

[0177] 2. Preprocessing

[0178] Pollen was screened to remove impurities, crushed to 80 mesh to increase the surface area, and soaked in 0.1% NaHCO3 solution (solid-liquid ratio 1:10, w / v) for 12 hours to remove the wax layer and some proteins on the pollen surface.

[0179] 2. Polysaccharide Extraction

[0180] 1. Enzymatic extraction

[0181] Reagents: cellulase and pectinase (mass ratio 2:1), the enzyme dosage is 1.5% of the pollen weight.

[0182] Conditions: temperature 45°C, pH 5.5, enzymatic hydrolysis time 3 hours.

[0183] Process: Extract with continuous stirring, and inactivate the enzyme in a 95℃ water bath for 10 minutes after enzymatic hydrolysis.

[0184] 2. Water extraction and alcohol precipitation

[0185] Water extraction: Heat extraction at 90℃ for 2 times, 2 hours each time, with a solid-liquid ratio of 1:12 (w / v).

[0186] The extracts were combined and filtered to remove impurities.

[0187] Alcohol precipitation: add anhydrous ethanol to a final concentration of 70%, let stand for 8 hours, centrifuge (8000 rpm, 10 minutes), and collect the precipitate.

[0188] 3. Deproteinization

[0189] Method: Sevag method (chloroform: n-butanol = 4:1, v / v), repeated 3 times.

[0190] 3. Polysaccharide modification

[0191] Carboxymethylation

[0192] Reagents: Monochloroacetic acid and NaOH.

[0193] The extracted polysaccharide was dissolved in an isopropanol solution, and then a sodium hydroxide solution was added and monochloroacetic acid was added dropwise (polysaccharide: monochloroacetic acid = 1:1.8, mass ratio).

[0194] Conditions: Stir the reaction at 55°C for 3 hours, adjust the pH to neutral, precipitate the modified polysaccharide with ethanol, centrifuge (8000 rpm, 10 minutes) and dry.

[0195] Test Example 1

[0196] 1. Performance Test

[0197] The pollen polysaccharides obtained in Examples 1-3 and Comparative Examples 1-4 were subjected to performance tests, including detection of substance types in the polysaccharide solution, basic properties of the polysaccharides, and plant experiments.

[0198] 1. Substance type detection

[0199] 2. Basic properties of polysaccharides

[0200] Including extraction rate (%), water solubility (expressed as dissolution rate and maximum dissolution concentration) and antioxidant capacity (DPPH free radical scavenging rate, %).

[0201] 3. Plant experiments include

[0202] (1) Seed treatment: Wheat, corn and soybean seeds were treated with 0.5% polysaccharide solution (immersed for 6 hours) and the germination rate (%) and germination speed (hours) were recorded.

[0203] (2) Foliar spraying: Plant the above seeds, dilute the polysaccharide solution to 0.05% and spray it on the seedlings, spray once every 7 days for 3 times, and measure the plant biomass (fresh weight, g) and plant height increase (%).

[0204] 2. Results

[0205] Table 1 shows the top 20 compounds of the pollen polysaccharides prepared in Example 1.

[0206] Table 1

[0207]

[0208] According to the contents of Table 1, the relative content of gluconic acid in the pollen polysaccharide prepared in Example 1 is the highest, reaching 49.658%, indicating that it is the main component of the polysaccharide. Gluconic acid, as a sugar acid, is a type of carbohydrate and may have an important influence on the water solubility and biological activity of the polysaccharide. The content of choline is 12.590%. As a quaternary amine, choline may play a key role in plant growth regulation. The content of trigonelline hydrochloride is 6.771%, which has anti-HSV-1, antibacterial and antifungal activities, which may provide additional biological protection functions for the polysaccharide. Citric acid, as an organic acid substance, has a content of 4.828%, which may help regulate the pH value of the polysaccharide solution, thereby affecting its stability and biological activity. Other compounds such as adenine, proline, mannitol, etc., although the content is low, their respective functions may also contribute to the comprehensive performance of the polysaccharide. Through the combined effects of these compounds, it can be expected that the pollen polysaccharide has potential application value in promoting and protecting plant growth.

[0209] The basic properties of polysaccharides and plant experimental results are shown in Table 2.

[0210] Table 2

[0211]

[0212] From the results in Table 2, it can be seen that the rapeseed pollen polysaccharide extracts of Examples 1-3 are superior to the extracts of Comparative Examples 1-4 in terms of extraction rate, water solubility, DPPH free radical scavenging rate, seed germination rate, seed germination speed, biomass increase and plant height increase. This indicates that the performance of rapeseed pollen polysaccharides can be significantly improved by enzymatic extraction and subsequent modification (sulfonation modification and carboxymethylation modification).

[0213] The extraction rates of Examples 1-3 are all above 70%, while the extraction rate of Comparative Example 1 is only 60%, indicating that enzymatic extraction can effectively improve the extraction efficiency of polysaccharides. In terms of water solubility, the water solubility of polysaccharides in Examples 1-3 is significantly higher than that in Comparative Example 1-2, which may be because the modification increases the hydrophilicity of the polysaccharides. In terms of antioxidant capacity, the DPPH free radical scavenging rates of Examples 1-3 are all above 80%, much higher than 55% of Comparative Example 1, indicating that the modification can effectively improve the antioxidant activity of polysaccharides.

[0214] In the plant experiment, the seed germination rate and germination speed of Examples 1-3 were better than those of Comparative Examples 1-4, indicating that the polysaccharide extract has a promoting effect on seed germination. The results of biomass increase and plant height increase also show that the polysaccharide extract of Examples 1-3 has a positive effect on plant growth.

[0215] In summary, the biological activity and application value of rapeseed pollen polysaccharides can be significantly improved by optimizing the extraction and modification processes. The extraction and modification methods of Examples 1-3 provide an effective technical path for the development and application of rapeseed pollen polysaccharides.

[0216] 3. Exploration of Principles and Mechanisms

[0217] 1. Microscopic mechanism of enzymatic extraction The enzymatic hydrolysis process selectively degrades the cellulose and pectin components of the outer wall of rapeseed pollen through the synergistic action of cellulase and pectinase, destroys the cell wall structure of pollen, and improves the release efficiency of intracellular polysaccharides. At the same time, mild enzymatic hydrolysis conditions (temperature and pH optimization) avoid polysaccharide degradation or structural damage caused by high temperature and strong acid and alkali, thereby retaining the activity and molecular integrity of polysaccharides to the greatest extent.

[0218] 2. Microscopic mechanism of modification

[0219] (1) Sulfonation modification

[0220] The sulfonation reaction increases the electronegativity and hydrophilicity of the molecule by introducing sulfonic acid groups (-SO3H) on the hydroxyl groups of the polysaccharide molecules. This modification not only improves the water solubility of the polysaccharide, but also forms more active sites on its surface, enhancing the ability of the polysaccharide to bind to DPPH free radicals, thereby improving the antioxidant activity. In addition, the sulfonated groups can bind to cations on the plant cell membrane to enhance biological activity.

[0221] (2) Carboxymethylation modification

[0222] Carboxymethylation introduces carboxymethyl groups (-CH2COOH) at the hydroxyl positions of polysaccharides to form more dissociable groups, further improving water solubility and solution stability. The introduction of carboxymethyl structures enhances the interaction between polysaccharides and polysaccharide molecules in plant cell walls, thereby promoting plant absorption and utilization and enhancing growth stimulation effects.

[0223] 3. Synergistic effect of combined modification (sulfonation and carboxymethylation)

[0224] The combined modification significantly improves the water solubility and biological functionality of polysaccharides through the synergistic effect of sulfonation and carboxymethylation. The dual introduction of sulfonic acid groups and carboxymethyl groups not only provides more active sites, but also improves the solution diffusion characteristics of polysaccharide molecules, making it easier to penetrate the plant epidermis or cell wall, improving plant absorption efficiency, and thus enhancing the plant growth promotion effect.

[0225] Example 2

[0226] The pollen polysaccharides obtained from Examples 1-3 and Comparative Examples 1-4 were subjected to stress resistance tests, including resistance to high temperature and plateau climate.

[0227] 1. Stress resistance test method

[0228] 1. Materials

[0229] Source of materials: The concentration of the rapeseed pollen polysaccharide samples extracted in Examples 1-3 and Comparative Examples 1-4 was unified to 0.3 mg / mL, and then subsequent treatment was performed.

[0230] Crops: Wheat and corn seeds.

[0231] 2. Processing method

[0232] Seed treatment: Soak wheat and corn seeds in polysaccharide solution for 6 hours, then take them out and dry them for subsequent use.

[0233] Foliar spraying treatment: After the seeds germinate and grow to the three-leaf stage, spray the plants once every 7 days for a total of 3 times.

[0234] 3. Test environment settings

[0235] High temperature resistance test: carried out in a greenhouse environment, the temperature was set at 42°C, the humidity was maintained at 50%, and the biological indicators were measured after 7 days of treatment.

[0236] Plateau climate resistance test: simulate plateau environmental conditions, with the air pressure set at 65kPa, the temperature at 15°C, and the humidity at 30%. The biological indicators are measured after 10 days of treatment.

[0237] 4. Determination indicators

[0238] Germination rate: refers to the proportion of seeds that germinate, expressed as a percentage (%).

[0239] Survival rate: refers to the proportion of plants that survive after treatment, expressed as a percentage (%).

[0240] Chlorophyll content: measured by SPAD value.

[0241] Biomass: refers to the dry weight of the plant after treatment, in milligrams per plant (mg / plant).

[0242] 2. Results

[0243] The test results are shown in Table 3.

[0244] Table 3

[0245]

[0246] It can be seen from the data in Table 2 that the rapeseed pollen polysaccharides of Examples 1-3 have a significant effect on improving the germination rate, survival rate, chlorophyll content and biomass of wheat and corn seeds under high temperature and plateau climate conditions. Specifically, the germination rate of Example 1 under high temperature environment is 88%, the survival rate is 82%, the chlorophyll content is 42.5SPAD value, and the biomass is 145mg / plant; under plateau environment, the germination rate is 85%, the survival rate is 80%, the chlorophyll content is 40.8SPAD value, and the biomass is 138mg / plant. The performance of Examples 2 and 3 in both environments is also better than that of Comparative Examples 1-4, which shows that the rapeseed pollen polysaccharides that have been optimized, extracted and modified can effectively improve the resistance of plants to adversity.

[0247] Under high temperature environment, the germination rate and survival rate of all examples were higher than those of the comparative example, indicating that the polysaccharide extract can enhance the survival ability of seeds under high temperature conditions. The increase in chlorophyll content indicates that the polysaccharide extract helps plants maintain normal photosynthesis under high temperature. The increase in biomass directly reflects the effect of promoting plant growth.

[0248] Under simulated plateau climate conditions, Examples 1-3 also showed a positive effect on plant growth. The increase in germination rate and survival rate indicates that the polysaccharide extract helps the growth of plants in low oxygen and low temperature environments. The increase in chlorophyll content and the increase in biomass further confirm the promotion of polysaccharide extract on plant photosynthesis and growth.

[0249] In summary, rapeseed pollen polysaccharide extract has a significant effect in improving the resistance of plants to high temperature and plateau adversity, which provides a strong scientific basis for the application of polysaccharide extracts in agricultural production and plant protection. Through further research and development, rapeseed pollen polysaccharide is expected to become a new type of plant growth promoter and adversity protector.

[0250] 3. Exploration of Principles and Mechanisms

[0251] After rapeseed pollen polysaccharides are modified, their structure and properties change significantly, thus improving their support for plant stress resistance. The specific mechanisms include the following aspects:

[0252] 1. Enhance water solubility and bioavailability

[0253] Sulfonation and carboxymethylation modification: By introducing sulfonic acid (-SO3H) and carboxymethyl (-CH2COOH) groups, the hydrophilicity of the modified polysaccharide is significantly enhanced, making it more soluble in water and increasing its bioavailability in plants. The increased water solubility makes it easier for the polysaccharide to be absorbed through the roots or leaves of plants, thereby providing plants with a more adequate source of nutrition and enhancing their ability to adapt to environmental stress.

[0254] Cell wall permeability: The modified polysaccharide molecular structure becomes more active and hydrophilic, enabling it to penetrate the plant cell wall more effectively, promoting water absorption and nutrient transport in plant cells, thereby enhancing the plant's stress resistance.

[0255] 2. Enhanced antioxidant properties

[0256] Sulfonation modification improves antioxidant activity: The introduction of sulfonation groups enhances the ability of polysaccharide molecules to react with free radicals and improves their antioxidant activity. In high temperature and plateau environments, the light is strong and the oxidative pressure increases. Plants are susceptible to oxidative stress, leading to cell membrane rupture and tissue damage. Modified polysaccharides can effectively remove free radicals and protect plant cells from oxidative damage, thereby improving the resistance of plants to extreme environments.

[0257] Ability to scavenge DPPH free radicals: The experimental results showed that the DPPH free radical scavenging rate of the modified polysaccharides was significantly higher than that of the unmodified samples, indicating that their enhanced antioxidant capacity can provide additional protection for plants, especially under high temperature or high altitude environments with high oxidative stress.

[0258] 3. Enhance the water retention capacity of plants

[0259] Water absorption and hydration of polysaccharides: Modified polysaccharides enhance their ability to absorb and retain water by increasing their hydrophilicity. This hydration helps improve the water supply of plants in high temperature environments, reduce water evaporation and dehydration, thereby maintaining the cell structure and function of plants and improving their ability to withstand high temperatures.

[0260] High temperature and plateau adaptability: High temperature and low humidity in plateau areas make plants face the challenges of water evaporation and soil drought. Modified polysaccharides improve the water retention capacity of plants, enhance the adaptability of plants to water deficiency, maintain the normal physiological processes of plants, and further improve their stress resistance.

[0261] 4. Promote the expression of plant stress resistance genes

[0262] Regulation of plant hormones: Modified polysaccharides may enhance the plant's ability to respond to adversity by regulating the plant's hormone balance (such as promoting the synthesis of auxin, gibberellins, etc.). In high temperature or high altitude environments, plants often adapt to environmental changes by adjusting hormone levels. As a natural plant growth regulator, modified polysaccharides can help plants maintain high growth activity and resistance in adversity.

[0263] Activation of stress resistance genes: Polysaccharides may enhance the plant's ability to respond to environmental stress by activating the expression of plant stress resistance genes, such as drought resistance, heat resistance and frost resistance genes.

[0264] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing rapeseed pollen polysaccharide, characterized in that: The following steps are involved: After defatting rapeseed pollen, a mixed enzyme solution of cellulase and pectinase is added for enzymolysis, and then the enzymolysis supernatant containing polysaccharides is obtained by centrifugation; The enzymatic supernatant is mixed with anhydrous ethanol, allowed to stand, and then centrifuged, and the precipitate is collected and dissolved with distilled water to obtain a polysaccharide solution; Adding a mixed solution of chlorosulfonic acid and pyridine to the polysaccharide solution for sulfonation modification reaction, and subjecting the polysaccharide solution after the sulfonation modification reaction to a mixed solution of monochloroacetic acid and NaOH for carboxymethyl modification reaction to obtain a modified polysaccharide solution; The modified polysaccharide solution is dialyzed to remove low molecular weight impurities, and the rapeseed pollen polysaccharide is obtained after freeze-drying.

2. The method for preparing rapeseed pollen polysaccharide according to claim 1, characterized in that: The mass ratio of the cellulase to the pectinase is 1:1-2, and the total enzyme addition amount is 1%-2% of the mass of the rapeseed pollen.

3. The method for preparing rapeseed pollen polysaccharide according to claim 1, characterized in that: The solid-liquid ratio of the enzyme solution during the enzymolysis process is 1:10-15 (g / mL).

4. The method for preparing rapeseed pollen polysaccharide according to claim 1, characterized in that: The mass ratio of chlorosulfonic acid to pyridine in the sulfonation reaction is 1:4-5.

5. The method for preparing rapeseed pollen polysaccharide according to claim 1, characterized in that: The mass ratio of monochloroacetic acid to NaOH in the carboxymethylation reaction is 1:2-2.

5.

6. The method for preparing rapeseed pollen polysaccharide according to claim 1, characterized in that: The molecular weight cut-off range of the dialysis was 1000-3000 Daltons.

7. A rapeseed pollen polysaccharide, characterized in that: The rapeseed pollen polysaccharide is prepared according to the method for preparing rapeseed pollen polysaccharide according to any one of claims 1-6.

8. Use of the rapeseed pollen polysaccharide according to claim 7 in promoting the growth of crops.

9. Use of the rape pollen polysaccharide according to claim 7 in anti-oxidation.

10. The use according to claim 8 or 9, characterized in that: The rapeseed pollen polysaccharide is applied by seed treatment and / or foliar spraying.

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