Mannan oligosaccharide fertilizer synergist with biological synergistic function and high-utilization-rate fertilizer

Abstract

The invention provides a mannan oligosaccharide fertilizer synergist with a biological synergistic function and a high-utilization-rate fertilizer. The mannan oligosaccharide fertilizer synergist comprises phosphorylated modified mannan oligosaccharide, mannan oligosaccharide, mannan oligosaccharide, mannan oligosaccharide, mannan oligosaccharide, mannan oligosaccharide, mannan oligosaccharide, mannan oligosaccharide and mannan oligosaccharide, the phosphorylated modified mannan oligosaccharide is obtained by carrying out phosphorylation reaction on mannan oligosaccharide through a phosphorylation reagent. According to the mannan oligosaccharide fertilizer synergist with the biological synergistic function and the high-utilization-rate fertilizer, mannan oligosaccharide is subjected to phosphorylation reaction to obtain phosphorylated modified mannan oligosaccharide which serves as the fertilizer synergist, compared with mannan oligosaccharide or other saccharides, the affinity of the phosphorylated modified mannan oligosaccharide with biomacromolecules such as films, enzymes and proteins is greatly improved, and the utilization rate of the fertilizer synergist is increased; while biological synergism is realized, absorption of nitrogen, phosphorus and potassium can be promoted, and the utilization rate of the fertilizer is increased.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to a mannan oligosaccharide fertilizer synergist with bio-enhancing function and a fertilizer with high utilization rate. Background Technology

[0002] Fertilizer is a crucial agricultural input, playing a vital role in ensuring bumper harvests and promoting agricultural development. my country is a major fertilizer consumer, but problems exist regarding excessive and indiscriminate fertilizer application. Excessive fertilizer application leads to increased accumulation of inorganic elements in the soil, soil compaction, reduced water and fertilizer retention capacity, decreased fertilizer utilization, and significant fertilizer loss into the soil, causing a series of problems such as environmental pollution, water quality deterioration, and agricultural product contamination. To address these issues, it is necessary to build an innovation system in the fertilizer field through fertilizer product innovation, technological innovation, organic substitution, and precision fertilization. Currently, the application of oligosaccharide synergists in fertilizers is one of the important directions for such innovation.

[0003] Oligosaccharide synergists are a class of carbohydrates with biological regulatory functions. Multiple studies have shown that oligosaccharides play an important role in improving plant resistance to abiotic stress and promoting plant growth. For example, Sun Lei (“Effects of Chitosan Treatment on Photosynthetic Characteristics and Thylakoid Membrane Fatty Acid Components of Hybrid Rice Leaves at Low Temperatures”, Nanjing: Nanjing Normal University, 2007.) found that after chitosan treatment, the chlorophyll content of rice seedlings increased, enhancing the cold resistance of rice and alleviating the damage caused by low temperatures. Xue Gaini et al. (“Study on the Application Effect of Amino Oligosaccharides on Wheat”, Modern Agricultural Science and Technology, 2012(18): 97-98.) studied the application effect of amino oligosaccharides on wheat. The results showed that seed dressing with amino oligosaccharides could improve the emergence rate and plant height of wheat seedlings, and could also increase the stress resistance of wheat. When applied at different times, the yield increased to a certain extent.

[0004] Mannooligosaccharides (MOS) are formed by the synthesis of mannose units through... β- Manno-oligosaccharides, formed by 1,4-glycosidic bonds, are a type of functional oligosaccharide. They are primarily derived from the hemicellulose components of plant cell walls. As a bioactive substance, manno-oligosaccharides possess various biological effects, including enhancing immune function, promoting growth and development, and improving gut health, thus attracting widespread attention and application in organic agriculture. In agricultural production, the functions of manno-oligosaccharides as a natural bioactive substance in increasing crop yield, enhancing crop disease resistance, and improving soil environment are gradually being developed and utilized.

[0005] To date, there are few reports on adding mannan oligosaccharides as a bioactive substance to fertilizers, therefore, research on their synergistic effects with fertilizers and other agricultural inputs remains to be deepened. How to achieve synergistic effects while ensuring the efficacy of each ingredient is an important direction for future research. Summary of the Invention

[0006] Based on the technical problems existing in the background art, the present invention proposes a mannan oligosaccharide fertilizer synergist with bio-enhancing function and a high-utilization fertilizer. By phosphorylating mannan oligosaccharide to obtain phosphorylated modified mannan oligosaccharide as a fertilizer synergist, compared with mannan oligosaccharide or other sugars, its affinity with biomacromolecules such as membranes, enzymes, and proteins is greatly improved. While achieving bio-enhancing, it can also promote the absorption of nitrogen, phosphorus, and potassium and improve fertilizer utilization.

[0007] The present invention proposes a mannan oligosaccharide fertilizer synergist with bio-enhancing function, comprising phosphorylated modified mannan oligosaccharides; The phosphorylated modified mannooligosaccharide is obtained by phosphorylating mannooligosaccharide with a phosphorylating agent.

[0008] In this invention, phosphorylated mannooligosaccharides are modified by introducing phosphate esters (-O-PO3H2) on the surface of mannooligosaccharides to improve hydrophilicity and solubility. More importantly, it enhances the affinity of mannooligosaccharides with biomolecules such as membranes, enzymes, and proteins, thereby improving the crop's ability to absorb and utilize mannooligosaccharides, ensuring the promotion of nitrogen, phosphorus, and potassium absorption, and improving fertilizer utilization.

[0009] Preferably, the phosphorylation agent comprises phosphorus pentoxide and pyridine; The weight ratio of mannan oligosaccharide to phosphorus pentoxide is 1:0.5-3.

[0010] In this invention, phosphorus pentoxide is used as a phosphorylation reagent and pyridine is used as an organic base. It can neutralize the acid generated in the reaction in a timely manner, push the reaction equilibrium in the positive direction, and greatly improve the efficiency and degree of phosphorylation.

[0011] Preferably, the phosphorylation reaction is carried out at a temperature of 10-30 °C for a time of 12-36 h.

[0012] Preferably, the mannan oligosaccharide is obtained by enzymatic hydrolysis of glucomannan with mixed enzyme reagents followed by centrifugation to obtain a mannan oligosaccharide mixture; the mannan oligosaccharide mixture is then purified by column and membrane filtration to obtain the mannan oligosaccharide. The mixed enzyme reagent includes β - Mannanase, cellulase, and xylanase.

[0013] This invention also proposes a high-utilization fertilizer, which includes the above-mentioned fertilizer synergist.

[0014] Preferably, the fertilizer comprises, by weight percentage: 1-6% fertilizer synergist, 0.5-2% microbial fermentation powder, and 92-97% mineral nutrients.

[0015] Preferably, the microbial fermentation powder comprises the fermentation product of *Pseudomonas fluorescens*.

[0016] In this invention, when the microbial fermentation powder relies on the function of Pseudomonas fluorescens and its bio-fermentation products, Pseudomonas fluorescens is a type of phosphate-solubilizing bacteria that can convert insoluble phosphorus and potassium in the soil into soluble nutrients, thereby improving fertilizer utilization.

[0017] Preferably, the fertilizer further includes: 1-5% modified attapulgite; The modified attapulgite is attapulgite with surface grafted cyclodextrin side-group polyacrylamide compounds. The attapulgite with surface-grafted cyclodextrin side-group polyacrylamide compounds is obtained by polymerizing acrylamide in the presence of a chain transfer agent of bis(carboxymethyl)trithiocarbonate and an initiator of azobisisobutyronitrile, followed by esterification condensation with attapulgite modified with cyclodextrin and silane coupling agent.

[0018] In this invention, acrylamide is polymerized under the chain transfer agent of bis(carboxymethyl)trithiocarbonate and the initiator of azobisisobutyronitrile to obtain polyacrylamide with two carboxyl groups at the end. Then, it is esterified and condensed with cyclodextrin and attapulgite in sequence to make polyacrylamide compounds with cyclodextrin side groups grafted on the surface of attapulgite. The specific structure is shown below.

[0019] On the one hand, cyclodextrin's excellent inclusion and adsorption capabilities can effectively adsorb and fix free metal ions in the soil, promoting crop absorption of micronutrients. It can also protect soil microbial agents, improving their tolerance and enhancing crop nutrient absorption. On the other hand, the porous structure and ion adsorption / displacement capacity of attapulgite soil can further improve fertilizer utilization. Furthermore, the amino groups in polyacrylamide can form ammonium salts with phosphorylated mannan oligosaccharides. This cross-linked composite structure not only exhibits excellent water absorption and retention but also adsorbs negative ions such as nitrate in the soil, further fixing the nutrients required by plants in the soil to improve fertilizer utilization and promote plant growth.

[0020] Preferably, the mineral nutrients include nitrogen, phosphorus, and potassium sources; The nitrogen source is at least one of urea, ammonium bicarbonate, ammonium sulfate, or ammonium chloride; the phosphorus source is at least one of monoammonium phosphate, diammonium phosphate, triammonium phosphate, monoammonium phosphate, or calcium phosphate; and the potassium source is at least one of potassium chloride, potassium sulfate, or potassium dihydrogen phosphate.

[0021] Preferably, the method for preparing the fertilizer includes: adding fertilizer synergist and mineral nutrients into a granulator for granulation, adding 5-8% water by weight as a binder, controlling the particle size to be 2-4 mm, drying the resulting masterbatch with hot air, cooling it to room temperature with cold air, adding microbial fermentation powder and continuing to stir and mix, and then sieving to obtain the fertilizer.

[0022] Compared with the prior art, the present invention has the following beneficial effects: In this invention, mannan oligosaccharides possess a variety of biological activities. Phosphorylation modification can further enhance their affinity for biomolecules such as membranes, enzymes, and proteins. The final experimental results show that the bio-enhancing function of phosphorylated mannan oligosaccharides is significantly improved, demonstrating the ability of phosphorylated mannan oligosaccharides to significantly improve fertilizer utilization in the fertilizer field. Attached Figure Description

[0023] Figure 1 The infrared spectrum of the modified attapulgite clay described in Example 3 is shown. Detailed Implementation

[0024] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0025] Example 1

[0026] A bio-enhancing mannan oligosaccharide fertilizer synergist, which is essentially a phosphorylated modified mannan oligosaccharide, is prepared by the following method: Glucomannan and ethanol were mixed at a ratio of glucomannan:ethanol = 1:10 (w / v), stirred at 50 °C for 1 h, centrifuged (4000 rpm), repeated 3 times, and then dried at 60 °C for later use. An enzyme solution was prepared by mixing β-mannanase, cellulase and xylanase with an activity of approximately 500 U / mg at a ratio of β-mannanase:cellulase:xylanase = 5:2:1 (w / w / w), dissolving in phosphate buffer at pH 6.0, and allowing to stand at 4°C for 30 min before use. The glucomannan substrate was pre-dissolved in pure water at a concentration of 10% (w / v), and then added at a mixed enzyme solution volume of 50 U / g. At 50-55 ℃ and pH 5.5-6.0, 5% substrate solution was added every 2 h, and the reaction was carried out for 10 h. After the reaction was completed, the mixture was boiled in a water bath for 10 min, cooled in an ice bath, and centrifuged (10000 rpm, 15 min). The supernatant was then collected for further separation and purification. Add 1% activated carbon (w / v) to the supernatant, stir at 70 °C for 30 min, filter, pass through a D301 resin column (flow rate 2 BV / h), and wash with water until neutral; use Millipore Pellicon membrane for ultrafiltration, with a molecular weight cutoff of 1-5 kDa, operate at 4 bar pressure, and collect the permeate. The mixture was chromatographically analyzed using a Bio-Gel P-2 column (2.5 × 100 cm) with ultrapure water elution (flow rate 0.5 mL / min), and fractions were collected. The product was then concentrated under reduced pressure at 40 °C using a rotary evaporator until the solid content was ≥20%. Finally, the concentrated product was spray-dried (inlet air temperature 160 °C, outlet air temperature 80 °C) to obtain a white mannan oligosaccharide powder (moisture content ≤5%). Under stirring, a mixed solution of phosphorus pentoxide (P2O5) and pyridine (P2O5 concentration 0.075 g / mL) was slowly added dropwise to a container containing white mannooligosaccharide powder. The addition was stopped when 30 times the amount of mannooligosaccharide powder was added. After stirring the reaction at room temperature for 24 h, the mixture was purified by chromatography using a Bio-Gel P-2 column (2.5 × 100 cm). After drying, the phosphorylated modified mannooligosaccharide was obtained.

[0027] Example 2

[0028] A high-utilization fertilizer comprises, by weight percentage: 4% phosphorylated modified mannan oligosaccharide, 21% urea, 26% monoammonium phosphate, 17% diammonium phosphate, 31% potassium sulfate, and 1% microbial fermentation powder; The phosphorylated modified mannan oligosaccharide was prepared according to the method described in Example 1; the microbial fermentation powder was prepared by the following method: Fluorescent Pseudomonas was inoculated into slant culture medium (10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, 15.0 g / L agar, pH 7.0-7.2) and cultured at 25 ℃ for 48 h until the colony diameter reached 1-2 mm. Then, single colonies were picked and inoculated into liquid seed culture medium (liquid volume ≤30%, 3% beef extract, 0.5% peptone, 0.5% sodium chloride, distilled water to make up the difference, pH 7.0-7.2) and cultured with shaking at 200 rpm and 25 ℃ for 24 h to obtain the seed culture. The seed culture was transferred to a fermenter at 10% (v / v) and fermentation broth (maltose 32.5 g / L, yeast extract 4.5 g / L, ammonium sulfate 4.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.06 g / L, NaCl 0.15 g / L) was added. The fermentation was carried out at 200 rpm and 25℃ for 48 h with shaking to obtain the fermentation broth. Centrifuge the fermentation broth at 6000 rpm for 10 min at 4 ℃, discard the supernatant, collect the wet cells, resuspend the cells in sterile physiological saline, and adjust the concentration to ≥5.0×10⁻⁶. 9 The concentration of CFU / g was then rapidly frozen at -80 °C for 2 h, and finally vacuum dried at -50 °C and 0.1 mbar for 24 h to obtain the microbial fermentation powder.

[0029] The preparation methods of the above fertilizers include: Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, and phosphorylated mannan oligosaccharide are added to a drum mixer and stirred at low speed for 10 minutes until uniform. Water of 6% of the total fertilizer weight is sprayed in, and the mixture is granulated in a rotary drum granulator for 20 minutes, controlling the particle size to 2-4 mm. The mixture is first pre-dried at 80 ℃ hot air for 30 minutes, then cooled to 60 ℃ and dried for another 20 minutes. After that, it is cooled to room temperature by cold air, microbial fermentation powder is added, and qualified particles of 2-4 mm are obtained by vibrating sieve.

[0030] Example 3

[0031] A high-utilization fertilizer comprises, by weight percentage: 4% phosphorylated modified mannan oligosaccharide, 17% urea, 26% monoammonium phosphate, 17% diammonium phosphate, 31% potassium sulfate, 1% microbial fermentation powder, and 4% modified attapulgite soil; The phosphorylated modified mannan oligosaccharide was prepared according to the method described in Example 1; the microbial fermentation powder was prepared according to the method described in Example 2; the modified attapulgite clay was prepared by the following method: Acrylamide, bis(carboxymethyl)trithiocarbonate, and azobisisobutyronitrile were added to ethanol in a ratio of acrylamide:bis(carboxymethyl)trithiocarbonate:azobisisobutyronitrile = 1:3:0.04 (w / w / w). The mixture was heated to 70°C under nitrogen protection and stirred for 20 h. The precipitate was washed in methanol, filtered, and dried to obtain polyacrylamide with two carboxyl groups at the end. Dicarboxyl-terminated polyacrylamide was added to an aqueous solution containing β-cyclodextrin and acidified attapulgite, with the ratio of dicarboxyl-terminated polyacrylamide:β-cyclodextrin:acidified attapulgite = 1:0.1:0.5 (w / w / w). Then EDC and NHS were added, with the ratio of dicarboxyl-terminated polyacrylamide:EDC:NHS = 1:0.02:0.015 (w / w / w). The mixture was heated to 50 °C and stirred for 6 h. After filtration, washing with water, and drying, the modified attapulgite was obtained. The acidified attapulgite is prepared by adding attapulgite to a sulfuric acid solution (50wt%), letting it stand for 8 hours, washing and drying it, then adding it to a sodium hydroxide solution (15wt%), adjusting the pH to 11, heating it to 50 ℃ and stirring the reaction for 2 hours to obtain the acidified attapulgite.

[0032] The infrared spectrum of the modified attapulgite is as follows: Figure 1 As shown, refer to Figure 1 It can be known that 3415cm -1 The peak of the non-stretching vibration of NH is at 2940 cm⁻¹. -1 and 2871cm -1 The peak at 1623 cm⁻¹ represents the stretching vibration of CH₄ on β-cyclodextrin. -1 The peak of the stretching vibration at C=O is 1428 cm⁻¹. -1 The peak at 1059 cm⁻¹ represents the stretching vibration of CH₂. -1 The point represents the stretching vibration peak of C=S.

[0033] The preparation methods of the above fertilizers include: Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, phosphorylated modified mannan oligosaccharide, and modified attapulgite soil are added to a drum mixer and stirred at low speed for 10 minutes until uniform. Water of 6% of the total weight of fertilizer is sprayed in, and the mixture is granulated in a rotary drum granulator for 20 minutes, controlling the particle size to 2-4 mm. The mixture is first pre-dried at 80 ℃ hot air for 30 minutes, then cooled to 60 ℃ and dried for another 20 minutes. After that, it is cooled to room temperature by cold air, microbial fermentation powder is added, and qualified particles of 2-4 mm are obtained by vibrating sieve.

[0034] Comparative Example 1 A high-utilization fertilizer comprises, by weight percentage: 4% mannan oligosaccharide, 21% urea, 26% monoammonium phosphate, 17% diammonium phosphate, 31% potassium sulfate, and 1% microbial fermentation powder; The mannan oligosaccharide was prepared according to the method described in Example 1; the microbial fermentation powder was prepared according to the method described in Example 2.

[0035] The preparation methods of the above fertilizers include: Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, and mannan oligosaccharide are added to a drum mixer and stirred at low speed for 10 minutes until uniform. Water of 6% of the total weight of fertilizer is sprayed in, and the mixture is rolled in a rotary drum granulator for 20 minutes to granulate, controlling the particle size to 2-4 mm. The mixture is first pre-dried at 80 ℃ hot air for 30 minutes, then cooled to 60 ℃ and dried for another 20 minutes. After that, it is cooled to room temperature by cold air, microbial fermentation powder is added, and qualified particles of 2-4 mm are obtained by vibrating sieve.

[0036] Comparative Example 2 A high-utilization fertilizer comprises, by weight percentage: 4% phosphorylated modified mannan oligosaccharide, 17% urea, 26% monoammonium phosphate, 17% diammonium phosphate, 31% potassium sulfate, 1% microbial fermentation powder, 3.3% acidified attapulgite clay, and 0.7% β-cyclodextrin; The phosphorylated modified mannan oligosaccharide was prepared according to the method described in Example 1; the microbial fermentation powder was prepared according to the method described in Example 2; and the acidified attapulgite clay was prepared according to the method described in Example 3.

[0037] The preparation methods of the above fertilizers include: Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, phosphorylated modified mannan oligosaccharide, acidified attapulgite clay, and β-cyclodextrin are added to a drum mixer and stirred at low speed for 10 minutes until homogeneous. Water at 6% of the total weight of the fertilizer is sprayed in, and the mixture is granulated in a rotary drum granulator for 20 minutes, controlling the particle size to 2-4 mm. The mixture is then pre-dried at 80 ℃ hot air for 30 minutes, then cooled to 60 ℃ and dried for another 20 minutes. After that, it is cooled to room temperature by cold air, microbial fermentation powder is added, and the mixture is vibrated and screened to obtain qualified particles of 2-4 mm, thus obtaining the fertilizer.

[0038] Comparative Example 3 A high-utilization fertilizer comprises, by weight percentage: 4% phosphorylated modified mannan oligosaccharide, 17% urea, 26% monoammonium phosphate, 17% diammonium phosphate, 31% potassium sulfate, 1% microbial fermentation powder, and 4% modified attapulgite clay; The phosphorylated modified mannan oligosaccharide was prepared according to the method described in Example 1; the microbial fermentation powder was prepared according to the method described in Example 2; and the modified attapulgite was prepared according to the method described in Example 3, except that acrylonitrile of the same mass was used instead of acrylamide.

[0039] The preparation methods of the above fertilizers include: Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, mannan oligosaccharide, and modified attapulgite soil are added to a drum mixer and stirred at low speed for 10 minutes until uniform. Water of 6% of the total fertilizer weight is sprayed in, and the mixture is granulated in a rotary drum granulator for 20 minutes, controlling the particle size to 2-4 mm. The mixture is first pre-dried at 80 ℃ hot air for 30 minutes, then cooled to 60 ℃ and dried for another 20 minutes. After that, it is cooled to room temperature by cold air, microbial fermentation powder is added, and the mixture is vibrated and screened to obtain qualified particles of 2-4 mm, thus obtaining the fertilizer.

[0040] Test Example 1 Experimental objective: To determine the phosphate content of phosphorylated mannan oligosaccharides in Example 1 using the molybdenum blue colorimetric method, to determine the degree of phosphorylation by measuring the amount of phosphate groups introduced before and after the reaction, and to determine the changes in reducing sugars using the 3,5-dinitrosalicylic acid method (DNS method) to assess the possible degradation of sugar chains during the reaction.

[0041] Materials and Equipment: Table 1. Reagents used in Test Example 1

[0042] Spectrophotometer: wavelength range 190-1100 nm, equipped with 1 cm glass cuvette (for molybdenum blue method at 690 nm and DNS method at 540 nm); Analytical balance: sensitivity 0.0001 g; Constant temperature water bath: temperature range room temperature to 100℃, accuracy ±0.5℃; pH meter: accuracy 0.01 pH unit; Centrifuge: speed adjustable from 0-12000 rpm; Ultrasonic cleaner: power 600W, frequency 40 kHz; Micropipette: range 10-1000 μL; Volumetric flasks: various sizes (10, 25, 50, 100, 250 mL); Colorimetric tubes: 25 mL stoppered graduated colorimetric tubes.

[0043] Experimental methods: Sample pretreatment: Ammonium molybdate-sulfuric acid solution: Slowly add 100 mL of concentrated sulfuric acid to 900 mL of distilled water, stirring and cooling continuously. Add 10 g of sodium molybdate, dissolve and mix well, then store in a brown glass bottle. Ascorbic acid solution (10%): Weigh 10.0 g of ascorbic acid and dissolve it in distilled water, then bring the volume to 100 mL. DNS reagent: Accurately weigh 6.3 g of 3,5-dinitrosalicylic acid, add 262 mL of 2 mol / L sodium hydroxide solution, stir until basically dissolved, then add to 500 mL of hot aqueous solution containing 182 g of potassium sodium tartrate, then add 5 g of phenol and 5 g of sodium sulfite, stir to dissolve, cool and make up to 1000 mL, store in a brown bottle protected from light for one week before use; Molybdenum blue method sample pretreatment: Accurately weigh 0.1000 g of the phosphorylated modified mannan oligosaccharide described in Example 1, dissolve it in a small amount of deionized water, quantitatively transfer it to a 100 mL volumetric flask, dilute to the mark, and shake well to obtain a stock solution with a concentration of 1 mg / mL; according to the preliminary experimental results, take 5.00 mL of this stock solution and place it in a 100 mL volumetric flask, dilute to the mark with deionized water to obtain a 20-fold diluted sample solution; DNS method sample pretreatment: Accurately weigh 0.1000 g each of humulin oligosaccharide and phosphorylated modified humulin oligosaccharide from Example 1, and prepare a 1 mg / mL stock solution according to the above method; based on the preliminary experiment, take 5.00 mL of each of the two stock solutions and place them in 100 mL volumetric flasks, and dilute to the mark with deionized water to obtain a 20-fold diluted sample solution.

[0044] Determination of phosphoric acid content by molybdenum blue colorimetric method: Accurately weigh 0.7165 g of potassium dihydrogen phosphate dried to constant weight at 105 °C, dissolve it in deionized water, and bring the volume to 500 mL to obtain a solution containing 1.00 mg per mL. The standard stock solution was prepared; 10.00 mL of this stock solution was transferred to a 500 mL volumetric flask and diluted to the mark to obtain a solution of 20.0 μg / mL. Prepare the working standard solution; take 7 25 mL stoppered colorimetric tubes and add the standard solution and reagents according to Table 2 below: Table 2 Phosphorus Standard Solutions

[0045] After adding reagents to each tube in sequence, mix immediately and bring the volume to 25.0 mL with deionized water. Shake well. Place all colorimetric tubes in a 40 ℃ constant temperature water bath for 15 min for color development, and then cool to room temperature. Using a 1 cm path length cuvette, zero the instrument at 690 nm wavelength with a blank tube (No. 1) and measure the absorbance of each tube. Perform three parallel measurements for each concentration, take the average value, and plot a standard curve. Take 2.00 mL of the molybdenum blue pretreatment sample solution (equivalent to 0.1 mg of phosphorylated mannan oligosaccharide) and place it in a 25 mL colorimetric tube. Add reagents, develop color, and measure absorbance according to the same operating steps as the standard curve described above. At the same time, set up a sample blank (using water instead of the sample) and a reagent blank (using water instead of all reagents) for calibration.

[0046] DNS method for determining reducing sugar content: Plotting the glucose standard curve: Accurately weigh 1.000 g of anhydrous glucose (dried to constant weight at 105 ℃), dissolve in deionized water and dilute to 1000 mL to obtain a 1 mg / mL glucose standard stock solution; take six 25 mL graduated test tubes and add the standard solution and reagents according to Table 3 below: Table 3 Glucose Standard Solutions

[0047] After adding DNS reagent to each tube, mix thoroughly and heat precisely in a boiling water bath for 5 min. Immediately cool to room temperature with running water. Adjust the volume to 15.0 mL with deionized water and mix well. Using a 1 cm path length cuvette, zero the instrument at 540 nm wavelength with a blank tube (No. 1) and measure the absorbance of each tube. Perform three parallel measurements for each concentration, take the average value, and plot a standard curve. Sample determination: Take 1.00 mL of each DNS pretreatment sample (equivalent to approximately 0.05 mg of mannan oligosaccharide or phosphorylated modified mannan oligosaccharide respectively) and place them in 25 mL graduated test tubes. Add DNS reagent, boil, cool, make up to volume and measure absorbance according to the same operating steps as the standard curve above; at the same time, set up a sample blank for calibration.

[0048] Data recording and calculation: Calculation of phosphate content: Based on the absorbance value of the sample, the phosphate concentration in the test solution is calculated from the regression equation of the phosphorus standard curve, and then the phosphate content of the sample is calculated using the following formula:

[0049] Where: C is obtained from the standard curve. Concentration (μg / mL); V is the final volume of the sample (mL); D is the dilution factor; m is the sample mass (g). is the conversion factor between μg and g.

[0050] Calculation of reducing sugar content: Based on the absorbance value of the sample, the concentration of reducing sugar in the test solution is calculated from the regression equation of the glucose standard curve. Then, the reducing sugar content of the sample is calculated using the following formula:

[0051] Where: C is the glucose concentration (mg / mL) obtained from the standard curve; V is the final volume of the sample (mL); D is the dilution factor; m is the sample mass (g); 10³ is the conversion factor between mg and g.

[0052] Phosphorylation level assessment: Phosphate group introduction amount: directly calculated from phosphate content (1% phosphate content is equivalent to 0.105 mmol phosphate groups / g oligosaccharide). Sugar chain retention rate:

[0053] Degree of phosphorylation:

[0054] Results and Data Analysis Phosphorus standard curve data: The absorbance values ​​of the phosphorus standard solutions were determined using the aforementioned method, and the results are shown in Table 4 below: Table 4 Absorbance data of phosphorus standard series solutions

[0055] by With the content (μg) as the abscissa (X) and the average absorbance value as the ordinate (Y), a linear regression analysis was performed, and the regression equation was obtained as: Y = 0.0408X-0.0024, with a correlation coefficient R² = 0.9998.

[0056] The results show that phosphorus content and absorbance value have a good linear relationship in the range of 0-40 μg, which conforms to Beer's Law; the linear range, sensitivity and precision all meet the analytical requirements.

[0057] Glucose standard curve data: The absorbance values ​​of the glucose standard series solutions were determined using the aforementioned method, and the results are shown in Table 5 below: Table 5 Absorbance data of glucose standard series solutions

[0058] Linear regression analysis was performed with glucose content (mg) on ​​the x-axis (X) and average absorbance value on the y-axis (Y). The regression equation was: Y = 1.074X + 0.001, with a correlation coefficient R² = 0.9996. This result indicates a good linear relationship between reducing sugar content and absorbance value within the range of 0-1.0 mg, conforming to Beer's Law. The linear range, sensitivity, and precision all meet the analytical requirements.

[0059] Actual sample measurement results: The phosphoric acid content of phosphorylated mannooligosaccharide samples and mannooligosaccharide raw materials was determined, and the results are shown in Table 6 below: Table 6. Results of phosphoric acid content determination of phosphorylated mannooligosaccharides

[0060] The results in Table 6 above show that the phosphate content in phosphorylated manno oligosaccharides reached 0.37%, while the raw manno oligosaccharides contained only 0.02% phosphate (possibly from the raw material itself or the background measurement). After deducting the background value, the actual introduced phosphate content was 0.35%, which is equivalent to introducing 0.39 mmol of phosphate groups per gram of manno oligosaccharides.

[0061] The reducing sugar content of phosphorylated mannooligosaccharide samples and mannooligosaccharide raw materials was determined, and the results are shown in Table 7 below: Table 7. Results of reducing sugar content determination of phosphorylated mannooligosaccharides

[0062] The results in Table 7 show that the reducing sugar content of the unmodified mannooligosaccharide raw material is 15.1%, while the reducing sugar content of the phosphorylated mannooligosaccharide is 13.5%, and the sugar chain retention rate is 89.4%. This indicates that 10.6% of the sugar chains were degraded or structurally changed during the phosphorylation modification process, resulting in a reduction of the reducing end.

[0063] The test results above show that phosphorylation successfully introduced phosphate groups into mannooligosaccharide molecules, and the amount introduced reached a high level. However, some sugar chain degradation inevitably occurred during the modification process, but the degree of degradation was controlled within an acceptable range (about 11%). Phosphorylation-modified mannooligosaccharides have significant advantages as fertilizer synergists. On the one hand, the introduced phosphate groups can enhance the water solubility and stability of oligosaccharides. On the other hand, the degree of sugar chain degradation during the modification of phosphorylation oligosaccharides is limited, and most of the biological activities are preserved.

[0064] Test Example 2 Experimental objective: To verify that phosphorylated mannooligosaccharides have a significantly enhanced affinity for biomolecules (cell membranes, enzymes, proteins) compared to mannooligosaccharides. This enhanced affinity is expected to promote the absorption and utilization of oligosaccharides by plants, thereby improving fertilizer efficiency.

[0065] Materials and Equipment: Table 8 Reagents used in Test Example 2

[0066] Test method: Membrane binding experiment: Liposome binding rate determination; Liposomes were used to simulate plant cell membranes, and membrane affinity was assessed by measuring the binding rate of oligosaccharides to liposomes; Liposome preparation: 100 mg of lecithin was dissolved in 10 mL of chloroform, and the solution was rotary evaporated to form a thin film. 10 mL of PBS (pH 7.4) was added for hydration, and the solution was sonicated to prepare a liposome suspension. Sample preparation: Prepare 1 mg / mL mannooligosaccharide and phosphorylated mannooligosaccharide solutions, and mix 2 mL of each with 2 mL of liposome suspension; Incubation conditions: 37 ℃ constant temperature water bath with shaking incubation for 1 h; Separation and detection: The reaction solution was centrifuged at 4 ℃ and 12000 rpm for 30 min, and the supernatant was collected; Content determination: The content of unbound oligosaccharides in the supernatant was determined by the DNS method; 10 g of 3,5-dinitrosalicylic acid was weighed, 200 mL of 2M NaOH and 500 mL of 30% sodium tartrate solution were added, and the volume was adjusted to 1000 mL; 0.5 mL of supernatant was added to 0.5 mL of DNS reagent, and the mixture was boiled in a water bath for 5 minutes. After cooling, the absorbance was measured at 540 nm.

[0067] Construction of the mannose standard curve: Table 9 Absorbance data of mannose standard series solutions

[0068] The standard curve equation is: Y = 1.432X + 0.012 (R² = 0.9987), where Y is the absorbance value and X is the mannose concentration (mg / mL).

[0069] Data recording and results analysis: Table 10. Membrane binding experimental data (liposome binding rate)

[0070] Membrane binding experiment: Phosphorylation modification significantly improved the affinity of mannooligosaccharides for membrane structures.

[0071] As shown in Table 10, the liposome binding rate of phosphorylated mannooligosaccharide (62.0%) was about 2.2 times higher than that of ordinary mannooligosaccharide (28.0%). This indicates that phosphorylation modification enhances the binding ability of oligosaccharide to biological membranes, possibly due to the enhanced electrostatic interaction between phosphate groups and membrane phospholipid heads.

[0072] Test Example 3 Experimental objective: To verify the promoting effect of the fertilizer of this invention on crop growth compared with ordinary fertilizers through a rigorous field trial design, with particular attention to the impact on crop protein absorption and accumulation.

[0073] Experimental materials: Ordinary fertilizer ( Example fertilizer: 15-15-15; Foliar fertilizer control: 0.2% potassium dihydrogen phosphate + 0.5% glucose; Crop: Rice (Oryza sativa L, variety: Daohuaxiang No. 2) Experimental Design: Four treatments were set up, each with three replicates, using a randomized block design. The cell area was 20 m² (4 m × 5 m). CK: Common base fertilizer (36g / m²) + 0.2% potassium dihydrogen phosphate control foliar fertilizer spray; Experimental fertilizer group 1: Base fertilizer of Example 2 (36g / m²); Experimental fertilizer group 2: Base fertilizer of Example 2 (36g / m²) + 0.2% foliar spray of fertilizer of Example 2 (sprayed once each during the tillering stage and the heading stage); Experimental fertilizer group 3: ordinary compound fertilizer base fertilizer (18g / m²) + fertilizer for spikes from Example 2 (18g / m²); Experimental fertilizer group 4: ordinary compound fertilizer base fertilizer (18g / m²) + fertilizer for spikes from Example 3 (18g / m²); Experimental fertilizer group 5: ordinary compound fertilizer base fertilizer (18g / m²) + comparative example 1 fertilizer spike fertilizer (18g / m²). Experimental fertilizer group 6: ordinary compound fertilizer base fertilizer (18g / m²) + comparative example 2 fertilizer top dressing (18g / m²). Experimental fertilizer group 7: ordinary compound fertilizer base fertilizer (18g / m²) + comparative example 3 fertilizer spike fertilizer (18g / m²). Fertilization method: Base fertilizer: Spread evenly one day before transplanting and rake it into the soil (0-10cm soil layer); Ear fertilizer: Apply during the young ear differentiation stage (45 days after transplanting); Foliar spraying: Spray evenly on sunny days after 4 pm during the tillering stage (25 days after transplanting) and the booting stage (55 days after transplanting), until both sides of the leaves are moist.

[0074] Table 11 Observation Indicators and Methods

[0075] Results and Analysis: Table 12 Effects on crop growth and yield

[0076] The fertilizer of this invention significantly increased rice yield compared to conventional fertilizer treatments. Among them, treatments 3 and 4 (optimized fertilization) showed the most significant yield increases, reaching 14.0% and 18.2%, respectively. This indicates that the fertilizer of this invention may increase yield by enhancing root vitality, improving photosynthetic efficiency, and improving nitrogen use efficiency.

[0077] Table 13 Effects on protein absorption and accumulation

[0078] The fertilizer of this invention significantly increased grain protein content compared to conventional fertilizer treatments, with treatments 3 and 4 showing the most significant effects. This fertilizer may promote protein synthesis and accumulation through two mechanisms: enhancing the activity of key nitrogen metabolism enzymes (such as nitrate reductase and glutamine synthase); and improving amino acid composition, including an increased proportion of essential amino acids and a higher percentage of gluten.

[0079] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A mannan oligosaccharide fertilizer synergist with bio-enhancing function, characterized in that, Including phosphorylated modified mannan oligosaccharides; The phosphorylated modified mannan oligosaccharide is obtained by phosphorylating mannan oligosaccharide with phosphorylation of phosphorus pentoxide.

2. The mannan oligosaccharide fertilizer synergist with bio-enhancing function according to claim 1, characterized in that, The phosphorylation reaction also includes the use of pyridine as a neutralizing agent; The weight ratio of mannan oligosaccharide to phosphorus pentoxide is 1:0.5-3.

3. The mannan oligosaccharide fertilizer synergist with bio-enhancing function according to claim 1 or 2, characterized in that, The phosphorylation reaction is carried out at a temperature of 10-30 °C for a time of 12-36 h.

4. The mannan oligosaccharide fertilizer synergist with bio-enhancing function according to claim 1 or 2, characterized in that, The mannan oligosaccharide is obtained by enzymatic hydrolysis of glucomannan with mixed enzyme reagents, followed by centrifugation to obtain a mannan oligosaccharide mixture; the mannan oligosaccharide mixture is then purified by column and membrane filtration to obtain the mannan oligosaccharide. The mixed enzyme reagent includes β - Mannanase, cellulase, and xylanase.

5. A high-utilization fertilizer, characterized in that, The fertilizer includes the fertilizer enhancer according to any one of claims 1-4.

6. The high-utilization fertilizer according to claim 5, characterized in that, The fertilizer comprises, by weight percentage: 1-6% fertilizer enhancer, 0.5-2% microbial fermentation powder, and 92-97% mineral nutrients.

7. The high-utilization fertilizer according to claim 6, characterized in that, The microbial fermentation powder includes the fermentation products of Pseudomonas fluorescens.

8. The high-utilization fertilizer according to claim 7, characterized in that, The fertilizer also includes: 1-5% modified attapulgite; The modified attapulgite is attapulgite with surface grafted cyclodextrin side-group polyacrylamide compounds. The attapulgite with surface-grafted cyclodextrin side-group polyacrylamide compounds is obtained by polymerizing acrylamide in the presence of a chain transfer agent of dicarboxymethyl trithiocarbonate and an initiator of azobisisobutyronitrile, followed by esterification condensation with cyclodextrin and attapulgite in sequence.

9. The high-utilization fertilizer according to any one of claims 6-8, characterized in that, The mineral nutrients include nitrogen, phosphorus and potassium sources; The nitrogen source is at least one of urea, ammonium bicarbonate, ammonium sulfate, or ammonium chloride; the phosphorus source is at least one of monoammonium phosphate, diammonium phosphate, triammonium phosphate, or calcium phosphate; and the potassium source is at least one of potassium chloride, potassium sulfate, or potassium dihydrogen phosphate.

10. The high-utilization fertilizer according to any one of claims 6-8, characterized in that, The preparation method of the fertilizer includes: adding fertilizer synergist and mineral nutrients into a granulator for granulation, adding 5-8% water by weight as a binder, controlling the particle size to 2-4 mm, drying the resulting masterbatch with hot air, cooling it to room temperature with cold air, adding microbial fermentation powder and continuing to stir and mix, and then sieving to obtain the fertilizer.

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