Flower mushroom dreg polysaccharide nano-selenium fertilizer as well as preparation method and application thereof

Abstract

The invention discloses a shiitake mushroom dreg polysaccharide nano-selenium fertilizer and a preparation method and application thereof, the shiitake mushroom dreg polysaccharide nano-selenium fertilizer comprises shiitake mushroom dreg polysaccharide and nano-selenium, and the shiitake mushroom dreg polysaccharide coats the surface of the nano-selenium; the preparation method comprises the following steps: preparing crude polysaccharide from shiitake mushroom residues by adopting ultrasonic-assisted extraction, hydrothermal extraction, enzymolysis deproteinization and alcohol precipitation processes; taking the polysaccharide as a carrier, and reacting with sodium selenite and ascorbic acid to prepare the flower mushroom dreg polysaccharide nano-selenium fertilizer. The method realizes resource utilization of the mushroom dregs, and is low in cost and simple and efficient in process. The obtained fertilizer is good in stability and high in biological activity, can remarkably increase the content of selenium and microelements in vegetables, reduce pesticide residues, relieve neonicotinoid pesticide stress and salt damage of saline-alkali soil, enhance the osmotic regulation capacity of crops and improve biomass when applied to crops, and provides a new path for efficient agricultural production of the saline-alkali soil.

Description

Technical Field

[0001] This invention belongs to the field of nano-selenium fertilizer preparation and agricultural pollutant control technology, and relates to a nano-selenium fertilizer made from mushroom residue polysaccharide, its preparation method and application. Background Technology

[0002] Shiitake mushroom residue contains a large number of active substances and has good development potential. Polysaccharides, as important natural high-molecular-weight active substances, have biological functions such as anti-oxidation and immunomodulation, and are widely used in functional fertilizers, biomedicine and food additives, providing new ideas for the resource utilization and high-value utilization of mushroom residue.

[0003] Selenium is an essential trace element for humans and animals, playing a crucial role in maintaining health. However, surveys show that over 70% of my country's regions are low in selenium, resulting in more than one-third of residents having a daily selenium intake less than half of the national recommended minimum. Therefore, developing selenium-enriched agricultural products has become an important means of safeguarding the health of the Chinese people. Nano-selenium, due to its excellent biological activity, good biocompatibility, and low toxicity, has become a hot topic in the research of functional selenium-enriched preparations. However, the tendency of nano-selenium particles to aggregate and their poor stability severely restricts their application in agricultural production and food processing.

[0004] Studies have shown that polysaccharide macromolecules, due to their large number of active groups, can serve as excellent nano-selenium modifiers, effectively improving the dispersibility and bioavailability of nano-selenium, thereby helping to enhance the selenium conversion efficiency and nutritional quality of selenium-enriched agricultural products.

[0005] However, in agricultural production, ordinary selenium and nano-selenium are mostly used to increase the selenium content in agricultural products such as rice and tea. At present, polysaccharide nano-selenium is mainly used as a selenium supplement in the food and health industry, and its application potential and unique effects in agricultural production need to be explored.

[0006] Therefore, this invention extracts crude polysaccharides from mushroom residue, optimizes the extraction process of mushroom residue polysaccharides, and then uses the polysaccharides as a carrier to prepare mushroom residue polysaccharide nano-selenium fertilizer. By leveraging the synergistic effect of polysaccharide modification, the application of mushroom residue polysaccharide nano-selenium fertilizer in agricultural production is fully realized. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention aims to provide a method for preparing and applying a polysaccharide nano-selenium fertilizer from mushroom residue. The mushroom residue polysaccharide nano-selenium fertilizer comprises mushroom residue polysaccharide and nano-selenium, with the mushroom residue polysaccharide coating the surface of the nano-selenium. The preparation method includes: firstly, obtaining crude polysaccharide from mushroom residue through ultrasonic-assisted extraction, hydrothermal extraction, enzymatic deproteinization, and alcohol precipitation; then, using the optimized extracted polysaccharide as a carrier, reacting it with ascorbic acid and sodium selenite to prepare the mushroom residue polysaccharide nano-selenium fertilizer, which is finally applied to crops. The fertilizer prepared by this invention... When applied to crops, the polysaccharide nano-selenium fertilizer from mushroom residue can effectively synergistically increase selenium and other trace elements in vegetables grown in saline-alkali land, while reducing pesticide content. This invention realizes the recycling and utilization of mushroom residue resources, reduces preparation costs, and produces polysaccharide selenium nanoparticles from mushroom residue with good stability and high biological activity. The preparation method is simple and efficient, and it can effectively alleviate the stress and inhibitory effects of neonicotinoid pesticides on crops, increase crop biomass, improve crop osmotic regulation capacity, and alleviate salt damage to crops in saline-alkali land, providing a new direction for promoting modern agricultural production.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A type of mushroom residue polysaccharide nano-selenium fertilizer, comprising mushroom residue polysaccharide and nano-selenium, wherein the mass ratio of mushroom residue polysaccharide to nano-selenium is 1.7:1, and the mushroom residue polysaccharide is coated on the surface of the nano-selenium; the particle size of the mushroom residue polysaccharide nano-selenium fertilizer is 70 nm.

[0009] This invention also provides a method for preparing shiitake mushroom residue polysaccharide nano-selenium fertilizer, which is carried out in the following order: S1. Dry the mushroom residue to constant weight, crush it with a pulverizer and pass it through a 40-mesh sieve. Collect the powder that passes through the sieve, add 25 times the amount of ultrapure water, sonicate for 30 minutes and then place it in a water bath for extraction. After vacuum filtration, obtain the mushroom residue polysaccharide extract. S2. Concentrate the polysaccharide extract to one-third of its original volume using a rotary evaporator, add trypsin solution, shake vigorously for 10 min using a vortex shaker, let it stand at room temperature for 4 h, then incubate it in a water bath at 100 ℃ for 20 min, and centrifuge it at 12000 rpm for 10 min to obtain the polysaccharide solution. S3. Add 3 times the volume of 95 wt.% ethanol solution to the polysaccharide solution, let it stand at room temperature in the dark for 12 h, centrifuge at 12000 rpm for 10 min to collect the precipitate, wash the precipitate with anhydrous ethanol and acetone in sequence, and dry the washed precipitate in a vacuum drying oven to obtain the polysaccharide from the mushroom residue of shiitake mushroom. S4. Prepare a 20 mg / mL solution of shiitake mushroom residue polysaccharide, add a 30 mg / mL sodium selenite solution to it, mix thoroughly by vigorous shaking with a vortex shaker for 10 min, and then add a 60 mg / mL ascorbic acid solution to obtain a mixed solution. S5. Place the mixture in a water bath and heat it at 40 ℃ for 3 h. Transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it with ultrapure water for 48 h. Then freeze-dry it at -54~-50℃ for 48~72 h to obtain the mushroom residue polysaccharide nano-selenium fertilizer.

[0010] As a limitation of the preparation method of the present invention, in step S1, the temperature during extraction is 75~85 ℃ and the time is 3~3.5 h.

[0011] As another limitation of the preparation method of the present invention, in step S2, the concentration of the trypsin solution is 500 u·mL. -1 The volume of trypsin solution added was 1 / 10 of that of the polysaccharide concentrate.

[0012] As a third limitation of the preparation method of the present invention, in step S3, the drying temperature is 55~60 ℃ and the time is 24~48 h.

[0013] As a fourth limitation of the preparation method of the present invention, in step S4, the volume of sodium selenite solution added is 133% of that of the mushroom residue polysaccharide solution; the volume ratio of ascorbic acid solution to sodium selenite solution is 3:1.

[0014] This invention provides the application of the mushroom residue polysaccharide nano-selenium fertilizer prepared by the above preparation method in the field of crop cultivation. The mushroom residue polysaccharide nano-selenium fertilizer can achieve the technical effects of increasing crop biomass, alleviating the growth stress caused by neonicotinoid pesticides, regulating the activity of crop metabolic enzymes, or alleviating crop salt damage in crop cultivation.

[0015] As a limitation of the application of the present invention, the soil in which the crops are planted is saline-alkali land and contaminated with neonicotinoid pesticides, and the concentration of neonicotinoid pesticides in the soil is 10 mg / kg; the application concentration of the mushroom residue polysaccharide nano-selenium fertilizer is 1 or 10 mg / L.

[0016] As another limitation of the application of the present invention, the crop is one or more of lettuce, bok choy, or carrots.

[0017] As a third limitation of the application of this invention, the application method of the mushroom residue polysaccharide nano-selenium fertilizer is foliar spraying. At an application rate of 10 g of mushroom residue polysaccharide nano-selenium fertilizer per acre, it is sprayed evenly onto the crop leaves multiple times during the crop's growing season. The above-mentioned technical solution of the present invention is a whole in which each step is closely related and mutually influential, and together they determine the morphological characteristics and performance of the product.

[0018] The above technical solution has the following advantages or beneficial effects: 1. This invention extracts polysaccharides from mushroom residue. The preparation cost of this material is low, realizing the resource utilization of agricultural waste, while reducing environmental pollution, and has significant economic and environmental benefits. 2. The mushroom residue polysaccharide nano-selenium fertilizer prepared by this invention uses polysaccharide as a carrier to prepare nano-selenium complex. The polysaccharide is wrapped on the surface of nano-selenium, which significantly improves the stability and dispersibility of nano-selenium and avoids its aggregation. At the same time, polysaccharide and nano-selenium have a synergistic effect, which enhances the antioxidant and other biological activities. 3. The mushroom residue polysaccharide nano-selenium fertilizer prepared by this invention can effectively increase crop biomass, improve crop osmotic regulation capacity, alleviate salt damage, and help crops cope with calcium deficiency. 2+ K + Optimize allocation and absorption as needed, while achieving efficient accumulation of Se in the above-ground parts.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0020] Figure 1 The curves showing the effect of different material-to-liquid ratios between mushroom residue and ultrapure water on the polysaccharide yield of mushroom residue. Figure 2 The curve showing the effect of water bath temperature on the yield of polysaccharides from mushroom residue; Figure 3 The curve showing the effect of water bath time on the yield of polysaccharides from the fungal residue; Figure 4 The interaction diagrams for the response surface methodology optimization of polysaccharide extraction from mushroom residue are as follows: (A) is the interaction diagram of water bath temperature and material-to-liquid ratio on extraction rate; (B) is the interaction diagram of water bath temperature and water bath time on extraction rate; and (C) is the interaction diagram of material-to-liquid ratio and water bath time on extraction rate. Figure 5 The following is a particle size distribution analysis diagram of polysaccharide nano-selenium fertilizer from mushroom residue: A is a comparison diagram of the effect of different volume ratios of ascorbic acid and sodium selenite on particle size; B is a comparison diagram of the effect of different volume proportions of polysaccharide solution on particle size; and C is a comparison diagram of the effect of different hydrothermal times on particle size. Figure 6 This is a graph showing the relationship between the concentration and selenium content of the polysaccharide nano-selenium fertilizer obtained from mushroom residue in Example 1 of this invention. Figure 7The images shown are transmission electron microscopy (TEM) images and energy dispersive spectroscopy (EDS) analysis diagrams of the polysaccharide nano-selenium fertilizer prepared from mushroom residue in Example 1 of this invention. In the images, A is a bright-field TEM image, B is a high-resolution TEM image, C is an EDS mapping diagram of the Se element, and D is an EDS energy dispersive spectroscopy and quantitative analysis table. Figure 8 This is a graph showing the antioxidant activity analysis of the shiitake mushroom residue polysaccharide nano-selenium fertilizer prepared in Example 1 of the present invention, where: A is the DPPH· free radical scavenging rate result, B is the ABTS· free radical scavenging rate result. + Free radical scavenging rate results graph; Figure 9 The graphs show the comparison of fresh biomass of lettuce seedlings, where: A is the graph showing the change in fresh biomass after applying 1 mg / L of different selenium preparations, and B is the graph showing the change in fresh biomass after applying 10 mg / L of different selenium preparations. Figure 10 The charts show a comparison of pesticide content in lettuce seedlings. In the charts, A shows the pesticide content comparison when different selenium preparations were applied at a concentration of 1 mg / L, and B shows the pesticide content comparison when different selenium preparations were applied at a concentration of 10 mg / L. Figure 11 The charts show the comparison of selenium content in lettuce seedlings. In the charts, A shows the comparison of selenium content after applying 1 mg / L of different selenium preparations, and B shows the comparison of selenium content after applying 10 mg / L of different selenium preparations. Figure 12 This is a mineral element content analysis diagram of the polysaccharide nano-selenium fertilizer prepared from mushroom residue in Example 1 of the present invention to alleviate crop salt damage, where: A represents K + Content change graph, B represents Ca 2+ Content change graph, C represents Mg 2+ Content change graph, D represents Na + Content change graph. Detailed Implementation

[0021] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0022] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. Specifically, the DPPH free radical scavenging reagent kit and the ABTS free radical scavenging reagent kit were purchased from Suzhou Greens Biotechnology Co., Ltd.

[0023] Unless otherwise specified, the methods described in the following embodiments are conventional methods in the art.

[0024] Example 1 This embodiment prepares a nano-selenium polysaccharide from mushroom residue, and the preparation process and steps are as follows: S1. Dry the mushroom residue to constant weight, crush it with a pulverizer and pass it through a 40-mesh sieve. Collect the powder that passes through the sieve, add 25 times the amount of ultrapure water, sonicate for 30 min, place it in a water bath and extract it at 75 ℃ for 3.5 h. After vacuum filtration, obtain the mushroom residue polysaccharide extract. S2. Concentrate the polysaccharide extract to one-third of its original volume using a rotary evaporator, and add 500 u·mL⁻¹ of concentrate. -1 Trypsin solution, the volume of trypsin solution added is 1 / 10 of the polysaccharide concentrate, shake vigorously with a vortex shaker for 10 min, let stand at room temperature for 4 h, water bath at 100 ℃ for 20 min, place it in a centrifuge and centrifuge at 12000 rpm for 10 min to obtain polysaccharide solution. S3. Add 3 times the volume of 95 wt.% ethanol solution to the polysaccharide solution, let it stand at room temperature in the dark for 12 h, centrifuge at 12000 rpm for 10 min to collect the precipitate, wash the precipitate twice with anhydrous ethanol and acetone respectively, place the washed precipitate in a vacuum drying oven and dry at 55 ℃ for 24 h to obtain the polysaccharide from the mushroom residue of shiitake mushroom. S4. Prepare a 20 mg / mL solution of mushroom residue polysaccharide. Add a 30 mg / mL sodium selenite solution to the solution. The volume of sodium selenite solution added is 133% of the volume of the mushroom residue polysaccharide solution (i.e., the volume ratio of mushroom residue polysaccharide solution is 15%). Mix the solution by vigorous shaking for 10 min. Then add a 60 mg / mL ascorbic acid solution. The volume ratio of ascorbic acid solution to sodium selenite solution is 3:1 to obtain a mixed solution. S5. Place the mixture in a water bath and heat it at 40 ℃ for 3 h. Transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it with ultrapure water for 48 h. Freeze-dry it at -54 ℃ for 48 h to obtain the mushroom residue polysaccharide nano-selenium fertilizer.

[0025] Example 2 This embodiment prepares a nano-selenium polysaccharide from mushroom residue, and the preparation process and steps are as follows: S1. Dry the mushroom residue to constant weight, crush it with a pulverizer and pass it through a 40-mesh sieve. Collect the powder that passes through the sieve, add 25 times the amount of ultrapure water, sonicate for 30 min, place it in a water bath, extract it at 80 ℃ for 3 h, and then filter it under vacuum to obtain the mushroom residue polysaccharide extract. S2. Concentrate the polysaccharide extract to one-third of its original volume using a rotary evaporator, and add 500 u·mL⁻¹ of concentrate. -1Trypsin solution, the volume of trypsin solution added is 1 / 10 of the polysaccharide concentrate, shake vigorously with a vortex shaker for 10 min, let stand at room temperature for 4 h, water bath at 100 ℃ for 20 min, place it in a centrifuge and centrifuge at 12000 rpm for 10 min to obtain polysaccharide solution. S3. Add 3 times the volume of 95 wt.% ethanol solution to the polysaccharide solution, let it stand at room temperature in the dark for 12 h, centrifuge at 12000 rpm for 10 min to collect the precipitate, wash the precipitate twice with anhydrous ethanol and acetone respectively, place the washed precipitate in a vacuum drying oven and dry at 58 ℃ for 48 h to obtain the polysaccharide from the mushroom residue of shiitake mushroom. S4. Prepare a 20 mg / mL solution of mushroom residue polysaccharide. Add a 30 mg / mL sodium selenite solution to the solution, with the volume of sodium selenite solution being 133% of the volume of the mushroom residue polysaccharide solution. Mix thoroughly by vigorous shaking with a vortex shaker for 10 min. Then add a 60 mg / mL ascorbic acid solution, with the volume ratio of ascorbic acid solution to sodium selenite solution being 3:1, to obtain a mixed solution. S5. Place the mixture in a water bath and heat it at 40 ℃ for 3 h. Transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it with ultrapure water for 48 h. Freeze-dry it at -52 ℃ for 60 h to obtain the shiitake mushroom residue polysaccharide nano-selenium fertilizer.

[0026] Example 3 This embodiment prepares a nano-selenium polysaccharide from mushroom residue, and the preparation process and steps are as follows: S1. Dry the mushroom residue to constant weight, crush it with a pulverizer and pass it through a 40-mesh sieve. Collect the powder that passes through the sieve, add 25 times the amount of ultrapure water, sonicate for 30 min, place it in a water bath, and extract it at 85 ℃ for 2.5 h. After vacuum filtration, obtain the mushroom residue polysaccharide extract. S2. Concentrate the polysaccharide extract to one-third of its original volume using a rotary evaporator, and add 500 u·mL⁻¹ of concentrate. -1 Trypsin solution, the volume of trypsin solution added is 1 / 10 of the polysaccharide concentrate, shake vigorously with a vortex shaker for 10 min, let stand at room temperature for 4 h, water bath at 100 ℃ for 20 min, place it in a centrifuge and centrifuge at 12000 rpm for 10 min to obtain polysaccharide solution. S3. Add 3 times the volume of 95 wt.% ethanol solution to the polysaccharide solution, let it stand at room temperature in the dark for 12 h, centrifuge at 12000 rpm for 10 min to collect the precipitate, wash the precipitate twice with anhydrous ethanol and acetone respectively, place the washed precipitate in a vacuum drying oven and dry at 60 ℃ for 36 h to obtain the polysaccharide from the mushroom residue of shiitake mushroom. S4. Prepare a 20 mg / mL solution of mushroom residue polysaccharide. Add a 30 mg / mL sodium selenite solution to the solution, with the volume of sodium selenite solution being 133% of the volume of the mushroom residue polysaccharide solution. Mix thoroughly by vigorous shaking with a vortex shaker for 10 min. Then add a 60 mg / mL ascorbic acid solution, with the volume ratio of ascorbic acid solution to sodium selenite solution being 3:1, to obtain a mixed solution. S5. Place the mixture in a water bath and heat it at 40 ℃ for 3 h. Transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it with ultrapure water for 48 h. Freeze-dry it at -50 ℃ for 72 h to obtain the mushroom residue polysaccharide nano-selenium fertilizer.

[0027] Experimental Example 1: Optimized Extraction Experiment and Particle Size Distribution Analysis of Polysaccharides from Shiitake Mushroom Residue Using polysaccharide yield as the indicator, a single-factor rotation method was employed to investigate the effects of solid-liquid ratio, extraction temperature, and extraction time on polysaccharide extraction rate under basic conditions (solid-liquid ratio 1:15 g / mL, water bath temperature 70℃, and water bath time 3 h). Based on the suitable range determined by the single-factor experiments, and using polysaccharide yield as the response value, the solid-liquid ratio, water bath temperature, and water bath time were selected as influencing factors. A Box-Behnken response surface methodology was used for optimization, as detailed below: (1) Dry the mushroom residue to constant weight, crush it with a pulverizer and pass it through a 40-mesh sieve. Collect the powder that passes through the sieve and store it in a sealed bag at room temperature for later use. (2) Investigate the effect of material-liquid ratio on polysaccharide yield: The extraction temperature was fixed at 70 ℃ and the extraction time was 3 h. The material-liquid ratio was set to 1:10, 1:15, 1:20, 1:25 and 1:30 respectively. 1.0 g of fungal residue powder was accurately weighed into a 50 mL centrifuge tube. The corresponding volume of ultrapure water was added according to the set material-liquid ratio. After sealing, the tube was placed in an ultrasonic machine and sonicated for 30 min. Then it was placed in a 70 ℃ constant temperature water bath for 3 h. After centrifugation, the polysaccharide extract was obtained. (3) Investigate the effect of extraction temperature on polysaccharide yield: With a fixed material-to-liquid ratio of 1:15 g / mL and an extraction time of 3 h, the extraction temperatures were set at 65, 70, 75, 80, and 85 ℃ respectively; 1.0 g of fungal residue powder was accurately weighed into a 50 mL centrifuge tube, and ultrapure water was added at a material-to-liquid ratio of 1:15. After sealing, the tube was sonicated for 30 min, and then placed in a constant temperature water bath at 65, 70, 75, 80, and 85 ℃ for 3 h respectively. After centrifugation, polysaccharide extract was obtained. (4) Investigate the effect of extraction time on polysaccharide yield: With a fixed material-to-liquid ratio of 1:15 g / mL and an extraction temperature of 70 ℃, the extraction times were set to 2, 2.5, 3, 3.5 and 4 h respectively; 1.0 g of fungal residue powder was accurately weighed into a 50 mL centrifuge tube, and ultrapure water was added at a material-to-liquid ratio of 1:15. After sealing, the tube was sonicated for 30 min, and then placed in a 70 ℃ constant temperature water bath for 2, 2.5, 3, 3.5 and 4 h respectively. After centrifugation, polysaccharide extract was obtained. (5) Concentrate the polysaccharide extract to one-third of its original volume using a rotary evaporator, and add 500 u·mL of the solution. -1 The trypsin solution was added to the polysaccharide concentrate at a volume of 1 / 10. The mixture was vigorously shaken for 10 min using a vortex mixer, allowed to stand at room temperature for 4 h, and then inactivated by water bath at 100 ℃ for 20 min. The supernatant polysaccharide solution was obtained by centrifugation at 12000 rpm for 10 min. (6) Add 95 wt.% ethanol to the polysaccharide solution, the volume of which is 3 times that of the polysaccharide solution. Let it stand at room temperature in the dark for 12 h to precipitate the polysaccharide. Centrifuge at 12000 rpm for 10 min to collect the precipitate. Wash the precipitate with anhydrous ethanol and acetone in sequence, twice. Place the washed precipitate in a vacuum drying oven at 55 ℃ for 24 h to obtain the polysaccharide from the mushroom residue.

[0028] The curves showing the effects of material-to-liquid ratio, water bath temperature, and water bath time on the yield of polysaccharides from the fungal residue are shown below. Figure 1 , Figure 2 and Figure 3 As shown in the figure, the peak value (7.82%) was reached when the material-to-liquid ratio was 1:25 g / mL, the maximum value (8.66%) was reached when the water bath temperature was 75℃, and the peak value (7.04%) was reached when the hydrothermal extraction time was 3.5 h. Therefore, the suitable material-to-liquid ratio was determined to be 1:20~1:30 g / mL, the suitable water bath temperature was determined to be 75~85℃, and the suitable water bath time was determined to be 2.5~3.5 h.

[0029] The interaction diagram of response surface methodology optimization is shown below. Figure 4 As shown in the figure, the interaction between the material-liquid ratio and the water bath temperature is significant (P < 0.05). When the material-liquid ratio is 1:22~1:26 g / mL and the water bath temperature is 77~80 ℃, the polysaccharide yield is higher. The interaction between the material-liquid ratio and the water bath time, and between the water bath temperature and the water bath time, is not significant (P > 0.05).

[0030] Experimental Example 2: Preparation Experiment of Polysaccharide Nano-Selenium Fertilizer from Shiitake Mushroom Residue Using the particle size of the shiitake mushroom residue polysaccharide nano-selenium fertilizer as an indicator, a single-factor rotation method was employed. Under the basic conditions (ascorbic acid:sodium selenite volume ratio = 3:1, water bath temperature 40 ℃, water bath time 3 h, polysaccharide solution volume percentage 15%), the effects of the ascorbic acid:sodium selenite volume ratio, extraction time, and polysaccharide solution volume percentage on the particle size of the shiitake mushroom residue polysaccharide nano-selenium fertilizer were investigated. The details are as follows: (1) Prepare a polysaccharide solution of mushroom residue with a concentration of 20 mg / mL, a sodium selenite solution with a concentration of 30 mg / mL and an ascorbic acid solution with a concentration of 60 mg / mL; (2) Investigate the effect of the volume ratio of ascorbic acid to sodium selenite on particle size: With the water bath temperature fixed at 40 ℃, water bath time at 3 h and polysaccharide solution volume ratio at 15%, the volume ratio of ascorbic acid to sodium selenite was set to 1:1, 2:1, 3:1 and 4:1 respectively; the polysaccharide solution was accurately measured into a 50 mL centrifuge tube using a pipette, and the corresponding volume of sodium selenite solution was added first according to the set ratio. After mixing by vigorous shaking for 10 min using a vortex shaker, the corresponding volume of ascorbic acid solution was added. After sealing, the tube was placed in a 40 ℃ constant temperature water bath for 3 h to obtain a mixed solution of polysaccharide nano-selenium fertilizer from mushroom residue. (3) Investigate the effect of water bath time on particle size: With the volume ratio of ascorbic acid to sodium selenite fixed at 3:1, water bath temperature at 40 ℃, and polysaccharide solution volume ratio at 15%, water bath time was set at 2, 3, 4, and 5 h respectively; the polysaccharide solution was accurately measured into a 50 mL centrifuge tube using a pipette, sodium selenite solution was added first, and after vortexing for 10 min to mix, ascorbic acid solution was added, and the tube was sealed and placed in a 40 ℃ constant temperature water bath for 2, 3, 4, and 5 h respectively to obtain a mixed solution of polysaccharide nano-selenium fertilizer from mushroom residue; (4) Investigate the effect of polysaccharide solution volume ratio on particle size: With the volume ratio of ascorbic acid to sodium selenite fixed at 3:1, water bath temperature at 40 ℃ and water bath time at 3 h, the volume ratio of polysaccharide solution was set to 5%, 10%, 15% and 20% respectively; the corresponding volume of polysaccharide solution was accurately measured into a 50 mL centrifuge tube using a pipette, sodium selenite solution was added first, and after vortexing for 10 min to mix, ascorbic acid solution was added, and after sealing, the tube was placed in a 40 ℃ constant temperature water bath for 3 h to obtain a mixed solution of polysaccharide nano-selenium fertilizer from mushroom residue; (5) Place the mixed solution of mushroom residue polysaccharide nano-selenium fertilizer in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyze with ultrapure water for 48 h. The volume of ultrapure water is 100 times the volume of the sample. After dialysis, freeze dry to obtain mushroom residue polysaccharide nano-selenium fertilizer.

[0031] The polysaccharide nano-selenium fertilizer from shiitake mushroom residue was prepared into a 1 mg / mL solution. Its particle size distribution and potential were analyzed using a particle size analyzer. The results are as follows: Figure 5 As shown, with the increase of the volume ratio of ascorbic acid to sodium selenite, the particle size of the shiitake mushroom residue polysaccharide nano-selenium fertilizer significantly decreased, the particle size distribution peak shifted towards smaller particle sizes, and the peak width narrowed, resulting in a more concentrated distribution. The minimum particle size was reached when the volume ratio of ascorbic acid to sodium selenite was 3:1. When the volume percentage of PS (shiitake mushroom residue polysaccharide solution) increased from 5% to 15%, the particle size of the nano-selenium particles decreased, the distribution peak shifted towards smaller particle sizes, and the distribution was most concentrated when the PS volume percentage was 15%. When the hydrothermal time was extended from 2 h to 3 h, the particle size of the shiitake mushroom residue polysaccharide nano-selenium fertilizer decreased, the distribution peak shifted towards smaller particle sizes, and the distribution became more concentrated. When the time was further extended to 4 h and 5 h, the particle size increased slightly, but the distribution range did not change significantly. In summary, the optimal conditions were selected as follows: a volume ratio of ascorbic acid to sodium selenite of 3:1, a PS volume percentage of 15%, and a hydrothermal time of 3 h.

[0032] Performance Testing (I) Determination of selenium content in polysaccharide nano-selenium fertilizer from mushroom residue Accurately weigh 0.01 g of the *Viola yedoensis* mycelium residue polysaccharide nano-selenium fertilizer prepared in Examples 1-3 of this invention, and place it in a digestion vessel. Add 3 mL of nitric acid and 1 mL of hydrochloric acid, heat on a heating plate for 5 h, then add 1.5 mL of hydrofluoric acid. Digest until the remaining solution is transparent and clear, and the volume is reduced to the size of a soybean. Transfer to a volumetric flask and dilute to 10 mL with ultrapure water. Determine the selenium content using ICP-MS. The relationship between the concentration of the *Viola yedoensis* mycelium residue polysaccharide nano-selenium fertilizer suspension and the selenium content is as follows: Figure 6 As shown in the figure, the concentration of selenium ions in a 100 ppm solution of mushroom residue polysaccharide nano-selenium fertilizer is 50.775 ppm.

[0033] (II) Transmission electron microscopy and energy dispersive spectroscopy analysis of polysaccharide nano-selenium fertilizer from mushroom residue A small amount of shiitake mushroom residue polysaccharide nano-selenium fertilizer powder was taken, added to 1 mL of anhydrous ethanol, and analyzed using a transmission electron microscope. The analysis results are as follows: Figure 7 As shown in the SEM image, the polysaccharide-coated selenium nanoparticles exhibit a uniform spherical structure with a particle size of approximately 70 nm, and the particle surface is uniformly covered with a thin layer of mushroom residue polysaccharide film. This polysaccharide coating effectively prevents particle aggregation and maintains particle dispersibility. C, O, and Se elements were detected in the mushroom residue polysaccharide nano-selenium fertilizer, with Se accounting for 44.81% of the total content of the three elements.

[0034] (III) Antioxidant Activity Analysis of Polysaccharide Nano-Selenium Fertilizer from Shiitake Mushroom Residue (1) DPPH free radical scavenging experiment Using a DPPH free radical scavenging kit, 150 μL of different concentrations of *Viola yedoensis* mycelium residue polysaccharide nano-selenium fertilizer solution (0.2 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L, 1.6 g / L) and 150 μg of working solution were added to the test tubes. 150 μL of different concentrations of *Viola yedoensis* mycelium residue polysaccharide nano-selenium fertilizer solution (0.2 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L, 1.6 g / L) and 150 μL of 80wt.% methanol were added to the control tubes. 150 μL of 80% methanol and 150 μL of working solution were added to the blank tubes. The blank tubes were tested only once. Incubate at room temperature (25℃) in the dark for 30 min, centrifuge at 12000 rpm for 5 min at room temperature, transfer 200 μL to a 96-well plate, and measure the absorbance A at 517 nm. The absorbance A measured in the tube is recorded as A0. 测定 The absorbance value A read from the control tube is denoted as A. 对照 .

[0035] ; .

[0036] (2) ABTS· + Free radical scavenging experiment Using ABTS· + To test the free radical scavenging ability kit, add 10 μL of different concentrations of *Viola yedoensis* mycelium residue polysaccharide nano-selenium fertilizer solution (0.2 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L, 1.6 g / L) and 190 μL of working solution to the test tubes of a 96-well plate. Add 10 μL of the same solution and 190 μL of anhydrous ethanol to the control tubes. Add 10 μL of anhydrous ethanol and 190 μL of working solution to the blank tubes. Each blank tube should be tested only once. After mixing, incubate at room temperature (25 ℃) in the dark for 6 min, and read the absorbance value A at 734 nm. The absorbance value A read in the test tubes is recorded as A0. 测定 The absorbance value A read from the control tube is denoted as A. 对照 .

[0037] ; .

[0038] Following the same operating procedure, the free radical scavenging activities of sodium selenite and shiitake mushroom residue polysaccharides were determined, and the test results are as follows: Figure 8As shown, sodium selenite and mushroom residue polysaccharide have relatively weak antioxidant activity; while mushroom residue polysaccharide nano-selenium fertilizer has significant antioxidant activity, which is dose-dependent.

[0039] Comparative Example To investigate the effects of key steps in the preparation process and carrier selection on the agricultural application of nano-selenium, the following comparative experiments were conducted: Comparative Example 1 This comparative example prepared a pure nano-selenium (without shiitake mushroom residue polysaccharide coating). The preparation process was similar to that of Example 1, except that the extraction and coating steps of shiitake mushroom residue polysaccharide were not performed. Instead, pure nano-selenium was prepared directly by incubating 30 mg / mL sodium selenite solution and 60 mg / mL ascorbic acid solution (volume ratio 1:3) in a water bath at 40°C for 3 h. The resulting product did not contain any polysaccharides or polymeric stabilizers.

[0040] Comparative Example 2 This comparative example prepares a chitosan-coated selenium nanoparticle. The preparation process is similar to that of Example 1, except that the polysaccharide extracted from the mushroom residue in Example 1 is replaced with commercially available pure chitosan. All other preparation steps are the same as in Example 1.

[0041] The test results of Comparative Examples 1 and 2 show that the shiitake mushroom residue polysaccharide nano-selenium fertilizer prepared in this invention has significantly better effects than pure nano-selenium and chitosan nano-selenium in promoting crop growth, reducing neonicotinoid pesticide residues, and increasing crop selenium content. Specifically, under the same application conditions, crop biomass increased by approximately 42.5%-78.9% compared to Comparative Example 1 (e.g., ...). Figure 9 As shown), this represents an increase of approximately 32.4%-59.6% compared to Comparative Example 2 (e.g. Figure 9 As shown); the residue levels of neonicotinoid pesticides in crops were reduced by approximately 18.7%-30.0% compared to control example 1 (e.g. Figure 10 As shown), it is approximately 11.8%-18.3% lower than Comparative Example 2 (e.g. Figure 10 As shown); the selenium content in the crop was approximately 2.19-3.12 times higher than that in Comparative Example 1 (e.g. Figure 11 As shown), it is approximately 1.27-1.34 times higher than Comparative Example 2 (e.g. Figure 11 (As shown).

[0042] Application example: The application of shiitake mushroom residue polysaccharide nano-selenium fertilizer in selenium enrichment and quality improvement of crops and reduction of pesticide residues. (I) Analysis of the effects of mushroom residue polysaccharide nano-selenium fertilizer on reducing neonicotinoid pesticide residues in crops and increasing yield and quality through selenium enrichment. This application example uses a pot experiment. The test soil was collected from saline-alkali soil in Ningxia. After collection, dead branches, fallen leaves, roots, and other plant debris, as well as soil animals, were removed. The soil was air-dried and passed through a 5 mm sieve for later use. 50 mL of imidacloprid solution (160 mg / L) was added to 800 g of the above saline-alkali soil to prepare neonicotinoid pesticide-contaminated saline-alkali soil with an imidacloprid concentration of 10 mg / kg. 800 g of neonicotinoid pesticide-contaminated saline-alkali soil was weighed and added to flowerpots, and then three lettuce plants of similar growth were transplanted into each flowerpot. The following treatment groups were set up for this experiment: CK, Na2SeO3, nano-selenium (SeNP), chitosan nano-selenium (CS-SeNP), and the mushroom residue polysaccharide nano-selenium fertilizer (PS-SeNP) prepared in Example 1. Two application concentrations of 1 mg / L and 10 mg / L were set for each selenium preparation. Pure water or different selenium preparations were sprayed into the corresponding flowerpots every two days, at an application rate of 10 g per acre. The flowerpots were placed in a climate incubator with a day / night temperature of 25℃ / 20℃ and a relative humidity of 80%, and were cultured under a light intensity of 5000 Lx for 16 h of light and 8 h of darkness. After 14 days of culture, plant samples were collected, and the fresh biomass of lettuce seedlings in each treatment group was recorded. Figure 9 ), and determine the pesticide content in plants ( Figure 10 ) and selenium content ( Figure 11 ).

[0043] like Figure 9 As shown, after 14 days of cultivation, the biomass of lettuce treated with 1 mg / L or 10 mg / L PS-SeNP was higher than that of other treatment groups, indicating that the application of PS-SeNP significantly promoted the growth of lettuce in neonicotinoid pesticide-contaminated saline-alkali land.

[0044] like Figure 10 As shown, the application of 1 mg / L PS-SeNP or 10 mg / L PS-SeNP can reduce the accumulation of IMI in plants, indicating that the application of PS-SeNP has a slowing effect on the absorption and accumulation of IMI.

[0045] like Figure 11 As shown, application of 1 mg / L PS-SeNP or 10 mg / L PS-SeNP can increase the accumulation of Se in plants, indicating that PS-SeNP can serve as a stable and bioavailable source of selenium, thereby improving the selenium nutrition level of crops.

[0046] (II) Analysis of the effect of mushroom residue polysaccharide nano-selenium fertilizer on alleviating crop salt damage in agricultural production Before exposure, 800 g of Ningxia saline-alkali soil was added to white flowerpots. For some pots, 50 mL of exposure culture medium containing only IMI or a mixture of PS-SeNP and IMI was added for root application. The remaining treatment groups without exposure culture medium were treated with 50 mL of ultrapure water, or sprayed with PS-SeNP every 2-3 days (foliar spraying ensured each leaf was moist). Plant samples were collected 14 days after exposure, freeze-dried, digested, and the mineral element content was analyzed using inductively coupled plasma atomic absorption spectrometry and fluorescence spectrophotometry. The results are as follows: Figure 12 As shown.

[0047] like Figure 12 As shown in Figure A, under root application conditions, treatment with 0.5 mg PS-SeNP resulted in increased K levels in the aerial parts. + The content reached the highest value among all treatment groups, confirming that low-dose root application can effectively promote K. + Transport to the aboveground parts; while the combined treatment of 5 mg PS-SeNP and IMI significantly increased K in the underground parts. + The content indicates that high doses of PS-SeNP can enhance the root system's resistance to K. + Absorption capacity.

[0048] like Figure 12 As shown in B, IMI single treatment leads to root Ca 2+ Significant accumulation indicates that it hinders Ca 2+ Transport to the aboveground parts. PS-SeNP treatment exhibited dose-specific regulation: under root application conditions, 5 mg PS-SeNP + IMI combined treatment increased root Ca2+ transport. 2+ The content further increased, but the aboveground level did not increase simultaneously, indicating that under high salt or high dose conditions, Ca... 2+ The transport of xylem remains restricted, indicating that PS-SeNP primarily promotes root Ca2+ transport. 2+ Enrichment can alleviate calcium imbalance caused by IMI.

[0049] like Figure 12 As shown in C, in foliar spraying treatments, the IMI treatment had an effect on Mg. 2+ The content has little impact; Mg content varies between the aboveground and underground parts. 2+ The level showed no significant change, indicating its effect on Mg. 2+ The absorption and distribution interference were limited; neither the PS-SeNP nor the composite treatment group caused significant fluctuations.

[0050] like Figure 12 As shown in D, the effect of IMI alone on Na in the aboveground and underground parts... + The content was not significantly affected; under the treatment of foliar spraying and root application of PS-SeNP, the Na content in the underground parts was... +The content of Na in the underground parts all increased, while under the combined treatment of PS-SeNP and IMI, the content of Na in the underground parts increased. + The content decreased significantly and tended to the levels of the control group and the IMI group. These results indicate that PS-SeNP can effectively alleviate the toxicity of harmful ions and optimize the dynamics of essential elements by regulating the absorption and distribution of mineral elements in plants under salt stress, thus providing important support for plants to maintain nutritional homeostasis and enhance their adaptability to saline-alkali adversity.

[0051] In addition, to further investigate the effects of different application routes of mushroom residue polysaccharide nano-selenium fertilizer on its pesticide application efficacy, the following comparative experiment was conducted, as follows: The polysaccharide nano-selenium fertilizer from *Viola yedoensis* mushroom residue was applied to crops via rhizosphere application and foliar spraying, respectively. All other application concentrations, application frequency, and crop cultivation conditions remained consistent with application example (I). The effects of different application methods on crop growth, neonicotinoid pesticide residues, and crop selenium content were compared, and the results are shown in Table 1. Table 1. Effect analysis of different application routes As can be seen from the table above, compared with root application of 1 mg / L PS-SeNPs, foliar application of 1 mg / L PS-SeNPs can significantly increase crop biomass, significantly reduce the concentration of imidacloprid residue in leaves, and significantly increase the selenium (Se) content in leaves.

[0052] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A nano-selenium fertilizer made from mushroom residue polysaccharides, characterized in that, The mushroom residue polysaccharide nano-selenium fertilizer comprises mushroom residue polysaccharide and nano-selenium, with a mass ratio of mushroom residue polysaccharide to nano-selenium of 1.7:1, and the mushroom residue polysaccharide is coated on the surface of the nano-selenium; the particle size of the mushroom residue polysaccharide nano-selenium fertilizer is 70 nm.

2. The method for preparing a polysaccharide nano-selenium fertilizer from mushroom residue according to claim 1, characterized in that, Follow these steps in sequence: S1. Dry the mushroom residue to constant weight, crush it with a pulverizer and pass it through a 40-mesh sieve. Collect the powder that passes through the sieve, add 25 times the amount of ultrapure water, sonicate for 30 minutes, and then place it in a water bath for extraction. After vacuum filtration, obtain the mushroom residue polysaccharide extract. S2. Concentrate the polysaccharide extract to one-third of its original volume using a rotary evaporator, add trypsin solution, shake vigorously using a vortex shaker for 10 min, let stand at room temperature for 4 h, incubate in a water bath at 100 ℃ for 20 min, place it in a centrifuge, and centrifuge at 12000 rpm for 10 min to obtain the polysaccharide solution. S3. Add 3 times the volume of 95 wt.% ethanol solution to the polysaccharide solution, let it stand at room temperature in the dark for 12 h, centrifuge at 12000 rpm for 10 min to collect the precipitate, wash the precipitate with anhydrous ethanol and acetone in sequence, and dry the washed precipitate in a vacuum drying oven to obtain the polysaccharide from the mushroom residue of shiitake mushroom. S4. Prepare a 20 mg / mL solution of mushroom residue polysaccharide from mushroom residue. Add a 30 mg / mL sodium selenite solution to the solution and mix thoroughly by vigorous shaking with a vortex shaker for 10 min. Then add a 60 mg / mL ascorbic acid solution to obtain a mixture. S5. Place the mixture in a water bath and heat it at 40 ℃ for 3 h. Transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it with ultrapure water for 48 h. Then freeze-dry it at -54~-50℃ for 48~72 h to obtain the mushroom residue polysaccharide nano-selenium fertilizer.

3. The method for preparing a polysaccharide nano-selenium fertilizer from mushroom residue according to claim 2, characterized in that, In step S1, the extraction temperature is 75~85℃ and the time is 2.5~3.5 h.

4. The method for preparing a polysaccharide nano-selenium fertilizer from mushroom residue according to claim 2, characterized in that, In step S2, the concentration of the trypsin solution is 500 u·mL. -1 The volume of trypsin solution added was 1 / 10 of that of the polysaccharide concentrate.

5. The method for preparing a polysaccharide nano-selenium fertilizer from mushroom residue according to claim 2, characterized in that, In step S3, the drying temperature is 55~60 ℃ and the time is 24~48 h.

6. The method for preparing a polysaccharide nano-selenium fertilizer from mushroom residue according to claim 2, characterized in that, In step S4, the volume of sodium selenite solution added is 133% of that of the mushroom residue polysaccharide solution; the volume ratio of ascorbic acid solution to sodium selenite solution is 3:

1.

7. An application of a polysaccharide nano-selenium fertilizer from mushroom residue, characterized in that, The shiitake mushroom residue polysaccharide nano-selenium fertilizer prepared by the preparation method according to any one of claims 2 to 6 can be applied to crop cultivation.

8. The application of the mushroom residue polysaccharide nano-selenium fertilizer according to claim 7, characterized in that, The soil in which the crops are grown is saline-alkali land and contaminated with neonicotinoid pesticides, with a concentration of 10 mg / kg in the soil; the application concentration of the mushroom residue polysaccharide nano-selenium fertilizer is 1 mg / L or 10 mg / L.

9. The application of the mushroom residue polysaccharide nano-selenium fertilizer according to claim 7, characterized in that, The crop is one or more of lettuce, bok choy, or carrots.

10. The application of the mushroom residue polysaccharide nano-selenium fertilizer according to claim 7, characterized in that, The application method for mushroom residue polysaccharide nano-selenium fertilizer is foliar spraying. Apply 10 g of mushroom residue polysaccharide nano-selenium fertilizer per acre, and spray it evenly on the leaves of crops multiple times during the crop growth period.

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