Water-soluble nano-selenium nitraria fruit polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to a water-soluble nano-selenium nitraria fruit polysaccharide as well as a preparation method and application thereof, and is technically characterized in that the water-soluble nano-selenium nitraria fruit polysaccharide is prepared from arabinose, galactose, mannose, xylose, fructose, glucose and galacturonic acid, the weight-average molecular weight of the nitraria fruit polysaccharide is 9932 Da, the number-average molecular weight of the nitraria fruit polysaccharide is 5885 Da, and the peak molecular weight of the nitraria fruit polysaccharide is 8897 Da. The selenium-rich foliar fertilizer can be used as a nano-selenium foliar fertilizer, is good in absorption performance, high in bioavailability and good in organic selenium conversion effect, solves the problems that crops are poor in selenium absorption rate and low in conversion rate, not only can effectively promote the absorption rate of the crops on exogenous selenium and the conversion rate of the organic selenium, but also can promote fruit growth, enhance oxidation resistance and improve crop quality, and has a wide application prospect. The method has a wide application prospect in selenium-enriched functional agricultural production.

Description

Technical Field

[0001] This invention relates to the field of foliar fertilizer technology, specifically to a water-soluble nano-selenium white thorn fruit polysaccharide, its preparation method and application, which is suitable for the cultivation of selenium-enriched functional strawberries and other berry crops. Background Technology

[0002] Selenium is an essential trace element for humans and animals. It participates in the formation of the body's antioxidant enzyme system, maintains the function of the immune system, and helps prevent cardiovascular diseases and tumors. Selenium deficiency can lead to diseases such as Kashin-Beck disease and Keshan disease. Humans and animals cannot synthesize selenium themselves and must obtain it from food. The selenium in food mainly comes from the soil. Sodium selenite and other selenium-containing pharmaceutical preparations are often used as selenium fortification agents, but because the threshold between selenium deficiency and excess is very narrow, organisms are prone to selenium deficiency or selenium poisoning. Plants are key carriers in the natural selenium ecological cycle and a direct source of selenium for the human body. Increasing the selenium content of agricultural products through exogenous selenium supplementation, thereby increasing the selenium intake of the target population, is a safe and effective way to supplement selenium. Therefore, improving the selenium content of agricultural products is crucial.

[0003] To date, the preparation of nano-selenium includes three methods: physical preparation, chemical reduction, and biosynthesis. Physical synthesis of nano-selenium requires the addition of catalysts and is carried out under high temperature and pressure conditions using large-scale equipment; the resulting elemental selenium nanoparticles tend to aggregate and turn brownish-black. Chemical reduction uses reagents such as sodium thiosulfate to reduce sodium selenite to elemental selenium; this method is energy-intensive and produces irritating compounds, and the synthesized nano-selenium nanoparticles tend to polymerize into grayish-brown elemental selenium. Biosynthesis of nano-selenium involves reducing selenite or selenate to elemental or organic selenium nanoparticles during the metabolic activities of plants or microorganisms. Among these methods, certain bacteria and fungi can synthesize nano-selenium extracellularly using sodium selenite, providing a new pathway for the green synthesis of nano-selenium and overcoming its commercialization bottlenecks.

[0004] Elemental selenium, with a molecular size in the nanometer range, exhibits poor stability in aqueous solutions, readily agglomerating into large, grayish-black, micron-sized particles, thus reducing its biological activity. Some microorganisms can synthesize selenium nanoparticles extracellularly. If biomolecules are present outside the microbial cells, their functional groups bind to the selenium nanoparticles via selenium-oxygen bonds, effectively maintaining their stability and enhancing their biological activity and bioavailability. Therefore, biomolecules are often used as dispersants in the preparation of functionalized selenium nanoparticles. Polysaccharides, composed of monosaccharide molecules linked by glycosidic bonds, contain branched structures and numerous functional groups (-OH, -CHO), which can modify and coat the surface of selenium nanoparticles. When bacteria or fungi ferment in a matrix containing sodium selenite and polysaccharides and synthesize elemental selenium extracellularly, the elemental selenium interacts with the branched chain polysaccharide molecules, achieving good dispersibility and thus being nano-sized. Simultaneously, its biological activity, bioavailability, stability, and sustained-release properties are all enhanced. Therefore, polysaccharides provide a new strategy for the design of functionalized selenium nanoparticles.

[0005] Crops can increase the content of intracellular organic selenium by applying exogenous selenium fertilizers, thereby enhancing their functional nutrition and increasing their economic value. Existing research shows that applying inorganic selenium, such as sodium selenite and selenium powder, can enrich crops with selenium. However, different crop varieties exhibit selective absorption of exogenous selenium fertilizers, and while large-scale use of inorganic selenium fertilizers can increase the amount of selenium absorbed by plants, it may inhibit plant growth and cause secondary pollution due to unabsorbed exogenous selenium entering the soil. Therefore, identifying suitable selenium sources and exploring ways to improve the conversion rate of organic selenium in crops are key development priorities for the selenium-enriched crop industry. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical defects of current exogenous selenium foliar fertilizers and provide a water-soluble nano-selenium white thorn fruit polysaccharide, its preparation method, and its application. The water-soluble nano-selenium white thorn fruit polysaccharide of this invention can be used as a nano-selenium foliar fertilizer. It has good absorption performance, high bioavailability, and excellent organic selenium conversion effect, solving the problem of poor absorption rate and low conversion rate of selenium element by crops. It can not only effectively promote the absorption rate of exogenous selenium element and the conversion rate of organic selenium by crops, but also promote fruit growth, enhance antioxidant properties, and improve crop quality. It has broad application prospects in selenium-enriched functional agricultural production.

[0007] The technical solution of this invention is:

[0008] A water-soluble nano-selenium white thorn fruit polysaccharide, the key technical features of which are: composed of arabinose, galactose, mannose, xylose, fructose, glucose and galacturonic acid, with a weight-average molecular weight of 9932 Da, a number-average molecular weight of 5885 Da, and a peak molecular weight of 8897 Da.

[0009] The above-mentioned water-soluble nano-selenium white thorn fruit polysaccharide has a nano-selenium particle size range of 200-300nm and a selenium content of 18.3-22.1%.

[0010] The water-soluble nano-selenium white thorn fruit polysaccharide mentioned above contains arabinose, galactose, mannose, xylose, fructose, glucose, and galacturonic acid in the following mass percentages: 10.74%, 4.76%, 20.86%, 2.56%, 13.42%, 20.36%, and 27.3%, respectively.

[0011] A method for preparing water-soluble nano-selenium white thorn fruit polysaccharide as described above, the key technical points of which include the following steps:

[0012] Step 1: Take the fruit of the white thorn and dry it, then grind it into white thorn powder;

[0013] Step 2: Using white thorn fruit powder as raw material, crude white thorn fruit polysaccharide is obtained by alkaline extraction, and then the protein in the crude polysaccharide is removed by protease-assisted Sevage method to obtain white thorn fruit polysaccharide.

[0014] Step 3: Synthesis of nano-selenium-rich white thorn fruit polysaccharide using filamentous fungi: Filamentous fungi were inoculated into PDA plate culture medium and activated by inverted culture at 28℃~30℃ for 3~4 days; after obtaining mycelial cakes from the PDA plate culture medium using a mycelial cake punch, the mycelial cakes were transferred to PDB culture medium for further culture at a rotation speed of 120~150 rpm and a culture temperature of 28℃~30℃ for 3-4 days; then the mycelia were filtered using sterile filter paper, and the filtrate was collected; the collected filtrate was mixed with sodium selenite solution and the white thorn fruit polysaccharide obtained in step 2, and reacted at 28℃~30℃ for 24h~30h; the reaction solution was collected, extracted by water-soluble alcohol precipitation, and freeze-dried to obtain the target product.

[0015] In the above-mentioned method for preparing water-soluble nano-selenium white thorn fruit polysaccharide, in step 2, the alkaline solution used in the alkaline extraction method is a 10% NaOH solution, and the mass ratio of white thorn fruit powder to the volume of 10% NaOH solution is 5.0g:50mL.

[0016] In the above-mentioned method for preparing water-soluble nano-selenium white thorn fruit polysaccharide, in step 2, the protease used in the protease-assisted Sevage method is papain. When removing protein, the freeze-dried powder of white thorn fruit crude polysaccharide needs to be completely dissolved in distilled water to form a crude polysaccharide solution. The mass ratio of the freeze-dried powder of white thorn fruit crude polysaccharide to the volume ratio of distilled water is 1g:4mL. Then, 1.5-2% papain is added to the crude polysaccharide solution to carry out the protein hydrolysis enzymatic reaction. After the reaction is completed, Sevage reagent is added to the reaction solution. The volume ratio of the reaction solution to Sevage reagent is 25:13. The protein removal is repeated 2-3 times.

[0017] In the above-mentioned method for preparing water-soluble nano-selenium white thorn fruit polysaccharide, the filamentous fungus used in step 3 is Trichoderma longibrachiatum TS269548.

[0018] In the above-mentioned method for preparing water-soluble nano-selenium white thorn fruit polysaccharide, in step 3, the mass-volume ratio of sodium selenite, white thorn fruit polysaccharide, and fermentation mycelium filtrate is 0.602 g: 5 g: 100 mL.

[0019] In the above-mentioned method for preparing water-soluble nano-selenium white thorn fruit polysaccharide, in step 3, the water-soluble alcohol precipitation method uses 95% ethanol, and the volume ratio of the reaction solution to 95% ethanol is 1:4.

[0020] The key technical point of the application of the water-soluble nano-selenium white thorn fruit polysaccharide as described above is: as a foliar fertilizer for selenium-enriched functional strawberries.

[0021] The beneficial effects of this invention are:

[0022] The *Nitraria tangutorum* used in this invention is a wild shrub belonging to the genus *Nitraria* in the family Zygophyllaceae. Its fruit has multiple medicinal and edible uses and is known as the "desert cherry," containing various vitamins, amino acids, and other physiologically active substances. my country has abundant *Nitraria tangutorum* fruit resources, with Xinjiang being one of the main producing areas. *Nitraria tangutorum* is endemic to China and possesses strong population dominance. Its fruit is used to treat spleen and stomach weakness, indigestion, and other ailments, and also has calming, digestive, and spleen-tonifying effects. *Trichoderma* is a widely used biocontrol fungus, primarily involved in inhibiting pathogens, promoting plant growth, degrading pollutants, and enhancing crop stress resistance. Its mechanisms of action are diverse, making it valuable in agriculture and environmental protection. The combination of these two factors plays a crucial role in the preparation of the water-soluble nano-selenium *Nitraria tangutorum* fruit polysaccharide described in this invention.

[0023] 1. To increase the organic selenium content in strawberry fruits, this invention extracts water-soluble white thorn fruit polysaccharide as a soft template. Using a filamentous fungus (Trichoderma) with biocontrol functions, the mycelial filtrate is obtained through fermentation. Sodium selenite is then reduced to nano-sized selenium, which is used as the main component in a foliar fertilizer. This invention not only avoids the instability inherent in chemically obtained nano-selenium but also enhances the water solubility of nano-selenium and its affinity for selenium transporters within plants, thereby promoting the absorption rate of exogenous selenium by crops.

[0024] 2. Compared with commonly used inorganic selenium-enriched foliar fertilizers, foliar fertilizers containing nano-selenium polysaccharides have low toxicity and high bioavailability. Moreover, the polysaccharide molecules contain a large number of functional groups with high affinity for selenium transport proteins, which can promote the transport of selenium in plants and significantly increase the content of organic selenium in crop fruits.

[0025] 3. The foliar fertilizer prepared by this invention can effectively improve the absorption and conversion of exogenous selenium by berry crops. Attached Figure Description

[0026] Figure 1 This is a spectrum showing the molecular weight of nano-selenium-rich white thorn polysaccharide determined by GPC method;

[0027] Figure 2 This is a GC-MS analysis of the monosaccharide composition of nano-selenium white thorn fruit polysaccharide;

[0028] Figure 3 This is an EDS analysis spectrum of the selenium content in nano-selenium white thorn fruit polysaccharide;

[0029] Figure 4 This is a scanning electron microscope image of the selenium polysaccharide from *Rhizoma Cirsium japonicum* obtained by this invention (in the image, the particle size of elemental selenium on the polysaccharide surface is between 200-300 nanometers). Detailed Implementation

[0030] To make the content of this invention easier to understand, the technical solution of this invention will be described in detail below with reference to specific embodiments.

[0031] Example 1.

[0032] 1. Raw material processing: The purchased Tangut white thorn fruits are initially washed with distilled water, the surface moisture is absorbed with gauze, and then placed in a 60℃ oven for 20 minutes. After that, they are crushed with a pulverizer and passed through a 20-mesh sieve. The sieved white thorn fruit powder is collected.

[0033] 2. Extraction of crude polysaccharides from white thorn fruit:

[0034] A certain amount of white thorn fruit powder was weighed and mixed with 10% sodium hydroxide solution at a mass-volume ratio of 1:10. The mixture was placed in a 70℃ water bath for 1.5 hours, then centrifuged at 4500 rpm for 5 minutes. The precipitate was discarded, and the supernatant was placed in a rotary evaporator and concentrated to 1 / 5 of its original volume at 50℃ and 30 rpm. The concentrated solution was mixed with anhydrous ethanol at a ratio of 1:3 and allowed to stand at 4℃ for 12 hours. The mixture was then centrifuged at 3500 rpm for 5 minutes, the supernatant was discarded, and the precipitate was freeze-dried to obtain crude white thorn fruit polysaccharide.

[0035] 3. Dissolve the crude polysaccharide freeze-dried powder in distilled water to a concentration of 25%. Add 2% papain and incubate for 2 hours. Mix the papain-treated polysaccharide solution, n-butanol, and chloroform in a ratio of 25:1:5 and stir magnetically for 20 minutes. Then centrifuge the mixture at 4000 rpm for 10 minutes to remove the middle protein layer and the bottom organic solvent layer. Repeat twice. Put the protein-removed polysaccharide solution into a dialysis bag with a molecular weight cutoff of 3500 and dialyze with distilled water for 48 hours. Freeze-dry the dialysate to obtain white thorn fruit polysaccharide.

[0036] 4. Inoculate *Trichoderma longifolia* TS269548 into PDA agar plates and incubate at 28℃ for 4 days to activate the mycelium (the incubation time can be shortened or extended appropriately depending on the growth of the mycelium). After forming mycelial cakes from the PDA agar plates using a mycelial cake punch, transfer them to PDB agar plates and incubate at 120 rpm and 28℃ for 3-4 days. Then filter the mycelium using sterile Whatman filter paper No. 1 and collect the filtrate. Mix the collected filtrate with sodium selenite solution and *Nitraria tangutorum* polysaccharide at a ratio of 100 mL, 0.602 g, and 25 g, and react at 50℃ for 12 hours. Collect the reaction solution, add 4 volumes of 95% ethanol, let it stand at 4℃ for 12 hours, centrifuge at 3500 rpm for 5 minutes, discard the supernatant, and freeze-dry the precipitate to obtain nano-selenium *Nitraria tangutorum* polysaccharide.

[0037] The obtained nano-selenium white thorn fruit polysaccharide was tested and analyzed:

[0038] 1. The obtained nano-selenium-rich white thorn fruit polysaccharide was analyzed for molecular weight and composition:

[0039] Among them, molecular weight was determined using GPC gel permeation chromatography, and the molecular weight determination results are as follows: Figure 1 As shown, the weight-average molecular weight of nano-selenium-rich white thorn polysaccharide is 9932 Da, the number-average molecular weight is 5885 Da, and the peak molecular weight is 8897 Da.

[0040] The monosaccharide composition of selenium polysaccharides was determined using gas chromatography-mass spectrometry (GC-MS). Figure 2 As shown in Table 6, the proportions of arabinose, galactose, mannose, xylose, fructose, glucose, and galacturonic acid are 10.74%, 4.76%, 20.86%, 2.56%, 13.42%, 20.36%, and 27.3%, respectively.

[0041] Scanning electron microscopy revealed that the selenium nanoparticles attached to the polysaccharide molecules were ellipsoidal, such as... Figure 4 As shown, its particle size ranges from 200 to 300 nm.

[0042] The selenium content in selenium polysaccharides was determined by EDS, such as... Figure 3As shown, the selenium content is 22.1%.

[0043] 2. Performance test: The selenium enrichment effect of white thorn fruit nano-selenium polysaccharide as a foliar fertilizer.

[0044] Experimental treatments: A randomized block design was adopted, with three treatments: 30 mg / L (T1), 50 mg / L (T2), and 70 mg / L (T3). Three different concentrations of *Rhizoma Citrus reticulatae* nano-selenium polysaccharide solution were applied, with water as a blank control (CK). The experiment was repeated four times, resulting in a total of 16 plots, with a protection row between each plot. Fertilization was performed by spraying the entire plant three times during flowering, fruit setting, and fruit enlargement stages. The treatment was carried out on one acre of land in a greenhouse. After fruit maturity, 300-400 fruits were randomly collected from each plot.

[0045] Quality Inspection: The weight of individual fruits was measured. Vitamin C content was determined using the 2,6-dichlorophenolindophenol titration method (GB6195-86), soluble sugar content using the 3,5-dinitrosalicylic acid colorimetric method, titratable acid content using the indicator titration method, selenium content using hydride atomic fluorescence spectrometry (GB 5009.93-2010), and soluble protein content using the Coomassie Brilliant Blue method. Individual fruit weight was measured using an electronic balance. Fruit color index (I) was measured using a CHNSpec Colorimeter CS-10 portable colorimeter. Fruit firmness was measured using a GY-1 handheld hardness tester. Fruit transverse and longitudinal diameters were measured using electronic vernier calipers, and the fruit shape index was calculated. Peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) were detected using corresponding kits (produced by Nanjing Jiancheng Biotechnology Co., Ltd.). The test results are shown in Tables 1-5.

[0046] Table 1. Different concentrations of Se 0 NP s Effects of solution on strawberry fruit weight and appearance indicators

[0047]

[0048] Table 2. Different concentrations of Se 0 NP s Effect of solution on the nutrient content of strawberry fruit

[0049]

[0050] Table 3. Different concentrations of Se 0 NP s The effect of solution on the taste of strawberry fruit

[0051]

[0052] Table 4. Different concentrations of Se 0 NP s Effects of selenium content in strawberry fruit

[0053]

[0054] Table 5. Effects of different concentrations of nano-selenium-enriched *Rhizoma Cirsium japonicum* polysaccharide on the antioxidant properties of strawberry fruit.

[0055]

[0056] As shown in Tables 1-5 above, compared with the blank control group, the total selenium content of strawberries increased and the organic selenium conversion rate improved after using the white thorn fruit nano-selenium polysaccharide prepared by this invention as a foliar fertilizer. At the same time, it improved the fruit weight, appearance indicators and fruit quality of strawberries, including increasing the average single fruit weight, color index, fruit firmness, fruit shape index, and the content of soluble protein, total phenols, vitamin C, soluble sugar and titratable acid in the fruit.

[0057] Table 6 Monosaccharide components of selenium polysaccharide from Rhizoma Cilantrosae (white thorn fruit)

[0058]

[0059] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A water-soluble nano-selenium white thorn fruit polysaccharide, characterized in that: It is composed of arabinose, galactose, mannose, xylose, fructose, glucose and galacturonic acid, with a weight-average molecular weight of 9932 Da, a number-average molecular weight of 5885 Da, and a peak molecular weight of 8897 Da.

2. The water-soluble nano-selenium white thorn fruit polysaccharide according to claim 1, characterized in that: The nano-selenium particles range in size from 200 to 300 nm, with a selenium content of 18.3% to 22.1%.

3. The water-soluble nano-selenium white thorn fruit polysaccharide according to claim 1, characterized in that: The mass percentages of arabinose, galactose, mannose, xylose, fructose, glucose, and galacturonic acid were 10.74%, 4.76%, 20.86%, 2.56%, 13.42%, 20.36%, and 27.3%, respectively.

4. A method for preparing water-soluble nano-selenium white thorn fruit polysaccharide as described in claim 1, characterized in that, Includes the following steps: Step 1: Take the fruit of the white thorn and dry it, then grind it into white thorn powder; Step 2: Using white thorn fruit powder as raw material, crude white thorn fruit polysaccharide is obtained by alkaline extraction, and then the protein in the crude polysaccharide is removed by protease-assisted Sevage method to obtain white thorn fruit polysaccharide. Step 3: Synthesis of nano-selenium-rich white thorn fruit polysaccharide using filamentous fungi: Filamentous fungi were inoculated into PDA plate culture medium and activated by inverted culture at 28℃~30℃ for 3~4 days; after obtaining mycelial cakes from the PDA plate culture medium using a mycelial cake punch, the mycelial cakes were transferred to PDB culture medium for further culture at a rotation speed of 120~150 rpm and a culture temperature of 28℃~30℃ for 3-4 days; then the mycelia were filtered using sterile filter paper, and the filtrate was collected; the collected filtrate was mixed with sodium selenite solution and the white thorn fruit polysaccharide obtained in step 2, and reacted at 28℃~30℃ for 24h~30h; the reaction solution was collected, extracted by water-soluble alcohol precipitation, and freeze-dried to obtain the target product.

5. The method for preparing water-soluble nano-selenium white thorn fruit polysaccharide according to claim 4, characterized in that: In step 2, the alkaline solution used in the alkaline extraction method is a 10% NaOH solution, and the mass ratio of white thorn fruit powder to the volume of 10% NaOH solution is 5.0g:50mL.

6. The method for preparing water-soluble nano-selenium white thorn fruit polysaccharide according to claim 4, characterized in that: In step 2, the protease used in the protease-assisted Sevage method is papain. When removing protein, the freeze-dried powder of crude polysaccharide from white thorn fruit must first be completely dissolved in distilled water to form a crude polysaccharide solution. The mass ratio of the freeze-dried powder of crude polysaccharide from white thorn fruit to the volume ratio of distilled water is 1g:4mL. Then, 1.5-2% papain is added to the crude polysaccharide solution to carry out the enzymatic reaction of protein hydrolysis. After the reaction is completed, Sevage reagent is added to the reaction solution. The volume ratio of the reaction solution to the Sevage reagent is 25:

13. The protein removal is repeated 2-3 times.

7. The method for preparing water-soluble nano-selenium white thorn fruit polysaccharide according to claim 4, characterized in that: In step 3, the filamentous fungus used is Trichoderma longibrachia tum TS269548.

8. The method for preparing water-soluble nano-selenium white thorn fruit polysaccharide according to claim 4, characterized in that: In step 3, the mass-to-volume ratio of sodium selenite, white thorn fruit polysaccharide, and fermentation mycelium filtrate is 0.602 g: 5 g: 100 mL.

9. The method for preparing water-soluble nano-selenium white thorn fruit polysaccharide according to claim 4, characterized in that: In step 3, the water-soluble alcohol precipitation method uses 95% ethanol, and the volume ratio of the reaction solution to 95% ethanol is 1:

4.

10. An application of the water-soluble nano-selenium white thorn fruit polysaccharide as described in claim 1, characterized in that: As a foliar fertilizer for selenium-enriched functional strawberries.

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