A macroelement water-soluble fertilizer of microbial-source phlorotannins and a preparation method thereof

Abstract

The application discloses a macroelement water-soluble fertilizer of microbial source fucoidan and a preparation method thereof. High-molecular-weight sodium alginate is produced through fermentation of brown nitrogen-fixing bacteria, and then is directionally degraded by mannuronic acid specific fucoidan lyase produced by Pseudoalteromonas or Bacillus, so that microbial source fucoidan with a polymerization degree of 3-8, a M / G molar ratio of 1.5-3.0 and a purity of greater than or equal to 85% is obtained through two-stage ultrafiltration membrane fractionation, and dependence on seaweed raw materials is completely eliminated.

Description

Technical Field

[0001] This invention belongs to the field of fertilizer manufacturing technology, specifically relating to a macro-element water-soluble fertilizer of brown algae oligosaccharides prepared by microbial double fermentation and its preparation method. Background Technology

[0002] Water-soluble fertilizers containing macronutrients have become an important product form in the field of precision fertilization in modern agriculture due to their advantages such as high solubility, high absorption rate, and compatibility with drip irrigation / fertigation technologies. With the deepening of agricultural supply-side structural reform, functional water-soluble fertilizers containing macronutrients, especially compound products that combine nutritional functions and biostimulatory activities, have broad market development prospects.

[0003] Fucoidan oligosaccharides are oligosaccharides composed of β-D-mannuronic acid and α-L-guluronic acid linked by 1,4-glycosidic bonds, typically with a degree of polymerization ranging from 2 to 20. Numerous studies have shown that fucoidan oligosaccharides possess multiple biostimulatory activities, including inducing systemic resistance in plants, promoting root development, activating plant antioxidant enzyme systems, and improving soil microbial community structure. Incorporating them into water-soluble fertilizer formulations can effectively enhance fertilizer functionality and crop yield.

[0004] However, existing patented technologies for water-soluble fertilizers containing brown algae oligosaccharides have the following main drawbacks in terms of preparation process and formulation: (1) Limitations of the preparation route of brown algae oligosaccharides: The existing technology starts from physical sources (seaweed raw materials) and uses a combination process of enzymatic hydrolysis-alcohol precipitation-acid hydrolysis to prepare brown algae oligosaccharides. This route is highly dependent on the seasonal supply and quality stability of seaweed raw materials; the alcohol precipitation step consumes a large amount of ethanol, increasing the cost of solvent recovery; the acid hydrolysis conditions are difficult to precisely control the degree of degradation, resulting in a wide distribution of the degree of polymerization of the product (DP 2~30). Among them, the high degree of polymerization component (DP>12) has significantly lower bioactivity than the low degree of polymerization component, and the proportion of effective active ingredients is unstable.

[0005] (2) Lack of targeted regulation of M / G ratio: The bioactivity of brown algae oligosaccharides is closely related to the ratio of their M / G structural units. Oligosaccharides rich in poly-M blocks are more active in inducing plant immune signaling pathways (such as salicylic acid and jasmonic acid pathways). However, the existing preparation process does not target the M / G ratio of the product, resulting in poor activity uniformity.

[0006] (3) Insufficient phosphorus utilization efficiency: Most existing water-soluble fertilizer formulas use orthophosphates such as potassium dihydrogen phosphate (MKP) and monoammonium phosphate (MAP) as phosphorus sources, while orthophosphate (HPO4²⁻) is insufficient. - / H2PO4 -Ammonium polyphosphate (APP) is easily fixed by calcium, iron, aluminum and other ions in the soil, which leads to a rapid decrease in phosphorus availability. At the same time, the existing technology has not made full use of the slow-release characteristics of ammonium polyphosphate (APP) in the soil by gradual hydrolysis into orthophosphate, and has not explored the synergistic effect mechanism between brown algae oligosaccharides and ammonium polyphosphate.

[0007] (4) Insufficient stability of active components: In the existing water-soluble fertilizer containing brown algae oligosaccharides, the brown algae oligosaccharides are easily degraded due to pH fluctuations or high ionic strength in the system, resulting in a decrease in biological activity during storage.

[0008] In view of the above-mentioned shortcomings, there is an urgent need to develop a water-soluble fertilizer containing macroelements that can stably prepare a specific degree of polymerization (DP 3-8), is rich in poly-M blocks, can be efficiently compounded with ammonium polyphosphate from phosphorus chemical industry, and has both high biostimulation activity and excellent storage stability, as well as its preparation method. Summary of the Invention

[0009] The purpose of this invention is to overcome the above-mentioned defects of the prior art and provide a water-soluble fertilizer containing macro-elements from microbial-derived brown algae oligosaccharides and its preparation method, comprising the following steps: Step S1: Inoculate the brown azotobacter chroococcum strain into LB medium and activate it at 28–32℃ and 150–180 rpm for 24 h to obtain seed culture. Then, transfer it at an inoculum of 1%–3% (v / v) to an alginate-producing bacteria fermentation medium with sucrose as the carbon source (30–50 g / L) and dipotassium hydrogen phosphate as the phosphorus source (1.0–2.0 g / L). Ferment and culture it at an aeration rate of 1.0–1.5 vvm, a temperature of 28–30℃, and a pH of 7.0–7.4 for 72–96 h to obtain fermentation broth A containing high molecular weight sodium alginate (weight average molecular weight Mw ≥ 200 kDa). Preferably, the alginate-producing bacteria fermentation medium consists of: 40 g sucrose, 1.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 0.01 g FeSO4·7H2O, 0.002 g Na2MoO4·2H2O, with the pH adjusted to 7.2. Step S2: Centrifuge the fermentation broth A obtained in step S1 at 4000–6000 rpm and 4℃ for 20–30 min, and collect the supernatant; add an equal volume of 0.1 mol / L NaCl solution to the supernatant, mix thoroughly, and then ultrafilter using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa at 0.1–0.2 MPa. The retentate is the crude extract B containing sodium alginate; dilute the crude extract B with deionized water to a sodium alginate concentration of 5–10 g / L, and set aside. Step S3: Inoculate either *Pseudoalteromonas* sp. or *Bacillus* sp., strains producing mannouronic acid-specific alginate lyase, into an enzyme-producing medium with sodium alginate (5–10 g / L) as the inducing carbon source. Incubate at 25–30°C and 160–200 rpm for 48–72 h to allow for sufficient secretion of extracellular mannouronic acid-specific alginate lyase. Filter through a 0.22 μm microporous membrane to remove bacterial cells, obtaining crude enzyme solution C containing alginate lyase. Detect enzyme activity, ensuring it is not less than 50 U / mL. 1 U is defined as the release of 1 μmol of unsaturated uronic acid from the substrate per minute at pH 7.0 and 37°C. Preferably, the enzyme-producing culture medium has the following composition (per liter): sodium alginate 8 g, peptone 10 g, NaCl 15 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, pH 7.5; Step S4: The crude extract B obtained in step S2 and the crude enzyme solution C obtained in step S3 are mixed at a substrate mass (sodium alginate) to enzyme activity ratio of (200-500) mg substrate / U enzyme activity. The mixture is then subjected to enzymatic hydrolysis at pH 7.0-7.5 and temperature 35-40℃ for 3-6 h. The reaction process is monitored in real time by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC, differential refractive index detector). When the oligosaccharide component with a degree of polymerization DP 3-8 accounts for ≥75% of the total sugar content, the enzyme is inactivated by heating to 90℃ and holding for 10 min to obtain the enzymatic hydrolysate D. Step S5: The enzymatic hydrolysate D is first ultrafiltered through a 30 kDa molecular weight cutoff ultrafiltration membrane, and the permeate is collected (to remove residual undegraded high molecular weight alginic acid and large molecular weight degradation fragments); the permeate is then ultrafiltered through a 1 kDa molecular weight cutoff ultrafiltration membrane, and the retentate is collected, retaining oligosaccharide components with a molecular weight >1 kDa, i.e., brown algae oligosaccharides with DP≥3, while removing small molecules such as monosaccharides, disaccharides, and inorganic salts; the retentate is decolorized by activated carbon (1% to 3% of the total solids), treated at 60℃ for 30 min, and desalted by D001 macroporous strong acid cation exchange resin, and then concentrated under reduced pressure (vacuum degree -0.06 to -0.08 MPa) at 55 to 65℃ to a solid content of 30% to 40%, to obtain brown algae oligosaccharide concentrate E; Preferably, the concentrated liquid E is spray-dried (inlet air temperature 150-160℃, outlet air temperature 75-85℃) to obtain alginate oligosaccharide powder with a moisture content ≤5%, a purity of ≥85% (calculated as glucuronic acid equivalent), a DP 3-8 component accounting for ≥80% of the total sugar, and an M / G molar ratio of 1.5-3.0.

[0010] The microbial-derived brown algae oligosaccharide macro-element water-soluble fertilizer contains the following components by weight: Ammonium polyphosphate 15-25 parts by weight, N≥14%, P2O5≥60%, polyphosphate to orthophosphate mass ratio of 3:1-5:1, average degree of polymerization n is 4-8; 20-30 parts by weight of urea; 5-15 parts by weight of ammonium nitrate or calcium nitrate; Potassium sulfate 15-58 parts by weight; 2-5 parts by weight of the microbial-derived brown algae oligosaccharide according to claim 4; Potassium humate 1-3 parts by weight, free humic acid content ≥70%; EDTA chelates trace elements in amounts of 0.1–0.5 parts by weight; pH buffer 0.5–1.5 parts by weight; Anti-caking agent: 0.1–0.5 parts by weight; The water-soluble fertilizer containing macronutrients has a total nutrient content (N+P2O5+K2O) ≥50%, water-insoluble matter ≤0.1%, and pH (1% aqueous solution) 5.5~6.5.

[0011] Preferably, the water-soluble fertilizer containing macro-elements according to claim 5 is characterized in that it further contains 0.3 to 0.8 parts by weight of vitamin C (L-ascorbic acid) as an antioxidant stabilizer to prevent the oxidative degradation of alginic oligosaccharides during storage.

[0012] Preferably, the water-soluble fertilizer containing macroelements according to claim 5 is characterized in that it further comprises 0.1 to 0.3 parts by weight of chitosan quaternary ammonium salt, wherein the degree of quaternization of the chitosan quaternary ammonium salt is ≥90%, which is used to improve the stability of the brown algae oligosaccharide in a high ionic strength system; the mass ratio of the brown algae oligosaccharide to potassium humate is (1.5 to 2.5):1.

[0013] Preferably, the NPK ratio of the water-soluble fertilizer containing macronutrients satisfies one of the following: N:P2O5:K2O = (25~30):(10~13):(8~12), with a content of 3%~4% for brown algae oligosaccharides; N:P2O5:K2O = (18~22):(13~17):(13~17), with a content of 2%~3% for brown algae oligosaccharides; N:P2O5:K2O = (13~17):(3~7):(28~32), with a content of 2%~3% of brown algae oligosaccharides; Preferably, the total nutrient content (N+P2O5+K2O) in the water-soluble fertilizer containing macronutrients is not less than 50%, of which the nitrogen (N) content is 13% to 26%, the phosphorus (P2O5) content is 5% to 20%, the potassium (K2O) content is 10% to 32%, the alginic oligosaccharide content is 2% to 5%, the humic acid content is not less than 1%, the water-insoluble matter is ≤0.1%, and the pH (1% aqueous solution) is 5.5 to 6.5.

[0014] The preparation method of the water-soluble fertilizer containing macro-elements includes the following steps: Step P1: Ammonium polyphosphate (APP) is prepared by dehydration and polycondensation of phosphoric acid using a wet-process thermal polymerization of phosphoric acid, or industrial-grade APP raw material is crushed and sieved (through a 100-mesh sieve) to ensure that N≥14%, P2O5≥60% and moisture≤0.5%; the degree of polymerization distribution is confirmed by phosphorus spectrum nuclear magnetic resonance, and adjusted to an average degree of polymerization n of 4 to 8 for ammonium polyphosphate. Step P2: Add urea, ammonium nitrate or calcium nitrate and potassium sulfate to a mixing tank according to the formula, and stir evenly at 40-60℃ to obtain macro-element premix F; Step P3: Mix the fucoidan powder obtained in step S5 of scheme one with potassium humate at a mass ratio of (1.5-2.5):1, and add 0.5%-1% of vitamin C as an antioxidant stabilizer. Grind and mix at room temperature (20-25℃) to obtain fucoidan-potassium humate composite stabilized powder G. The multifunctional groups of humic acid can coat fucoidan through hydrogen bonding and electrostatic interaction to form a composite stable structure, preventing the hydrolytic degradation of fucoidan in high ionic strength fertilizer systems. Vitamin C acts as an antioxidant to prevent the oxidative degradation of fucoidan during storage. Step P4: Add and mix the ammonium polyphosphate obtained in step P1, the premix F obtained in step P2, and the composite stabilizer powder G obtained in step P3 in the following order: First, mix ammonium polyphosphate with premix F at 40-50℃ until homogeneous (3-5 min). After the temperature of the mixture drops below 35°C, slowly add the composite stabilizer powder G and mix at low speed (30-60 rpm) for 5-8 minutes. Add EDTA-chelated trace element premix and continue stirring for 3–5 minutes; Finally, add the pH buffer and anti-caking agent, and mix thoroughly; the entire process should be carried out in an environment with a relative humidity of ≤60%; Step P5: Sample and test the product's pH (1% aqueous solution), water-insoluble matter, content of various nutrients, and content of brown algae oligosaccharides. Seal and package the product in an environment with relative humidity ≤60%.

[0015] Preferably, in step P1, ammonium polyphosphate and dipotassium hydrogen phosphate (K2HPO4) are compounded in a mass ratio of (3:1) to (5:1) to ensure that the product has both the slow-release properties of polyphosphate and a certain amount of readily available orthophosphate to meet the peak phosphorus requirements of crops during the seedling stage; in the total amount of polyphosphate and orthophosphate, the proportion of polyphosphate is ≥75%; Preferably, in step P3, 0.1% to 0.3% by mass of chitosan quaternary ammonium salt (HACC, degree of quaternization ≥90%) is added as an auxiliary stabilizer to enhance the stability of fucoidan in high-concentration salt solutions. Preferably, the product obtained in step P4 is dissolved in an appropriate amount of deionized water to prepare a liquid water-soluble fertilizer with a total nutrient content of ≥20%, and 0.1% to 0.3% xanthan gum is added as a suspension stabilizer. The product has a density of 1.20 to 1.35 g / cm³ and a pH of 5.5 to 6.5, and is suitable for irrigation fertilization systems.

[0016] Beneficial effects Compared with the prior art, the present invention has the following significant advantages: (1) Innovative source of brown algae oligosaccharides, independent of seaweed raw materials: This invention adopts a dual microbial synergistic fermentation system of "alginate-lyase bacteria" and uses inexpensive carbon source as substrate. Sodium alginate is synthesized by microorganisms and then degraded by microbial mannuronic acid-specific alginate lyase, which completely eliminates the dependence on seaweed raw materials and can achieve a stable supply throughout the year, reducing raw material costs by 20% to 30%. (2) Controllable oligosaccharide structure and excellent bioactivity: The present invention prepares target oligosaccharides of DP 3-8 through a two-stage ultrafiltration membrane fractionation technology, with the M / G ratio precisely controlled between 1.5 and 3.0, and rich in poly-M blocks; it has been verified that the poly-M-rich brown algae oligosaccharides of DP 3-8 have a 35%-55% higher induction activity for plant systemic resistance than existing technology products, and a 20%-40% higher promotion rate for crop root length; (3) High phosphorus utilization rate of ammonium polyphosphate: This invention uses ammonium polyphosphate as the main source of phosphorus. The polyphosphate ions are gradually hydrolyzed into orthophosphate in the soil, which effectively slows down the rate at which phosphorus is fixed by the soil. At the same time, the brown algae oligosaccharides can activate the activity of rhizosphere phosphorus-solubilizing microorganisms, further improving the phosphorus utilization efficiency. Experiments show that after applying the product of this invention, the phosphorus absorption and utilization rate of crops is 15% to 25% higher than that of ordinary orthophosphate salt-soluble fertilizer. (4) High storage stability of active ingredients: The potassium humate composite coating technology and vitamin C antioxidant protection system are adopted to effectively prevent the degradation of brown algae oligosaccharides in high ionic strength fertilizer systems and during storage; after 24 months of storage at room temperature of 25℃, the bioactivity retention rate of brown algae oligosaccharides is ≥85%. Detailed Implementation

[0017] The present invention is further illustrated below through specific embodiments, but these embodiments do not constitute a limitation on the scope of protection of the present invention. Unless otherwise specified, the materials and equipment used in the embodiments are all commercially available; the brown nitrogen-fixing bacteria and alginate-lysin-producing strains used are all commercially available strains.

[0018] Example 1: Preparation of microbial-derived brown algae oligosaccharides Step S1: Take commercially available brown azotocin bacteria, streak them onto LB solid medium, incubate at 30℃ for 48 h, then pick a single colony and inoculate it into LB liquid medium. Incubate at 30℃ and 160 rpm for 24 h to obtain OD. 600 The seed culture was prepared at a concentration of 0.8–1.2. The seed culture was inoculated into a 10 L fermenter (working volume 7 L) at a 2% (v / v) inoculation rate. The fermentation medium consisted of: 40 g / L sucrose, 1.5 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 0.1 g / L NaCl, 0.01 g / L FeSO₄·7H₂O, 0.002 g / L Na₂MoO₄·2H₂O, and pH 7.2. Fermentation conditions were: temperature 28℃, aeration rate 1.2 vvm, stirring speed 300 rpm, pH maintained at 7.0–7.4 (adjusted with ammonia), and fermentation for 84 h. After fermentation, the sodium alginate concentration in fermentation broth A was measured to be 8.6 g / L, the weight-average molecular weight Mw = 285 kDa, and the M / G ratio = 2.3.

[0019] Step S2: Centrifuge fermentation broth A at 5000 rpm and 4℃ for 25 min, take the supernatant, add an equal volume of 0.1 mol / L NaCl solution and mix well. Use a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 50 kDa at 0.15 MPa (membrane area 0.5 m²). The sodium alginate concentration in the retentate (crude extract B) is about 15 g / L, and the volume is 1.2 L. Dilute with deionized water to a sodium alginate concentration of 7 g / L and set aside.

[0020] Step S3: Commercially available *Pseudomonas alterniflora* strain was inoculated into an enzyme-producing medium (sodium alginate 8 g / L, peptone 10 g / L, NaCl 15 g / L, K₂HPO₄ 1.0 g / L, MgSO₄·7H₂O 0.5 g / L, pH 7.5) and cultured at 28℃ and 180 rpm for 60 h with shaking. After culture, the bacterial cells were removed by filtration through a 0.22 μm microporous membrane to obtain crude enzyme solution C. The activity of extracellular mannouronic acid-specific alginate lyase (EC 4.2.2.3) was determined to be 68 U / mL (pH 7.0, 37℃, substrate: sodium alginate).

[0021] Step S4: The sodium alginate solution (300 mg dry weight) obtained in Step S2 was mixed with crude enzyme solution C at a substrate / enzyme activity ratio of 350 mg / U. Enzymatic hydrolysis was performed at pH 7.2 and 37℃ for 4.5 h. Samples were taken every 1 h, and the degree of polymerization distribution was detected by HPLC (RI detector). After 4.5 h, DP 3–8 fractions accounted for 78.3% of the total sugar. The temperature was raised to 90℃ and held for 10 min to inactivate the enzyme, yielding enzymatic hydrolysate D.

[0022] Step S5: Enzyme hydrolysate D is sequentially subjected to: ① Use an ultrafiltration membrane with a molecular weight cutoff of 30 kDa for ultrafiltration, collect the permeate, and remove residual high molecular weight alginic acid and macromolecular degradation debris; ② Use an ultrafiltration membrane with a molecular weight cutoff of 1 kDa for ultrafiltration, collect the retentate, retain oligosaccharide components with a DP ≥ 3, and remove small molecules such as monosaccharides, disaccharides, and inorganic salts; ③ Add 1.5% activated carbon (w / v) to the retentate, decolorize at 60℃ for 30 min, and filter; ④ The decolorized solution is desalted using D001 macroporous strong acid cation exchange resin; ⑤ Concentrate under reduced pressure at 60℃ and -0.07 MPa to a solid content of 35%; ⑥ Spray drying (inlet air 160℃, outlet air 80℃) yields brown algae oligosaccharide powder.

[0023] Test results: The brown algae oligosaccharide powder has a moisture content of 4.2%, a purity of 87.6%, a DP 3-8 component ratio of 82.4%, an M / G molar ratio of 2.1, and is a light yellow to white powder that is easily soluble in water with high transparency in aqueous solution.

[0024] Example 2: Preparation of a microbial-derived brown algae oligosaccharide macronutrient water-soluble fertilizer (N-P2O5-K2O≈20-15-15) Prepare a balanced macro-element water-soluble fertilizer according to the following formula (for producing 100 kg of product) (see Table 2):

[0025] Table 2. Raw material formulation for Example 2 (balanced type, 100 kg) Nutrient calculation: N = (24.5×14%+25.0×46.4%+14.5×34.4%) / 100 = (3.43+11.60+4.99) / 100 = 20.0%; P2O5 = (24.5×61.3%+0.8×40.7%) / 100 = (15.02+0.33) / 100 = 15.4%; K2O = (28.8×52%+0.8×54.1%) / 100 = (14.98+0.43) / 100 = 15.4%; Total nutrients = 50.8%.

[0026] Preparation steps: Step P1: Ammonium polyphosphate was pulverized and passed through a 100-mesh sieve. The results showed that N=14.2%, P2O5 content was 61.3%, polyphosphate / orthophosphate mass ratio was 4.1:1 (confirmed by ³¹P-NMR), average degree of polymerization n = 5.8, and moisture content was 0.4%. The sample was deemed qualified and ready for use.

[0027] Step P2: Urea (25.0 kg), ammonium nitrate (14.5 kg), and potassium sulfate (28.8 kg) were added to a horizontal ribbon mixer. The jacket temperature was set to 50°C and the speed to 20 rpm. The mixture was mixed for 10 min to obtain macro-element premix F. The material temperature was controlled at 49°C.

[0028] Step P3: Microbial-derived brown algae oligosaccharide powder (3.0 kg) and potassium humate (1.5 kg) were ground together in a ball mill for 15 min until fully homogeneous. 0.5 kg of vitamin C was added, and the mixture was mixed for another 5 min to obtain brown algae oligosaccharide-potassium humate composite stabilized powder G, which was then packaged and sealed for later use.

[0029] Step P4: Add ammonium polyphosphate (24.5 kg) to the mixer, then add premix F, set the jacket temperature to 45℃, and mix for 8 minutes; After the mixture cools to 32°C, slowly add the composite stabilizer powder G, reduce the rotation speed to 40 rpm, and mix for 10 min; Add EDTA to chelate trace elements (0.3 kg) and mix for 5 min; Add dipotassium hydrogen phosphate (0.8 kg) and citric acid (0.8 kg), and mix for 5 min; Add 0.3 kg of silica and mix for 3 min; the entire process should be carried out in an environment with a relative humidity of ≤55%.

[0030] Step P5: Sampling and testing; results are shown in Table 3.

[0031] Table 3 Product testing results of Example 2 All indicators meet the requirements of NY / T 1107-2020 standard. The product is a light yellow powder, easily soluble in water, and a 1% aqueous solution is clear and transparent.

[0032] Example 3: Preparation of a microbial-derived brown algae oligosaccharide macronutrient water-soluble fertilizer (N-P2O5-K2O≈15-5-30) Prepare a high-potassium water-soluble fertilizer containing macronutrients according to the following formula (for producing 100 kg of product) (see Table 4):

[0033] Table 4. Raw material formulation for Example 3 (high-potassium type, 100 kg) Nutrient calculation: N = (8.2×14%+29.8×46.4%) / 100 = (1.15+13.83) / 100 = 15.0%; P2O5 = (8.2×61.3%+0.6×40.7%) / 100 = (5.03+0.24) / 100 = 5.3%; K2O = (57.1×52%+0.6×54.1%) / 100 = (29.69+0.32) / 100 = 30.0%; Total nutrients = 50.3%.

[0034] The preparation steps are the same as in Example 2. In step P3, brown algae oligosaccharide (2.0 kg), potassium humate (1.0 kg) and chitosan quaternary ammonium salt (0.1 kg) are ground and mixed together. In step P2, only urea and potassium sulfate are used for macro-element premixing (excluding ammonium nitrate). In step P1, the amount of ammonium polyphosphate is 8.2 kg.

[0035] Test results: Total nutrients (N+P2O5+K2O) = 50.3%, N = 15.0%, P2O5 = 5.3%, K2O = 30.0%, water-insoluble matter 0.04%, pH 6.0, brown algae oligosaccharide content 2.0%, humic acid content 0.9%, all indicators meet the requirements of NY / T 1107-2020 standard.

[0036] Example 4: Storage Stability Verification Test The balanced product obtained in Example 2 (E2) and a commercially available similar water-soluble fertilizer containing brown algae oligosaccharides (control, processed using enzymatic hydrolysis-alcohol precipitation-acid hydrolysis, CK) were sealed and stored in a constant temperature and humidity chamber at 25°C and 60% relative humidity. Samples were taken at 0, 6, 12, 18, and 24 months. The bioactivity of brown algae oligosaccharides was evaluated by promoting the vitality of wheat seedling roots (hydroponics, treatment concentration 100 mg / L, cultured for 7 days, and measuring the main root length). Simultaneously, the content of DP3–8 oligosaccharide components was detected by HPLC (results are shown in Table 5). The formula for calculating the activity retention rate is: Activity retention rate = (Main root length of the treated group - Main root length of the blank control) / (Initial main root length of the treated group - Main root length of the blank control) × 100%.

[0037]

[0038] Table 5. Results of storage stability test (Blank control, main root length: 5.2 ± 0.4 cm; 6 parallel samples per group, mean ± standard deviation) Results analysis: After 24 months of storage, the root viability retention rate of the product E2 of this invention was (7.5-5.2) / (7.9-5.2) = 2.3 / 2.7 = 85.2% (≥85%), and the retention rate of DP 3-8 components was 88.6%; while the activity retention rate of the control product CK was (6.0-5.2) / (7.7-5.2) = 0.8 / 2.5 = 32.0%, and the retention rate of DP 3-8 components was only 51.8%, indicating a severe decline in activity. The stability of this invention is significantly better than that of the prior art (p<0.01, independent samples t-test).

[0039] Example 5: Field Trial Verification A field comparative experiment of cucumber (variety: Jinyou 35) was conducted at a vegetable planting base in Jingmen City, with the following treatment groups set up: T1 (Product of this invention N-P2O5-K2O≈20-15-15): Product of Example 2 for fertigation throughout the entire growth period, application rate 450g / mu·time, for a total of 4 applications; T2 (commercially available reference product): Apply commercially available water-soluble fertilizer containing brown algae oligosaccharides (enzymatic hydrolysis-alcohol precipitation-acid hydrolysis process) with the same nutrient content, and apply at the same rate and frequency; T3 (Conventional water-soluble fertilizer): Apply ordinary water-soluble fertilizer with equal nutrients and no brown algae oligosaccharides, with the same application amount and frequency; CK0 (blank control): Only an equal amount of water was applied, without fertilizer.

[0040] Each treatment consisted of 3 replicates, with each replicate having a plot area of ​​30 m², arranged in a randomized block design. The main results are shown in Table 6.

[0041] Table 6. Main results of the cucumber field experiment The results showed that cucumbers treated with product T1 of this invention yielded 3087 kg / mu, which was 359 kg / mu higher than T2 (commercial control) (an increase of 13.2%) and 543 kg / mu higher than T3 (conventional water-soluble fertilizer) (an increase of 21.3%). Furthermore, the incidence of downy mildew (8.3%) was significantly lower than that of T2 (15.7%) and T3 (18.2%), indicating that the product of this invention has significant advantages in promoting crop yield and inducing systemic resistance.

Claims

1. A method for preparing microbial-derived brown algae oligosaccharides, characterized in that, Includes the following steps: Step S1: Inoculate alginate-producing bacteria into a fermentation medium with sucrose as the carbon source, and ferment at an aeration rate of 1.0–1.5 vvm, a temperature of 28–30℃, and a pH of 7.0–7.4 for 72–96 h to obtain fermentation broth A containing sodium alginate; wherein the sodium alginate has a weight-average molecular weight Mw ≥ 200 kDa; Step S2: Centrifuge fermentation broth A to collect the supernatant, then ultrafilter it through a 50 kDa molecular weight cutoff ultrafiltration membrane. Dilute the retentate to a sodium alginate concentration of 5-10 g / L to obtain crude extract B. Step S3: Inoculate the microorganisms that produce mannuronic acid-specific alginate lyase into the enzyme-producing medium with sodium alginate as the inducing carbon source, and culture at 25-30℃ for 48-72 h. Filter to remove the bacterial cells and obtain crude enzyme solution C with enzyme activity ≥50 U / mL. Step S4: Mix crude extract B and crude enzyme solution C at a substrate to enzyme activity ratio of (200-500) mg substrate / U enzyme activity, and enzymatically hydrolyze for 3-6 h at pH 7.0-7.5 and temperature 35-40℃ until the oligosaccharide component of DP 3-8 accounts for ≥75% of the total sugar, and then inactivate the enzyme to obtain enzymatic hydrolysate D; Step S5: The enzymatic hydrolysate D is first ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the permeate is collected; the permeate is then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the retentate is collected. After decolorization, desalting, and concentration, the alginate oligosaccharide is obtained, with DP 3-8 components accounting for ≥80% and M / G molar ratio of 1.5-3.

0.

2. The preparation method according to claim 1, characterized in that, The alginate-producing bacteria mentioned in step S1 are brown nitrogen-fixing bacteria; The microorganisms that produce mannuronic acid-specific alginate lyase in step S3 are *Pseudomonas aeruginosa* or *Bacillus*.

3. The preparation method according to claim 1, characterized in that, The brown algae oligosaccharide obtained in step S5 is spray-dried to obtain a powder with a moisture content of ≤5%, a purity of ≥85%, a DP 3-8 component ratio of ≥80%, and an M / G molar ratio of 1.5-3.

0.

4. Microbial-derived brown algae oligosaccharides prepared by the preparation method according to any one of claims 1 to 3.

5. A water-soluble fertilizer containing macro-elements derived from microbial brown algae oligosaccharides, characterized in that, It contains the following components in parts by weight: Ammonium polyphosphate 15-25 parts by weight, N≥14%, P2O5≥60%, polyphosphate to orthophosphate mass ratio of 3:1-5:1, average degree of polymerization n is 4-8; 20-30 parts by weight of urea; 5-15 parts by weight of ammonium nitrate or calcium nitrate; Potassium sulfate 15-58 parts by weight; 2-5 parts by weight of the microbial-derived brown algae oligosaccharide according to claim 4; Potassium humate 1-3 parts by weight, free humic acid content ≥70%; EDTA chelates trace elements in amounts of 0.1–0.5 parts by weight; pH buffer 0.5–1.5 parts by weight; Anti-caking agent: 0.1–0.5 parts by weight; The water-soluble fertilizer containing macronutrients has a total nutrient content (N+P2O5+K2O) ≥50%, water-insoluble matter ≤0.1%, and pH (1% aqueous solution) 5.5~6.

5.

6. The water-soluble fertilizer containing macro-elements according to claim 5, characterized in that, It also contains 0.3 to 0.8 parts by weight of vitamin C, which acts as an antioxidant stabilizer to prevent the oxidative degradation of alginic oligosaccharides during storage.

7. The water-soluble fertilizer containing macro-elements according to claim 5, characterized in that, It also contains 0.1 to 0.3 parts by weight of chitosan quaternary ammonium salt, wherein the degree of quaternization of chitosan quaternary ammonium salt is ≥90%, which is used to improve the stability of fucoidan oligosaccharide in a high ionic strength system; the mass ratio of fucoidan oligosaccharide to potassium humate is (1.5 to 2.5):

1.

8. The water-soluble fertilizer containing macro-elements according to any one of claims 5 to 7, characterized in that, The NPK ratio of the water-soluble fertilizer containing macronutrients meets one of the following requirements: N:P2O5:K2O = (25~30):(10~13):(8~12), with a content of 3%~4% for brown algae oligosaccharides; N:P2O5:K2O = (18~22):(13~17):(13~17), with a content of 2%~3% for brown algae oligosaccharides; N:P2O5:K2O = (13~17):(3~7):(28~32), with a brown algae oligosaccharide content of 2%~3%.

9. A method for preparing the water-soluble fertilizer containing macroelements according to any one of claims 5 to 8, characterized in that, Includes the following steps: Step P1: Crush ammonium polyphosphate through a 100-mesh sieve and confirm the average degree of polymerization n as 4-8 and the polyphosphate / orthophosphate ratio by ³¹P-NMR for later use; Step P2: Mix urea, ammonium nitrate or calcium nitrate and potassium sulfate at 40-60℃ until homogeneous to obtain macro-element premix F; Step P3: Mix microbial-derived brown algae oligosaccharides and potassium humate at a mass ratio of (1.5-2.5):1, add 0.5%-1% of vitamin C to the total amount of the mixture, grind and mix well to obtain brown algae oligosaccharide-potassium humate composite stabilized powder G; Step P4: First, mix ammonium polyphosphate and premix F evenly at 40-50℃. After the temperature drops to below 35℃, add composite stabilizer powder G and mix. Then, add EDTA chelated trace elements, pH buffer and anti-caking agent in sequence and mix evenly. The entire step P4 is carried out in an environment with relative humidity ≤60%. Step P5: Seal the packaging.

10. The preparation method according to claim 9, characterized in that, In step P4, after mixing ammonium polyphosphate with premix F, the mixture is cooled to below 35°C before adding composite stabilizer powder G to prevent the degradation of brown algae oligosaccharides caused by high temperature. The maximum feeding temperature shall not exceed 35°C.

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