A manno-oligosaccharide fertilizer synergist with bio-enhancing function and a fertilizer

Abstract

The present application provides a mannose oligosaccharide fertilizer synergist with biological synergistic function and a high utilization rate fertilizer, the mannose oligosaccharide fertilizer synergist comprises: phosphorylated modified mannose oligosaccharide; the phosphorylated modified mannose oligosaccharide is obtained by phosphorylating mannose oligosaccharide with a phosphorylating agent.The present application provides a mannose oligosaccharide fertilizer synergist with biological synergistic function and a high utilization rate fertilizer, by obtaining phosphorylated modified mannose oligosaccharide through phosphorylation reaction of mannose oligosaccharide as a fertilizer synergist, compared with mannose oligosaccharide or other saccharides, the affinity with biological macromolecules such as membrane, enzyme and protein is greatly improved, biological synergistic effect is realized, and the absorption of nitrogen, phosphorus and potassium is promoted, and the fertilizer utilization rate is improved.

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] Up to now, there are few reports on adding manno-oligosaccharide as a bioactive substance to fertilizers, and thus the synergistic effect of manno-oligosaccharide and agricultural inputs such as fertilizers still needs to be further studied. How to realize synergistic effect under the premise of ensuring the respective effects is an important direction for future research. SUMMARY

[0006] Based on the technical problems in the background art, the present application provides a manno-oligosaccharide fertilizer synergist with biological synergistic function and a high utilization rate fertilizer, which uses phosphorylated modified manno-oligosaccharide obtained by phosphorylation reaction of manno-oligosaccharide as a fertilizer synergist. Compared with manno-oligosaccharide or other sugars, the phosphorylated modified manno-oligosaccharide has greatly improved affinity with biological macromolecules such as membranes, enzymes and proteins, realizes biological synergistic effect, and promotes the absorption of nitrogen, phosphorus and potassium, and improves the utilization rate of fertilizer.

[0007] The manno-oligosaccharide fertilizer synergist with biological synergistic function provided by the present application comprises phosphorylated modified manno-oligosaccharide.

[0008] The phosphorylated modified manno-oligosaccharide is obtained by phosphorylation reaction of manno-oligosaccharide with a phosphorylation reagent.

[0009] In the present application, the phosphorylated modified manno-oligosaccharide is obtained by introducing phosphate (-O-PO3H2) on the surface of manno-oligosaccharide to improve hydrophilicity and solubility, and more importantly, to improve the affinity of manno-oligosaccharide with biological macromolecules such as membranes, enzymes and proteins, thereby improving the absorption and utilization capacity of crops to manno-oligosaccharide, ensuring the promotion of the absorption of nitrogen, phosphorus and potassium, and improving the utilization rate of fertilizer.

[0010] Preferably, the phosphorylation reagent comprises phosphorus pentoxide and pyridine.

[0011] The weight ratio of manno-oligosaccharide to phosphorus pentoxide is 1:0.5-3.

[0012] In the present application, phosphorus pentoxide is used as a phosphorylation reagent, and pyridine is used as an organic base. It can timely neutralize the acid generated in the reaction, push the reaction equilibrium to the positive direction, and greatly improve the efficiency and degree of phosphorylation.

[0013] Preferably, the phosphorylation reaction temperature is 10-30 ℃, and the time is 12-36 h.

[0014] Preferably, the manno-oligosaccharide is obtained by centrifugation of a manno-oligosaccharide mixed solution obtained by enzymatic hydrolysis reaction of glucomannan with a mixed enzyme reagent.

[0015] The mixed enzyme reagent comprises β - mannanase, cellulase and xylanase.

[0016] The application also provides a high utilization rate fertilizer, which comprises the fertilizer synergist.

[0017] Preferably, the fertilizer comprises, by weight percentage, 1-6% of the fertilizer synergist, 0.5-2% of the microbial fermentation powder, and 92-97% of the mineral nutrient element.

[0018] Preferably, the microbial fermentation powder comprises a fermentation product of Pseudomonas fluorescens.

[0019] In the application, when the microbial fermentation powder relies on the functions of Pseudomonas fluorescens and its biological fermentation product, the Pseudomonas fluorescens is a kind of phosphorus solubilizing bacteria, which can convert insoluble phosphorus and potassium in the soil into soluble nutrients, thereby improving the utilization rate of the fertilizer.

[0020] Preferably, the fertilizer further comprises 1-5% of modified attapulgite.

[0021] The modified attapulgite is attapulgite grafted with a cyclodextrin side group polyacrylamide compound.

[0022] The attapulgite grafted with the cyclodextrin side group polyacrylamide compound is obtained by esterification condensation of attapulgite modified with cyclodextrin and a silane coupling agent in sequence after polymerization of acrylamide under a chain transfer agent of bis(carboxymethyl)trithiocarbonate and an initiator of azobisisobutyronitrile.

[0023] In the application, after polymerization of acrylamide under a chain transfer agent of bis(carboxymethyl)trithiocarbonate and an initiator of azobisisobutyronitrile, carboxyl-terminated polyacrylamide is obtained, and then esterification condensation is performed in sequence with cyclodextrin and attapulgite, so that the attapulgite is grafted with the cyclodextrin side group polyacrylamide compound, and the specific structure is shown in the following schematic diagram.

[0024]

[0025] On the one hand, the excellent inclusion and adsorption capacities of the cyclodextrin can effectively adsorb and fix free metal ions in the soil, promote the absorption of trace elements by crops, protect the soil bacteria, improve the tolerance, and enhance the ability of the crops to absorb nutrients; on the other hand, the porous structure and ion adsorption and replacement capacity of the attapulgite can further improve the utilization rate of the fertilizer; and on the other hand, the amino groups contained in the polyacrylamide can form ammonium salt with the phosphatized modified mannose oligosaccharide, and the cross-linked composite structure of the two can not only exhibit excellent water absorption and water retention, but also can adsorb negative ions such as nitrate in the soil, further fix the nutrient elements and nutrients required by plants in the soil to improve the utilization rate of the fertilizer, and promote the growth of plants.

[0026] Preferably, the mineral nutrient elements include a nitrogen source, a phosphorus source and a potassium source.

[0027] 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.

[0028] Preferably, the preparation method of the fertilizer comprises: adding the fertilizer synergist and the mineral nutrient elements into a granulator for granulation, adding 5-8% of water as a binder, controlling the particle size of the granules to be 2-4 mm, and adding microbial fermentation powder after the obtained master batch is first dried by hot air and then cooled to room temperature by cold air, and then stirring and uniformly mixing, and sieving, to obtain the fertilizer.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] In the present application, the mannose oligosaccharide has multiple biological activities, and the phosphorylation modification thereof can further improve the affinity of the mannose oligosaccharide to biological macromolecules such as membranes, enzymes and proteins. The final test results show that the biological synergistic function of the phosphorylated modified mannose oligosaccharide is significantly improved, which shows the ability of the phosphorylated modified mannose oligosaccharide to greatly improve the fertilizer utilization rate in the field of fertilizers. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The infrared spectrum of the modified attapulgite described in Example 3. DETAILED DESCRIPTION

[0032] In the following, the technical solutions of the present application will be described in detail through specific examples, but it should be clear that these examples are used for illustration, but not to be interpreted as limiting the scope of the present application.

[0033] Example 1

[0034] A mannose oligosaccharide fertilizer synergist with biological synergistic function, which is essentially a phosphorylated modified mannose oligosaccharide, is prepared by the following method:

[0035] Glucomannan and ethanol are mixed in a ratio of Glucomannan:ethanol = 1:10 (w / v), stirred at 50 ℃ for 1 h, centrifuged (4000 rpm), repeated 3 times, and then dried at 60 ℃ for standby use;

[0036] β-mannanase, cellulase and xylanase with an activity of about 500 U / mg are mixed in a ratio of β-mannanase:cellulase:xylanase = 5:2:1 (w / w / w) to prepare an enzyme solution, which is dissolved with a pH = 6.0 phosphate buffer, and then placed at 4 ℃ for 30 min for standby use.

[0037] 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.

[0038] 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.

[0039] 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%).

[0040] 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.

[0041] Example 2

[0042] 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;

[0043] 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:

[0044] Pseudomonas fluorescens was inoculated into slant culture medium (10.0 g / L proteose 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, a single colony was inoculated into liquid seed culture medium (liquid volume ≤30%, 3% beef extract, 0.5% proteose peptone, 0.5% sodium chloride, distilled water, pH 7.0-7.2) and cultured at 200 rpm and 25 ℃ for 24 h to obtain a seed solution;

[0045] The seed solution was transferred into a fermentation tank at 10% (v / v) and fermentation liquid (32.5 g / L maltose, 4.5 g / L yeast extract, 4.5 g / L ammonium sulfate, 0.5 g / L K2HPO4, 0.06 g / L MgSO4·7H2O, 0.15 g / L NaCl) was added. The mixture was subjected to fermentation at 200 rpm and 25 ℃ for 48 h to obtain a fermentation liquid.

[0046] The fermentation liquid was centrifuged at 6000 rpm and 4 ℃ for 10 min, and the supernatant was discarded. The wet bacterial cells were collected and resuspended in sterile saline. The concentration of the bacterial cells was adjusted to ≥5.0×10 9 CFU / g. Then, the bacterial cells were rapidly frozen at -80 ℃ for 2 h, and finally, vacuum dried at -50 ℃ and 0.1 mbar for 24 h to obtain the microbial fermentation powder.

[0047] The preparation method of the above-mentioned fertilizer comprises the following steps:

[0048] Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, and phosphatized modified mannose oligosaccharide were put into a drum mixer and stirred at low speed for 10 min until uniform. Water accounting for 6% of the total weight of the fertilizer was sprayed, and granulation was performed in a rotary drum granulator for 20 min. The particle size was controlled to be 2-4 mm. The granules were first dried at 80 ℃ under hot air conditions for 30 min, then the temperature was reduced to 60 ℃ for drying for another 20 min, and then cooled to room temperature by cold air. The microbial fermentation powder was added, and the 2-4 mm qualified granules were screened by a vibrating screen to obtain the fertilizer.

[0049] Example 3

[0050] A high-utilization-rate fertilizer comprises, by weight percentage, phosphatized modified mannose oligosaccharide 4%, urea 17%, monoammonium phosphate 26%, diammonium phosphate 17%, potassium sulfate 31%, microbial fermentation powder 1%, and modified attapulgite 4%.

[0051] The phosphatized modified mannose oligosaccharide is prepared by the method described in Example 1. The microbial fermentation powder is prepared by the method described in Example 2. The modified attapulgite is prepared by the following method:

[0052] Acrylamide, bis(carboxymethyl)trithiocarbonate and azobisisobutyronitrile were added into ethanol according to acrylamide: bis(carboxymethyl)trithiocarbonate: azobisisobutyronitrile = 1:3:0.04 (w / w / w), stirred for 20 h after heating to 70 ℃ under nitrogen protection, precipitated and washed in methanol, suction filtered and dried to obtain the carboxyl-terminated polyacrylamide;

[0053] The carboxyl-terminated polyacrylamide was added into an aqueous solution in which β-cyclodextrin and acidized attapulgite were dispersed, and the carboxyl-terminated polyacrylamide: β-cyclodextrin: acidized attapulgite = 1:0.1:0.5 (w / w / w), then EDC and NHS were added, and the carboxyl-terminated polyacrylamide: EDC: NHS = 1:0.02:0.015 (w / w / w), stirred for 6 h after heating to 50 ℃, filtered, washed with water and dried to obtain the modified attapulgite.

[0054] The acidized attapulgite was prepared by adding attapulgite into a sulfuric acid solution (50wt%), standing for 8 h, washing, drying, adding a sodium hydroxide solution (15wt%) and adjusting the pH to 11, and stirring for 2 h after heating to 50 ℃.

[0055] The infrared spectrum of the modified attapulgite is shown in Figure 1 , and it can be known from Figure 1 that the peak at 3415 cm -1 is the inextensible vibration peak of N-H, the peaks at 2940 cm -1 and 2871 cm -1 are the stretching vibration peaks of C-H on β-cyclodextrin, the peak at 1623 cm -1 is the stretching vibration peak of C=O, the peak at 1428 cm -1 is the stretching vibration peak of CH2, and the peak at 1059 cm -1 is the stretching vibration peak of C=S.

[0056] The preparation method of the above-mentioned fertilizer comprises:

[0057] Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, phosphatized modified mannose oligosaccharide and modified attapulgite were put into a drum mixer and stirred at low speed for 10 min until uniform, and 6% of the total weight of the fertilizer was sprayed into water, and granulated in a drum granulator for 20 min, the particle size was controlled to be 2-4 mm, and then the granules were first dried at 80 ℃ under hot air for 30 min, then cooled to room temperature after being dried at 60 ℃ for 20 min, and then microbial fermentation powder was added, and the 2-4 mm qualified granules were screened by a vibrating screen to obtain the fertilizer.

[0058] Comparative Example 1

[0059] A high utilization rate fertilizer comprising, by weight percentage, mannose oligosaccharide 4%, urea 21%, monoammonium phosphate 26%, diammonium phosphate 17%, potassium sulfate 31%, and microbial fermentation powder 1%;

[0060] The mannose oligosaccharide is prepared according to the method described in Reference Example 1; and the microbial fermentation powder is prepared according to the method described in Reference Example 2.

[0061] The preparation method of the above-mentioned fertilizer comprises:

[0062] Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, and mannose oligosaccharide are put into a drum mixer and stirred at low speed for 10 min until uniform, 6% of water by total weight of the fertilizer is sprayed, granulation is performed in a rotary drum granulator for 20 min, the particle size of the granules is controlled to be 2-4 mm, initial drying is performed at 80 ℃ hot air for 30 min, the temperature is then lowered to 60 ℃ for further drying for 20 min, and then cold air cooling is performed to room temperature, microbial fermentation powder is added, and the 2-4 mm qualified granules are screened by a vibrating screen, thereby obtaining the fertilizer.

[0063] Comparative Example 2

[0064] A high utilization rate fertilizer comprising, by weight percentage, phosphatized modified mannose oligosaccharide 4%, urea 17%, monoammonium phosphate 26%, diammonium phosphate 17%, potassium sulfate 31%, microbial fermentation powder 1%, acidified attapulgite 3.3%, and β-cyclodextrin 0.7%;

[0065] The phosphatized modified mannose oligosaccharide is prepared according to the method described in Reference Example 1; the microbial fermentation powder is prepared according to the method described in Reference Example 2; and the acidified attapulgite is prepared according to the method described in Reference Example 3.

[0066] The preparation method of the above-mentioned fertilizer comprises:

[0067] Urea, monoammonium phosphate, diammonium phosphate, potassium sulfate, phosphatized modified mannose oligosaccharide, acidified attapulgite, and β-cyclodextrin are put into a drum mixer and stirred at low speed for 10 min until uniform, 6% of water by total weight of the fertilizer is sprayed, granulation is performed in a rotary drum granulator for 20 min, the particle size of the granules is controlled to be 2-4 mm, initial drying is performed at 80 ℃ hot air for 30 min, the temperature is then lowered to 60 ℃ for further drying for 20 min, and then cold air cooling is performed to room temperature, microbial fermentation powder is added, and the 2-4 mm qualified granules are screened by a vibrating screen, thereby obtaining the fertilizer.

[0068] Comparative Example 3

[0069] A high utilization rate fertilizer comprising, by weight percentage, phosphatized modified mannose oligosaccharide 4%, urea 17%, monoammonium phosphate 26%, diammonium phosphate 17%, potassium sulfate 31%, microbial fermentation powder 1%, modified attapulgite 4%;

[0070] 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.

[0071] The preparation methods of the above fertilizers include:

[0072] 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.

[0073] Test Example 1

[0074] 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.

[0075] Materials and Equipment:

[0076] Table 1. Reagents used in Test Example 1

[0077]

[0078] 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.

[0079] Experimental methods:

[0080] Sample pretreatment:

[0081] Ammonium molybdate-sulfuric acid solution: 100 mL of concentrated sulfuric acid was slowly added to 900 mL of distilled water, and stirred and cooled, 10 g of sodium molybdate was added, dissolved and mixed, and stored in a brown glass bottle;

[0082] Ascorbic acid solution (10%): 10.0 g of ascorbic acid was weighed into distilled water, and diluted to 100 mL;

[0083] DNS reagent: 6.3 g of 3,5-dinitrosalicylic acid was accurately weighed, 262 mL of 2 mol / L sodium hydroxide solution was added, and stirred until dissolved, and then added to 500 mL of a hot aqueous solution containing 182 g of potassium sodium tartrate, 5 g of phenol and 5 g of sodium sulfite were added, and stirred and dissolved, and then diluted to 1000 mL after cooling, and stored in a brown bottle and used after storing in the dark for one week;

[0084] Mo blue method sample pretreatment: 0.1000 g of the phosphatized modified mannose oligosaccharide described in Example 1 was accurately weighed, dissolved with a small amount of deionized water, and then quantitatively transferred to a 100 mL volumetric flask, and diluted to the mark, and shaken to obtain a stock solution with a concentration of 1 mg / mL; according to the results of the pre-experiment, 5.00 mL of the stock solution was taken and placed in a 100 mL volumetric flask, and diluted to the mark with deionized water to obtain a 20-fold diluted sample solution to be tested;

[0085] DNS method sample pretreatment: 0.1000 g of each of the mannose oligosaccharide and the phosphatized modified mannose oligosaccharide of Example 1 was accurately weighed, and a stock solution with a concentration of 1 mg / mL was prepared according to the above method; according to the pre-experiment, 5.00 mL of each of the two stock solutions was taken and placed in a 100 mL volumetric flask, and diluted to the mark with deionized water to obtain a 20-fold diluted sample solution to be tested.

[0086] Mo blue colorimetric method for determining phosphoric acid content:

[0087] 0.7165 g of potassium dihydrogen phosphate dried to constant weight at 105°C was accurately weighed, dissolved with deionized water and diluted to 500 mL to obtain a standard stock solution containing 1.00 mg of phosphoric acid per mL; 10.00 mL of the stock solution was taken and placed in a 500 mL volumetric flask, and diluted to the mark to obtain a working standard solution of 20.0 μg / mL ; 7 25 mL stoppered colorimetric tubes were prepared, and the standard solution and reagent were added according to the following Table 2:

[0088] Table 2 Phosphorus standard solution

[0089]

[0090] After adding reagents to each tube in order, mix immediately, adjust volume to 25.0 mL mark with deionized water, shake thoroughly; place all colorimetric tubes in a 40 ℃ constant temperature water bath for color development for 15 min, then cool to room temperature; use 1 cm light path cuvette, at 690 nm wavelength, zero with blank tube (No. 1), measure absorbance value of each tube; measure 3 times in parallel for each concentration, take average value, draw standard curve;

[0091] Take 2.00 mL of molybdenum blue method pretreated sample solution (equivalent to 0.1 mg of phosphated mannose oligosaccharide) in a 25 mL colorimetric tube, add reagents, develop color and measure absorbance according to the same operation steps of the above standard curve, and set up sample blank (replace sample with water) and reagent blank (replace all reagents with water) for correction.

[0092] DNS method for determining reducing sugar content:

[0093] Glucose standard curve drawing:

[0094] Accurately weigh 1.000 g of anhydrous glucose (dried to constant weight at 105 ℃), dissolve with deionized water and dilute to 1000 mL to obtain a 1 mg / mL glucose standard stock solution; take 6 25 mL graduated test tubes, add standard solution and reagent according to the following Table 3:

[0095] Table 3 Glucose standard solution

[0096]

[0097] After adding DNS reagent to each tube, mix thoroughly, accurately heat in a boiling water bath for 5 min, immediately cool to room temperature with running water; accurately adjust volume to 15.0 mL with deionized water, mix thoroughly; use 1 cm light path cuvette, at 540 nm wavelength, zero with blank tube (No. 1), measure absorbance value of each tube; measure 3 times in parallel for each concentration, take average value, draw standard curve;

[0098] Sample determination: take 1.00 mL of each DNS pretreated sample (equivalent to about 0.05 mg of mannose oligosaccharide or phosphated modified mannose oligosaccharide, respectively) in 25 mL graduated test tubes, add DNS reagent, boil, cool, dilute and measure absorbance according to the same operation steps of the above standard curve; at the same time, set up sample blank for correction.

[0099] Data recording and calculation:

[0100] Phosphate content calculation:

[0101] According to the absorbance value of the sample, the phosphate concentration in the determination solution is calculated from the phosphate standard curve regression equation, and then the phosphate content in the sample is calculated according to the following formula:

[0102]

[0103] 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.

[0104] Calculation of reducing sugar content:

[0105] 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:

[0106]

[0107] 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.

[0108] Phosphorylation level assessment:

[0109] Phosphate group introduction amount: directly calculated from phosphate content (1% phosphate content is equivalent to 0.105 mmol phosphate groups / g oligosaccharide).

[0110] Sugar chain retention rate:

[0111] Degree of phosphorylation:

[0112] Results and Data Analysis

[0113] Phosphorus standard curve data:

[0114] The absorbance values ​​of the phosphorus standard solutions were determined using the aforementioned method, and the results are shown in Table 4 below:

[0115] Table 4 Absorbance data of phosphorus standard series solutions

[0116]

[0117] 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.

[0118] The results show that the phosphorus content and the absorbance value have a good linear relationship in the range of 0-40 μg, which meets the Beer's law; the linear range, sensitivity and precision meet the analysis requirements.

[0119] Glucose standard curve data:

[0120] The absorbance values of the glucose standard series solutions were determined according to the aforementioned method, and the results are shown in Table 5 below:

[0121] Table 5 Absorbance data of glucose standard series solutions

[0122]

[0123] The glucose content (mg) was taken as the horizontal coordinate (X), and the average absorbance value was taken as the vertical coordinate (Y) to perform linear regression analysis, and the regression equation was Y = 1.074X + 0.001, and the correlation coefficient R² = 0.9996. The results show that the reducing sugar content and the absorbance value have a good linear relationship in the range of 0-1.0 mg, which meets the Beer's law. The linear range, sensitivity and precision meet the analysis requirements.

[0124] Actual sample determination results:

[0125] The phosphoric acid content of the phosphated mannose oligosaccharide sample and the mannose oligosaccharide raw material was determined, and the results are shown in Table 6 below:

[0126] Table 6 Determination results of phosphoric acid content of phosphated mannose oligosaccharide

[0127]

[0128] The determination results in Table 6 above show that the phosphate content in the phosphated mannose oligosaccharide reaches 0.37%, while the raw material mannose oligosaccharide contains only 0.02% of phosphate (may come from the raw material itself or the determination background). After deducting the background value, the actual introduced phosphate content is 0.35%, which is equivalent to introducing 0.39 mmol of phosphate groups per g of mannose oligosaccharide.

[0129] The reducing sugar content of the phosphated mannose oligosaccharide sample and the mannose oligosaccharide raw material was determined, and the results are shown in Table 7 below:

[0130] Table 7 Determination results of reducing sugar content of phosphated mannose oligosaccharide

[0131]

[0132] The determination results of Table 7 show that the reducing sugar content of the unmodified mannose oligosaccharide raw material is 15.1%, and the reducing sugar content of the phosphated modified mannose oligosaccharide is 13.5%, and the sugar chain retention rate is 89.4%, indicating that 10.6% of the sugar chains are degraded or structurally changed during the phosphating modification process, resulting in a decrease in the reducing end.

[0133] The test results show that the phosphating modification successfully introduces phosphate groups into the mannose oligosaccharide molecules, and the introduction amount reaches a high level, but partial sugar chain degradation inevitably occurs during the modification process, but the degradation degree is controlled within an acceptable range (about 11%); the phosphated mannose oligosaccharide as a fertilizer synergist has significant advantages, on the one hand, the introduced phosphate groups can enhance the water solubility and stability of the oligosaccharide, and on the other hand, the sugar chain degradation degree during the phosphating oligosaccharide modification process is limited, and most of the biological activity is retained.

[0134] Test Example 2

[0135] Purpose of the experiment: to verify that the affinity of the phosphated modified mannose oligosaccharide to biological macromolecules (cell membrane, enzyme, protein) is significantly improved compared with the mannose oligosaccharide, and this enhanced affinity is expected to promote the absorption and utilization of oligosaccharide by plants, thereby improving the fertilizer efficiency.

[0136] Materials and equipment:

[0137] Table 8 Reagents used in Test Example 2

[0138]

[0139] Test method:

[0140] Membrane binding experiment: liposome binding rate determination; liposomes are used to simulate plant cell membranes, and the membrane affinity is evaluated by determining the binding rate of oligosaccharide to liposomes;

[0141] Preparation of liposomes: 100 mg of lecithin was dissolved in 10 mL of chloroform, rotary evaporation to form a thin film, addition of 10 mL of PBS (pH 7.4) for hydration, and ultrasonic treatment to prepare a liposome suspension;

[0142] Sample treatment: prepare 1 mg / mL solutions of mannose oligosaccharide and phosphated modified mannose oligosaccharide, and mix 2 mL of each with 2 mL of liposome suspension;

[0143] Incubation conditions: incubate in a constant temperature water bath at 37°C for 1 h with shaking;

[0144] Separation and detection: centrifuge the reaction solution at 12000 rpm for 30 min at 4°C, and take the supernatant;

[0145] 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.

[0146] Preparation of the mannose standard curve:

[0147] Table 9 Absorbance data of mannose standard series solutions

[0148]

[0149] 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).

[0150] Data recording and results analysis:

[0151] Table 10. Membrane binding experimental data (liposome binding rate)

[0152]

[0153] Membrane binding experiment: Phosphorylation modification significantly improved the affinity of mannooligosaccharides for membrane structures.

[0154] 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.

[0155] Test Example 3

[0156] 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.

[0157] Experimental materials:

[0158] 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)

[0159] Experimental Design:

[0160] Four treatments were set up with three replicates each, arranged in randomized block design, plot area 20 m2(4 m x 5 m):

[0161] CK: common fertilizer base fertilizer (36 g / m2) + 0.2% potassium dihydrogen phosphate control foliar spray;

[0162] Test fertilizer group 1: Example 2 fertilizer base fertilizer (36 g / m2);

[0163] Test fertilizer group 2: Example 2 fertilizer base fertilizer (36 g / m2) + 0.2% Example 2 fertilizer foliar spray (sprayed once at the tillering stage and the booting stage);

[0164] Test fertilizer group 3: common compound fertilizer base fertilizer (18 g / m2) + Example 2 fertilizer ear fertilizer (18 g / m2);

[0165] Test fertilizer group 4: common compound fertilizer base fertilizer (18 g / m2) + Example 3 fertilizer ear fertilizer (18 g / m2);

[0166] Test fertilizer group 5: common compound fertilizer base fertilizer (18 g / m2) + Comparative Example 1 fertilizer ear fertilizer (18 g / m2);

[0167] Test fertilizer group 6: common compound fertilizer base fertilizer (18 g / m2) + Comparative Example 2 fertilizer ear fertilizer (18 g / m2);

[0168] Test fertilizer group 7: common compound fertilizer base fertilizer (18 g / m2) + Comparative Example 3 fertilizer ear fertilizer (18 g / m2);

[0169] Fertilization method:

[0170] Base fertilizer: evenly spread and rake into the soil (0-10 cm soil layer) one day before transplanting;

[0171] Ear fertilizer: applied at the young ear differentiation stage (45 days after transplanting);

[0172] Foliar spray: evenly sprayed after 4 o'clock on sunny days at the tillering stage (25 days after transplanting) and the booting stage (55 days after transplanting), with the leaf surface wetted as the standard.

[0173] Table 11 Observation index and method

[0174]

[0175] Results and analysis:

[0176] Table 12 Effect on crop growth and yield

[0177]

[0178] The fertilizer of the present application can significantly improve the yield of rice compared with common fertilizer treatment, wherein the yield increase of the test fertilizer groups 3 and 4 (optimum fertilization) is the most significant, and the yield increase reaches 14.0% and 18.2% respectively. It is indicated that the fertilizer of the present application can realize yield increase by enhancing root activity, improving photosynthetic efficiency and improving nitrogen utilization rate.

[0179] Table 13 Influence on protein absorption and accumulation

[0180]

[0181] The fertilizer of the present application can significantly improve the grain protein content compared with common fertilizer treatment, wherein the treatment effect of the test fertilizer groups 3 and 4 is the most significant. The fertilizer of the present application can promote protein synthesis and accumulation through two mechanisms: enhancing the activity of key enzymes in nitrogen metabolism (such as nitrate reductase and glutamine synthetase); and improving amino acid composition, including increasing the proportion of essential amino acids and increasing the proportion of glutelin.

[0182] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can make equivalent replacement or change according to the technical scheme and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. A manno-oligosaccharide fertilizer synergist having a bio-potentiation function, characterized in that, The phosphorization modified mannose oligosaccharide comprises a phosphorization modified mannose oligosaccharide; The phosphorization modified mannose oligosaccharide is obtained by phosphorization reaction of mannose oligosaccharide with phosphorus pentoxide; The mannose oligosaccharide is obtained by centrifugation of a mannose oligosaccharide mixture obtained by enzymatic hydrolysis reaction of glucosan with a mixed enzyme reagent; the mannose oligosaccharide mixture is purified by column and membrane filtration to obtain the mannose oligosaccharide. The mixed enzyme reagent comprises β - mannanase, cellulase and xylanase.

2. The manno-oligosaccharide fertilizer synergist with bio-enhancing function according to claim 1, characterized in that, The phosphorization reaction further comprises using pyridine as a neutralizing agent. The weight ratio of the mannose oligosaccharide to phosphorus pentoxide is 1:0.5-3.

3. The manno-oligosaccharide fertilizer synergist with bio-enhancing function according to claim 1 or 2, characterized in that, The phosphorization reaction temperature is 10-30 ℃, and the reaction time is 12-36 h.

4. A fertilizer, characterized by The fertilizer comprises the fertilizer synergist according to any one of claims 1-3.

5. The fertilizer of claim 4, wherein the fertilizer is a granular fertilizer. The fertilizer comprises, by weight percentage, 1-6% of the fertilizer synergist, 0.5-2% of microbial fermentation powder, and 92-97% of mineral nutrient elements.

6. The fertilizer of claim 5, wherein, The microbial fermentation powder comprises a fermentation product of Pseudomonas fluorescens.

7. The fertilizer of claim 6, wherein the fertilizer is a granular fertilizer. The fertilizer further comprises 1-5% of modified attapulgite. The modified attapulgite is attapulgite grafted with a cyclodextrin side group polyacrylamide compound. The attapulgite grafted with the cyclodextrin side group polyacrylamide compound is obtained by esterification condensation of acrylamide, cyclodextrin and attapulgite in turn after polymerization reaction of acrylamide under a chain transfer agent of bis(carboxymethyl)trithiocarbonate and an initiator of azobisisobutyronitrile.

8. The fertilizer according to any one of claims 5 to 7, wherein the fertilizer is a granular fertilizer. The mineral nutrient elements comprise a nitrogen source, a phosphorus source and a potassium source. 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 dihydrogen potassium phosphate.

9. The fertilizer according to any one of claims 5-7, characterized in that, The preparation method of the fertilizer comprises: adding the fertilizer synergist and the mineral nutrient elements into a granulator for granulation, adding 5-8% of water as a binder, controlling the particle size of the granules to be 2-4 mm, drying the obtained master batch by hot air, cooling to room temperature by cold air, adding the microbial fermentation powder for further stirring and mixing, and sieving to obtain the fertilizer.

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