A zinc and selenium-rich microbial fertilizer and a preparation method thereof

Abstract

The present application relates to a kind of zinc selenium-rich microbial fertilizer and its preparation method, belong to the technical field of microbial fertilizer, the present application with the bacillus subtilis with selenite reduction capacity and zinc ion conversion capacity as production strain, using the preparation method of zinc-rich pre-culture and selenium-rich fermentation culture in the same fermentation system in turn, using the glutathione induced secretion in selenium reduction process drive zinc ion in situ nanometer, extracellular polysaccharide is simultaneously coated on the surface of nanometer selenium and nanometer zinc to form composite nanoparticles, and supplemented with selenium reduction rate monitoring-based dynamic zinc supplement feedback regulation.The present application makes selenium and zinc complete collaborative bioconversion and composite nanometer in the same fermentation system, selenium zinc in the obtained fertilizer exists in the form of composite nanoparticles, significantly improves the absorption and utilization efficiency of selenium and zinc for crops, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial fertilizer technology, specifically to a zinc-selenium-enriched microbial fertilizer and its preparation method. Background Technology

[0002] Selenium is an essential trace element for both plants and animals, participating in various metabolic pathways and possessing important physiological functions such as antioxidation and immune enhancement. In recent years, nano-selenium has attracted widespread attention as a novel form of selenium product, its nanoscale size endowing it with a higher specific surface area and biological activity. However, the traditional preparation of nano-selenium mainly relies on chemical reduction methods, which involve adding chemical reducing agents such as vitamin C to selenate or selenite solutions and synthesizing them under conditions of additional stabilizers such as bovine serum albumin. This process is not only resource-intensive but also prone to leaving chemical residues in the product, posing potential toxicity risks. Microbial transformation technology provides a new green synthesis pathway for nano-selenium—microorganisms can reduce toxic selenite to non-toxic nano-elemental selenium. This process requires no external reducing agents or stabilizers, offering significant advantages such as low energy consumption, high safety, and environmental friendliness. Currently, several microorganisms have been reported to possess the ability to reduce selenite to selenium nanoparticles, including Bacillus licheniformis, Bacillus subtilis, Bacillus cereus, Lactobacillus plantarum, and Bacillus sonorensis. For example, Bacillus subtilis SR41 can efficiently reduce sodium selenite to selenium nanoparticles with an average particle size of approximately 65 nm; Bacillus sonorensis NL4 can reduce 2.5 mmol / L sodium selenite by 85.94% within 24 hours, and the resulting selenium nanoparticles are coated with proteins and polysaccharides, exhibiting good stability.

[0003] Although significant progress has been made in the microbial synthesis of nano-selenium, existing technologies still have significant shortcomings. Firstly, current research primarily focuses on the microbial transformation of selenium as a single element, with little attention paid to the synergistic transformation of selenium with other micronutrients such as zinc within the same fermentation system. Zinc is also an essential micronutrient for plants and animals, playing an irreplaceable role in crop growth and development, enzyme activity regulation, and stress resistance. In existing microbial fertilizer products, selenium and zinc are typically added separately as inorganic salts, or first transformed separately by microorganisms before physical mixing; in-situ synergistic biotransformation and composite nano-sizing of selenium and zinc within the same fermentation system has not yet been achieved. Secondly, selenite has a significant toxic inhibitory effect on microorganisms; high concentrations of selenite inhibit strain growth and metabolic activity, limiting the yield and conversion efficiency of nano-selenium. Existing technologies often address this by screening for highly selenium-tolerant strains or optimizing fermentation conditions, but there is currently no technical solution to improve selenium conversion efficiency by introducing zinc to pre-enhance strain tolerance. Thirdly, during the reduction of selenite by microorganisms, metabolites such as glutathione and extracellular polysaccharides are secreted. In a single selenium conversion system, these products only act as coating stabilizers for nano-selenium. However, in a system where selenium and zinc coexist, the effects of these metabolites on zinc ions have not been fully understood and utilized, failing to construct a cascade mechanism for zinc conversion driven by selenium reduction products. Therefore, developing a microbial fertilizer and its preparation method that can achieve synergistic bioconversion of selenium and zinc in the same fermentation system, utilize selenium reduction metabolites to drive zinc nanoparticle formation, and form composite nanoparticles is of great significance for improving the product performance and production efficiency of microbial fertilizers containing trace elements. Summary of the Invention

[0004] In order to solve the problems of the prior art, the present invention provides a zinc-selenium-enriched microbial fertilizer and its preparation method.

[0005] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: In a first aspect, a zinc-selenium-enriched microbial fertilizer comprises bio-nano selenium and bio-nano zinc formed by a microbial strain capable of reducing selenite to nano-elemental selenium and converting zinc ions into nano-zinc particles in the same fermentation system through sequential biotransformation of an inorganic selenium source and an inorganic zinc source; the surface of the bio-nano selenium is coated with extracellular polysaccharides and proteins produced by microbial metabolism, and the surface of the bio-nano zinc is coated with glutathione produced by microbial metabolism.

[0006] In one specific embodiment of the first aspect, the microbial strain is Bacillus subtilis; the particle size of the bio-nano selenium is 70-500 nm, and the particle size of the bio-nano zinc is 20-200 nm.

[0007] In one specific embodiment of the first aspect, the bio-nano selenium, bio-nano zinc and extracellular polysaccharide together form composite nanoparticles, the average particle size of the composite nanoparticles being 50-300 nm.

[0008] Secondly, a method for preparing a zinc-selenium-enriched microbial fertilizer includes the following steps: S1: Strain activation: The microbial strain is inoculated into the activation medium and cultured at a temperature of 25-40℃ and a rotation speed of 100-250r / min for 12-48h to obtain the activated bacterial solution; S2: Zinc-enriched pre-culture: The activated bacterial solution obtained in step S1 is inoculated into a fermentation medium containing an inorganic zinc source at an inoculation rate of 1% to 15%, and pre-cultured for 12 to 48 hours at a temperature of 25 to 40°C, a pH of 5.5 to 8.0, and a rotation speed of 100 to 250 r / min; the concentration of the inorganic zinc source in the fermentation medium is 0.1 to 5 g / L. S3: Selenium-enriched fermentation culture: An inorganic selenium source is added to the culture system obtained in step S2 to make the concentration of the inorganic selenium source in the fermentation medium 0.5-10 mmol / L. Fermentation culture is continued for 24-96 h at a temperature of 25-40℃, pH 5.5-8.0, and a rotation speed of 100-250 r / min. The inorganic selenium source is sodium selenite and / or sodium selenate, and the inorganic zinc source is zinc sulfate, zinc chloride, and / or zinc acetate. During fermentation, the microorganisms reduce selenite to nano-selenium and secrete glutathione and extracellular polysaccharides. The glutathione reduces inorganic zinc to nano-zinc, and the extracellular polysaccharides simultaneously coat the surface of nano-selenium and nano-zinc. S4: Product collection: Collect the fermentation broth obtained in step S3 to obtain the liquid product of bio-nano selenium zinc microbial fertilizer; or after drying, obtain the solid product of bio-nano selenium zinc microbial fertilizer.

[0009] In one specific embodiment of the second aspect, the fermentation culture medium in step S2 comprises the following components by weight: 10-50 parts carbon source, 5-30 parts nitrogen source, 1-10 parts inorganic salt, 0.01-10 parts inorganic zinc source, and 50-90 parts water; the carbon source is selected from one or more of glucose, sucrose, starch, and molasses; the nitrogen source is selected from one or more of yeast extract, peptone, soybean meal powder, ammonium sulfate, and urea.

[0010] In one specific embodiment of the second aspect, during the fermentation process described in step S3, the concentration of selenite in the fermentation broth is monitored in real time. When the reduction rate of selenite is lower than 60% of the initial reduction rate, an inorganic zinc source is added to the fermentation system to maintain the total concentration of zinc ions in the fermentation system at 0.1–1.0 g / L. At the same time, the free zinc ions in the generated nano-selenium complex system maintain the concentration of free zinc ions in the system at 0.05–0.5 mmol / L.

[0011] In a specific embodiment of the second aspect, the fermentation culture described in step S3 is a stepwise fed-batch fermentation: first, an inorganic selenium source is added to make the initial concentration of the selenium source in the fermentation culture medium 0.5-2 mmol / L, and when the selenite reduction rate reaches more than 80%, an inorganic selenium source is added to make the final concentration 5-10 mmol / L.

[0012] In one specific embodiment of the second aspect, the microbial strain has a reduction rate of more than 65% for selenite and a conversion rate of more than 70% for zinc ions.

[0013] In one specific embodiment of the second aspect, the drying process in step S4 is spray drying, freeze drying or vacuum drying; a protective agent is added to the fermentation broth before the drying process, the protective agent being selected from one or more of trehalose, skim milk powder, and glycerin, and the amount of protective agent added is 1% to 10% of the mass of the fermentation broth.

[0014] Thirdly, the application of a zinc-selenium-enriched microbial fertilizer or a zinc-selenium-enriched microbial fertilizer prepared according to a method thereof in promoting crop growth and increasing the selenium and zinc content in crop grains; wherein the crop is rice, wheat, corn, soybean, tomato, pepper, strawberry or apple.

[0015] The beneficial effects of this invention are as follows: 1. This invention employs a sequential transformation strategy of "zinc-enriched pre-culture – selenium-enriched fermentation culture" within the same fermentation system. Zinc ions are introduced into the culture system beforehand, serving as a cofactor for the microbial antioxidant enzyme system. This strengthens the cell wall integrity and cell membrane stability of the strain before the introduction of selenite stress, significantly enhancing the strain's tolerance to subsequent high concentrations of selenite. Under this enhanced tolerance, the efficiency of the selenium reduction process is maximized. While the microorganisms efficiently reduce selenite to nano-level elemental selenium, they also secrete large amounts of glutathione and extracellular polysaccharides in response to stress. The in-situ generated glutathione, with its thiol group as a reduction-driving group, reduces zinc ions to zero-valent nano-zinc in the same system, achieving direct chemical driving of the zinc conversion process by the selenium reduction product. This constructs a cascade linkage mechanism of "selenium reduction → glutathione secretion → zinc nano-sizing." Meanwhile, the large amount of extracellular polysaccharides induced by selenium reduction stress, with their active functional groups such as carboxyl and carbonyl groups, simultaneously coordinate with the surfaces of nano-selenium and nano-zinc, forming a composite structure in which the extracellular polysaccharides simultaneously coat both types of nanoparticles. This allows nano-selenium and nano-zinc to coexist stably and synergistically in the same system, effectively inhibiting the aggregation and sedimentation of nanoparticles and significantly improving the dispersibility and storage stability of the product. Furthermore, this invention monitors the reduction rate of selenite in the fermentation broth in real time. When the reduction rate decreases, zinc ions are added to the system to activate the strain's antioxidant enzyme system and restore selenium reduction activity. Simultaneously, the in-situ generated nano-selenium complexes the free zinc ions in the system to maintain them within a suitable concentration range, forming a closed-loop dynamic equilibrium of "zinc supplementation activation—activity recovery—reduction recovery—zinc ion complexation," further ensuring the continuous and efficient operation of the fermentation process. 2. The bio-nano selenium-zinc microbial fertilizer prepared by the above process has an active ingredient of bio-nano selenium and bio-nano zinc composite nanoparticles coated with extracellular polysaccharides. This composite form allows both selenium and zinc to exist in a nano-zero valence state with higher bioavailability, and the microbial metabolites coated on the surface of the nanoparticles endow them with excellent hydrophilicity and dispersibility. After being applied to the rhizosphere of crops, the composite nanoparticles easily bind to the root surface due to the affinity of extracellular polysaccharides. Nano-selenium and nano-zinc enter the crop through the root absorption channels, significantly improving the efficiency of selenium and zinc migration and accumulation in the edible parts of the crop. The Bacillus subtilis live bacteria also present in the fertilizer colonize the rhizosphere soil and promote crop root development and nutrient absorption by continuously secreting metabolites such as auxins and siderophores, forming a synergistic promoting effect of "biofortification-nutrient supplementation" with nano-selenium and zinc. Furthermore, the extracellular polysaccharides in the microbial fertilizer act as natural cementing agents for soil aggregates, improving the rhizosphere microecological environment and increasing the stability of soil aggregate structure. Meanwhile, the antioxidant activity of nano-selenium can scavenge reactive oxygen species in the crop rhizosphere, reducing the inhibitory effects of abiotic stresses such as continuous cropping obstacles on crop growth. Applying this microbial fertilizer not only enables the simultaneous supplementation of selenium and zinc in the same fertilization operation, but also significantly improves the effective utilization rate of trace elements through the dual pathways of nano-scale processing and biotransformation, reducing the environmental residue risks associated with inorganic selenium and zinc application, thus possessing excellent economic and ecological benefits. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the preparation process of the present invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] like Figure 1 This invention relates to a zinc-selenium-enriched microbial fertilizer and its preparation method.

[0019] I. Strains Source and Culture The microbial strain used in this invention is *Bacillus subtilis*, which can be obtained from commercial collection institutions such as the China General Microbiological Culture Collection Center (CGMCC) or the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), or isolated and screened from selenium-rich soil or rhizosphere soil of farmland that has been treated with selenium-containing fertilizers for a long time. This strain should possess the ability to reduce selenite to nano-elemental selenium and to convert zinc ions into nano-zinc particles. *Bacillus subtilis* is a known microbial species, and those skilled in the art can isolate and purify strains with the above-mentioned functions from nature using conventional microbiological methods.

[0020] The strain was activated using LB medium (Luria-Bertani medium) with the following formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0–7.2. Activation conditions included a temperature of 25–40℃, a rotation speed of 100–250 rpm, and a culture time of 12–48 h, yielding an activated bacterial suspension in the logarithmic growth phase. The viable count of the activated suspension should reach 10-1. 8 ~10 9 CFU / mL.

[0021] II. Fermentation Culture Medium The fermentation medium comprises the following components by weight: 10-50 parts carbon source, 5-30 parts nitrogen source, 1-10 parts inorganic salt, 0.01-10 parts inorganic zinc source, and 50-90 parts water.

[0022] The carbon source is selected from one or more of glucose, sucrose, starch, and molasses, preferably glucose or sucrose, and more preferably glucose. The carbon source provides energy and carbon skeleton for microbial growth. If the concentration is too low, it will lead to slow cell growth and insufficient metabolites, while if the concentration is too high, it may cause substrate inhibition or increase the osmotic pressure of the culture medium. Therefore, the above range can ensure the normal growth and metabolic activity of the strain.

[0023] The nitrogen source is selected from one or more of yeast extract, peptone, soybean meal, ammonium sulfate, and urea, preferably a combination of yeast extract and peptone. Nitrogen sources are fundamental for the synthesis of proteins, nucleic acids, and other vital substances by microorganisms, and their type and concentration directly affect the growth rate and metabolic product synthesis of the strain.

[0024] The inorganic salts are selected from one or more of phosphates, magnesium salts, calcium salts, and potassium salts, specifically including dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), magnesium sulfate (MgSO4·7H2O), calcium chloride (CaCl2), etc., to provide essential mineral elements for microorganisms and maintain the buffering capacity and osmotic pressure of the culture medium.

[0025] The inorganic zinc source is selected from zinc sulfate (ZnSO4·7H2O), zinc chloride (ZnCl2), and / or zinc acetate (Zn(CH3COO)2), preferably zinc sulfate. The concentration of the inorganic zinc source in the fermentation medium is 0.1–5 g / L (calculated as zinc ions). If the zinc ion concentration is too low, the enrichment and conversion efficiency of zinc by the strain will be insufficient; if the concentration is too high, it may have toxic inhibitory effects on the strain.

[0026] The inorganic selenium source is selected from sodium selenite (Na₂SeO₃) and / or sodium selenate (Na₂SeO₄), preferably sodium selenite. The concentration of sodium selenite in the fermentation medium is 0.5–10 mmol / L. The concentration of sodium selenite is one of the key control parameters of this process: if the concentration is too low, the yield of nano-selenium will be insufficient; if the concentration is too high, the toxic effect of selenite on microorganisms will be significantly enhanced, inhibiting the growth and metabolic activity of the strain. Studies have shown that Bacillus subtilis has good reduction efficiency in the initial selenite concentration range of 1–4 mmol / L.

[0027] III. Strain Activation (Step 1) The preserved Bacillus subtilis strain was inoculated onto LB solid agar plates and activated by incubation at 30–37°C for 24–48 h. Single colonies were picked and inoculated into LB liquid agar and incubated in a shaker at 25–40°C and 100–250 r / min for 12–48 h to obtain the activated bacterial solution.

[0028] Input: Bacillus subtilis strain, LB medium.

[0029] Output: Activated bacterial solution, viable count 10-1 8 ~10 9 CFU / mL.

[0030] Key parameters: activation temperature 25-40℃, preferably 30-37℃; shaking speed 100-250 r / min, preferably 150-200 r / min; activation time 12-48 h, preferably 18-24 h.

[0031] IV. Zinc-enriched pre-culture (Step 2) The activated bacterial solution obtained in step 1 was inoculated into a fermentation medium containing an inorganic zinc source at an inoculation rate of 1% to 15%, and pre-cultured with zinc for 12 to 48 hours at a temperature of 25 to 40°C, a pH of 5.5 to 8.0, and a rotation speed of 100 to 250 r / min.

[0032] Technical Principle: The core function of the zinc-enriched pre-culture stage is "zinc protection"—allowing the strain to adapt and grow in a zinc-containing environment before introducing toxic selenite. Appropriate amounts of zinc ions can enhance the integrity of microbial cell walls and cell membranes, improving the strain's tolerance to subsequent selenite stress. Specifically, zinc ions act as cofactors for many microbial enzymes, participating in the synthesis and activation of antioxidant enzyme systems such as superoxide dismutase (SOD), thereby enhancing the strain's ability to resist oxidative stress. After zinc pre-enrichment, the cell membrane permeability and integrity of the strain are improved, enabling it to better resist oxidative damage caused by selenite, providing "pre-protection" for subsequent high-concentration selenium conversion.

[0033] Input: activated bacterial solution, zinc-containing fermentation medium.

[0034] Output: Zinc-enriched pre-culture medium (containing zinc-enhanced strains and zinc components initially transformed by the strains).

[0035] Key parameters: Inoculation amount: 1%–15%, preferably 5%–10%; Temperature: 25–40℃, preferably 30–37℃; pH: 5.5–8.0, preferably 6.5–7.5; Rotational speed: 100–250 r / min, preferably 150–200 r / min; Time: 12–48 hours, preferably 18–30 hours; Inorganic zinc source concentration: 0.1–5 g / L (calculated as zinc ions), preferably 0.5–2 g / L.

[0036] V. Selenium-enriched fermentation culture (step 3) Add an inorganic selenium source (sodium selenite and / or sodium selenate) to the culture system obtained in step 2 to make the concentration of the inorganic selenium source in the fermentation medium 0.5-10 mmol / L, and continue fermentation for 24-96 h at a temperature of 25-40℃, pH 5.5-8.0, and a rotation speed of 100-250 r / min.

[0037] Technical principle: This step is the core of the entire process, involving the coordinated transformation of two levels: (I) Bioreduction and Nanostructuring of Selenium. Bacillus subtilis bioreduced and nanostructured selenite (SeO3) in the culture medium. 2- ) is reduced to elemental selenium (Se) 0 Selenite exists in the form of nanoparticles. This reduction process involves the microbial nitrate reductase system and sulfur redox system, where selenite is reduced intracellularly to red selenium nanoparticles, which are then secreted extracellularly. During the reduction process, the microorganisms secrete metabolites such as glutathione (GSH) and extracellular polysaccharides (EPS) as a stress response.

[0038] (II) GSH-mediated zinc nanostructuring. Glutathione (GSH), induced during selenium reduction, has strong reducing properties and can reduce zinc ions (Zn) in the system. 2+ ) reduced to nano zinc (Zn 0 The thiol group (-SH) in the GSH molecule provides reducing electrons, reducing zinc ions to zero-valent zinc nanoparticles. Simultaneously, GSH and its oxidation product GSSG coat the surface of the zinc nanoparticles, stabilizing and dispersing them. This creates a positive cascade effect of "selenium reduction, GSH secretion, and zinc nanostructuring."

[0039] (III) Two-way coating of EPS. Selenium reduction stress induces microorganisms to secrete large amounts of extracellular polysaccharides (EPS). The polysaccharide chains in EPS contain carboxyl groups (-COO). - The extracellular polysaccharide (EPS) contains functional groups such as α and β (carbonyl, C=O), which can simultaneously interact with the surfaces of nano-selenium and nano-zinc to form a coating layer. EPS simultaneously coats the surfaces of nano-selenium and nano-zinc, forming a three-layer composite nanoparticle structure of "nano-selenium-nano-zinc-extracellular polysaccharide". This structure allows the two types of nanoparticles to coexist stably and synergistically in the same system, avoiding particle aggregation and sedimentation.

[0040] Input: Zinc-rich pre-culture medium, inorganic selenium source.

[0041] Output: Selenium-enriched fermentation broth (containing bio-nano selenium, bio-nano zinc and EPS-coated composite nanoparticles).

[0042] Key parameters: Inorganic selenium source concentration: 0.5–10 mmol / L, preferably 1–5 mmol / L Temperature: 25–40℃, preferably 30–37℃ pH: 5.5–8.0, preferably 6.5–7.5 Rotational speed: 100–250 r / min, preferably 150–200 r / min Time: 24–96 hours, preferably 48–72 hours Under these conditions, the microbial strain can achieve a reduction rate of over 65% for selenite and a conversion rate of over 70% for zinc ions.

[0043] Stepwise fed-batch fermentation (preferred method): Selenium-enriched fermentation can employ a stepwise fed-batch fermentation strategy. First, an inorganic selenium source is added to bring the initial concentration of selenium in the fermentation medium to 0.5–2 mmol / L. Once the selenite reduction rate reaches over 80%, the inorganic selenium source is added to a final concentration of 5–10 mmol / L. The advantage of this strategy is that the low-concentration selenium initiation phase allows the strain to gradually adapt to the selenium stress environment and activate the relevant reductase system. After the strain's selenium reduction capacity is fully induced, a high concentration of selenium is added to achieve high yield, thus balancing strain adaptability and final yield.

[0044] VI. Dynamic Feedback Control During the selenium-enriched fermentation process, the concentration of selenite in the fermentation broth was monitored in real time. When the reduction rate of selenite was lower than 60% of the initial reduction rate, an inorganic zinc source was added to the fermentation system to maintain the total zinc ion concentration in the fermentation system at 0.1–1.0 g / L.

[0045] Technical Principle: A decreased selenite reduction rate usually indicates inhibited metabolic activity in the bacterial strain, possibly stemming from oxidative stress caused by the cumulative toxicity of selenium. Supplementing with zinc ions at this time, as a cofactor for antioxidant enzymes such as superoxide dismutase (SOD), activates the strain's antioxidant enzyme system, scavenges reactive oxygen species (ROS), and restores the strain's reducing activity. Simultaneously, the nano-selenium continuously generated during fermentation has the ability to complex free zinc ions in the system—the active sites (such as oxygen-containing functional groups) on the surface of the nano-selenium can coordinate with free zinc ions, maintaining the free zinc ion concentration in the system at 0.05–0.5 mmol / L, preventing secondary inhibition of microorganisms due to excessively high zinc ion concentrations. This forms a closed-loop dynamic balance of "zinc supplementation activating antioxidant enzymes, restoring selenium reducing activity, nano-selenium complexing free zinc, and preventing zinc toxicity."

[0046] VII. Product Collection (Step 4) Collect the fermentation broth obtained in step 3 to obtain the biological nano-selenium zinc microbial fertilizer liquid product.

[0047] To prepare a solid product, the fermentation broth can be dried, such as by spray drying, freeze drying, or vacuum drying. Before drying, a preservative can be added to the fermentation broth. This preservative is selected from one or more of trehalose, skim milk powder, and glycerol, and the amount added is 1% to 10% of the fermentation broth's mass. The preservative's function is to protect the activity of the microbial cells and the structural integrity of the nanoparticles during the drying process, preventing cell damage and nanoparticle aggregation caused by high temperature or dehydration.

[0048] Input: Selenium-enriched fermentation broth (containing composite nanoparticles and live bacteria).

[0049] Output: Bio-nano selenium zinc microbial fertilizer liquid product (directly collected) or solid product (after drying).

[0050] VIII. Product Features The bio-nano-selenium-zinc microbial fertilizer prepared by the above method comprises bio-nano-selenium and bio-nano-zinc formed by the sequential biotransformation of inorganic selenium and inorganic zinc sources by Bacillus subtilis in the same fermentation system. The surface of the bio-nano-selenium is coated with extracellular polysaccharides and proteins produced by microbial metabolism, while the surface of the bio-nano-zinc is coated with glutathione produced by microbial metabolism. The bio-nano-selenium, bio-nano-zinc, and extracellular polysaccharides together form composite nanoparticles. The particle size of the bio-nano-selenium is 70–500 nm, the particle size of the bio-nano-zinc is 20–200 nm, and the average particle size of the composite nanoparticles is 50–300 nm.

[0051] Example Example 1 1. Strain activation Bacillus subtilis was inoculated onto LB agar plates and activated by incubation at 37°C for 24 hours. A single colony was then picked and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium and incubated at 37°C and 180 rpm for 18 hours to obtain the activated bacterial solution. The viable count of the activated solution was determined to be 5.2 × 10⁻⁶ cells / mL using the plate count method. 8 CFU / mL.

[0052] LB liquid culture medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0.

[0053] 2. Zinc-enriched pre-culture Prepare the fermentation medium (per 1L): 20g glucose, 10g yeast extract, 10g peptone, 2g K₂HPO₄, 1g KH₂PO₄, 0.5g MgSO₄·7H₂O, 2.2g ZnSO₄·7H₂O (equivalent to 0.5g / L zinc ions), with the remainder being water. Adjust the pH to 7.0. Sterilize at 121℃ for 20 minutes.

[0054] The activated bacterial solution was inoculated into the above fermentation medium at an inoculation rate of 8% (volume fraction), and pre-cultured in zinc-rich medium at 37℃ and 180r / min for 24h.

[0055] 3. Selenium-enriched fermentation culture Sodium selenite (Na₂SeO₃) was added to the zinc-enriched pre-culture system to bring the final concentration of sodium selenite in the fermentation medium to 3.4 mmol / L. Fermentation was continued for 72 h at 37 °C, pH 7.0, and 180 r / min.

[0056] During fermentation, the color of the fermentation broth gradually changed from light yellow to red, indicating that selenite was reduced to nano-elemental selenium. Sampling and measurement at 24 hours of fermentation showed a selenite reduction rate of 52.3%; at 48 hours, the reduction rate was 78.6%; and at 72 hours, the reduction rate reached 91.2%. Simultaneously, the total concentration of zinc ions in the fermentation broth gradually decreased from an initial 0.5 g / L to 0.32 g / L, with the free zinc ion concentration maintained at 0.08–0.15 mmol / L after complexation with nano-selenium, indicating that zinc ions were effectively converted into nano-zinc and stabilized by EPS coating.

[0057] 4. Dynamic zinc supplementation control (optional) At 48 hours of fermentation, the reduction rate of selenite was observed to decrease to 55% of the initial reduction rate (below the 60% threshold). ZnSO4·7H2O solution was then added to the fermentation system to restore the total zinc ion concentration to 0.5 g / L. Two hours after zinc supplementation, the reduction rate recovered to 78% of the initial reduction rate, indicating that zinc supplementation effectively activated the metabolic activity of the strain.

[0058] 5. Product collection After fermentation, the fermentation liquid is collected to obtain the biological nano-selenium zinc microbial fertilizer liquid product.

[0059] 6. Product Characterization Fermentation broth samples were collected and centrifuged at low speed (500 r / min, 5 min) to remove large particulate impurities. The morphology of the nanoparticles was then observed using transmission electron microscopy (TEM). The results showed that a large number of spherical nanoparticles were present in the fermentation broth. Among them, selenium nanoparticles had a diameter of 80–350 nm and an average diameter of 186 nm; zinc nanoparticles had a diameter of 30–150 nm and an average diameter of 85 nm. Both nanoparticles and extracellular polysaccharides formed composite nanoparticles with an average diameter of 120–250 nm. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of Se and Zn elements in the particles.

[0060] Fourier transform infrared spectroscopy (FTIR) was used to analyze the surface functional groups of the composite nanoparticles, and the results showed that at 3420 cm⁻¹... -1 (OH stretching vibration), 2925cm -1 (CH stretching vibration), 1630cm -1 (C=O stretching vibration), 1400cm -1 (COO) - Symmetric stretching vibration) and 1080cm -1 The presence of a characteristic absorption peak at the (COC stretching vibration) confirms that the extracellular polysaccharide was successfully coated onto the surface of the nanoparticles.

[0061] The extracellular protein content in the fermentation broth was determined to be 2.31 g / L using the Coomassie Brilliant Blue method; the extracellular polysaccharide content was determined to be 4.67 g / L using the phenol-sulfuric acid method.

[0062] 7. Determination of viable bacteria count in microbial fertilizer The viable count of Bacillus subtilis in the fermentation broth was determined using the plate count method, and the result was 3.8 × 10⁻⁶. 8 CFU / mL.

[0063] Experimental Example Experimental Example 1: Effects of different zinc-enriched pre-culture times on the selenium tolerance and nano-selenium production of bacterial strains Experimental Objective The effects of zinc-enriched pre-culture time on the selenite tolerance and nano-selenium yield of Bacillus subtilis were investigated to verify the synergistic effect of "zinc pre-culture, improved selenium tolerance, and improved selenium conversion rate".

[0064] Experimental Design Five treatment groups were set up, with zinc-enriched pre-culture times of 0 h (control, no zinc-enriched pre-culture), 6 h, 12 h, 24 h, and 48 h, with three replicates per group. After the pre-culture, sodium selenite was added to a final concentration of 3.4 mmol / L, and fermentation was continued for 72 h. Other conditions were the same as in Example 1.

[0065] Determination methods The reduction rate of selenite was determined using the diaminobenzidine spectrophotometric method: Fermentation broth samples were centrifuged at 8000 r / min for 10 min. 2 mL of 0.1% diaminobenzidine solution was added to the supernatant, and the mixture was heated in a boiling water bath at pH 2.0 for 10 min. After cooling, the absorbance was measured at 530 nm. The remaining selenite concentration was calculated based on the selenium standard curve. Reduction rate (%) = (Initial selenium concentration - Remaining selenium concentration) / Initial selenium concentration × 100%.

[0066] Determination of nano-selenium yield: The fermentation broth was centrifuged at 8000 r / min for 15 min to collect the precipitate, washed 3 times with deionized water, freeze-dried and weighed.

[0067] Experimental results ; Compared with the control group without zinc-enriched pre-culture, after 24 hours of zinc-enriched pre-culture, the selenite reduction rate increased from 68.5% to 91.2%, an increase of 22.7 percentage points; the nano-selenium yield increased from 1.68 mmol / L to 2.38 mmol / L, an increase of 41.7%; and the bacterial OD... 600 The increase from 1.52 to 2.15 indicates a significant increase in bacterial growth. When the pre-culture time was extended to 48 hours, there were no significant differences in any of the indicators compared to 24 hours (P>0.05).

[0068] The above results indicate that zinc-enriched pre-culture significantly improves the tolerance and reduction efficiency of Bacillus subtilis to selenite. The mechanism lies in the absorption of zinc ions by the strain during pre-culture and their participation in the synthesis of antioxidant enzymes, thereby enhancing the strain's ability to cope with selenium stress. 24 hours is the optimal pre-culture time, which fully realizes the "pre-protection" of the strain by zinc while ensuring the economic efficiency of the process.

[0069] Experimental Example 2: Effects of different sodium selenite concentrations on nano-selenium yield and zinc conversion rate Experimental Objective The effects of sodium selenite concentration on nano-selenium yield and zinc conversion rate were investigated to determine the optimal range of selenium source concentration.

[0070] Experimental Design Six treatment groups were set up with sodium selenite concentrations of 0.5, 1.0, 2.0, 3.4, 5.0, and 10.0 mmol / L, with three replicates per group. After 24 h of zinc-enriched pre-culture, different concentrations of sodium selenite were added, and fermentation was continued for another 72 h. All other conditions were the same as in Example 1.

[0071] Determination methods The yield of nano-selenium was determined in the same manner as in Example 1. Zinc conversion rate was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) to measure the concentration of residual zinc ions in the fermentation broth supernatant. Zinc conversion rate (%) = (initial zinc concentration - residual zinc concentration) / initial zinc concentration × 100%.

[0072] Experimental results ; Results Analysis As the sodium selenite concentration increased from 0.5 mmol / L to 3.4 mmol / L, the yield of nano-selenium gradually increased from 0.38 mmol / L to 2.38 mmol / L, with the selenium reduction rate remaining above 70% and the zinc conversion rate increasing from 52.3% to 71.2%. When the sodium selenite concentration increased to 5.0 mmol / L, although the absolute yield of nano-selenium continued to increase to 2.85 mmol / L, the selenium reduction rate significantly decreased to 57.0%, and the bacterial OD... 600 The decrease from 2.15 to 1.68 indicates that high concentrations of selenite had a significant toxic inhibitory effect on the strain. When the concentration reached 10.0 mmol / L, the selenium reduction rate was only 35.2%, and bacterial growth was severely inhibited.

[0073] Taking into account the yield of nano-selenium, reduction rate and strain activity, the optimal concentration of sodium selenite is 2.0–5.0 mmol / L, with 3.4 mmol / L being the optimal balance point, which balances high yield and strain activity.

[0074] Experiment Example 3: Verification Experiment of GSH-Mediated Zinc Nanostructuring Experimental Objective The study verified the promoting effect of glutathione (GSH) induced during selenium reduction on zinc ion nano-sizing, demonstrating the cascade linkage mechanism of "selenium reduction, GSH secretion, and zinc nano-sizing".

[0075] Experimental Design Set up 3 processing groups, with 3 replicates in each group: Group A (the present invention group): zinc-enriched pre-culture + selenium-enriched fermentation (selenium-induced GSH secretion) was carried out according to the method of Example 1.

[0076] Group B (GSH-added control group): No sodium selenite was added. After zinc-enriched pre-culture, GSH was added directly (the final concentration was the same as the GSH concentration at 48h of fermentation in Group A, which was measured to be 0.85 mmol / L), and culture was continued for 72h.

[0077] Group C (blank control group): No sodium selenite was added, no GSH was added, and all other conditions were the same as in Group A.

[0078] Determination methods The GSH content was determined using the DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) method: the supernatant of the fermentation broth was taken, DTNB solution was added, and the absorbance was measured at 412 nm. The content was calculated according to the GSH standard curve.

[0079] Nano zinc particle size and yield determination: TEM was used to observe the morphology of nano zinc and the particle size was counted; ICP-OES was used to determine the zinc content in the precipitate.

[0080] Experimental results ; Results Analysis In group A (this invention), the selenium-induced reduction strain secreted a large amount of GSH (0.85 mmol / L). Under these conditions, the average particle size of the zinc nanoparticles was 85 nm, the zinc nanoparticle yield reached 186 mg / L, and the zinc conversion rate reached 71.2%. In group B, although the same concentration of GSH (0.85 mmol / L) was added, the zinc nanoparticle yield (142 mg / L) and zinc conversion rate (54.6%) were significantly lower than those in group A (P<0.05), and the zinc nanoparticle size was slightly larger (92 nm). In group C, due to the lack of selenium induction and GSH supply, the zinc nanoparticle yield was extremely low (28 mg / L), and the zinc conversion rate was only 10.8%.

[0081] The above results indicate that GSH generated in situ during selenium reduction significantly promotes zinc nanostructuring. Compared with simple exogenous GSH, the in situ process of "selenium reduction and GSH secretion" not only provides the reducing agent GSH but also involves the synergistic secretion of other metabolites such as EPS, collectively creating a microenvironment conducive to zinc nanostructuring and achieving a superior zinc conversion effect compared to simple exogenous GSH. This confirms the scientific validity and effectiveness of the "selenium-zinc" cascade linkage mechanism in this invention.

[0082] Experiment Example 4: Stability Experiment of Composite Nanoparticles Experimental Objective The stability of the composite nanoparticles (nano-selenium-zinc-EPS) prepared by this invention was investigated under different pH and temperature conditions to verify the enhancing effect of bidirectional EPS coating on the stability of nanoparticles.

[0083] Experimental Design The fermentation broth obtained in Example 1 was centrifuged at low speed to remove large particles and then divided into two groups: Experimental group: Composite nanoparticles coated with EPS (original solution of this invention).

[0084] Control group: The composite nanoparticles were washed three times with deionized water to remove the EPS coating layer and then resuspended.

[0085] Two groups of samples were placed in buffer solutions with pH values ​​of 3.0, 5.0, 7.0, and 9.0, and at temperatures of 4℃, 25℃, 40℃, and 60℃ for 72 hours, respectively, and the particle size change and sedimentation rate of the nanoparticles were measured.

[0086] Determination methods Particle size was determined using dynamic light scattering (DLS). Sedimentation rate was determined by taking the supernatant suspension after standing for 72 hours and measuring the concentration of nanoparticles. Sedimentation rate (%) = (initial concentration - supernatant concentration) / initial concentration × 100%.

[0087] Experimental results ; Results Analysis Under the protection of EPS coating (experimental group), the composite nanoparticles exhibited excellent stability under various pH and temperature conditions: the particle size variation did not exceed 13%, and the sedimentation rate did not exceed 19%. Especially under acidic (pH 3.0) and high temperature (60℃) conditions, the particle size variation (+8.2% and +12.5%) and sedimentation rate (12.3% and 18.6%) of the experimental group were significantly better than those of the control group (particle size variation +45.6% and +68.2%, sedimentation rate 58.7% and 76.4%).

[0088] The above results indicate that the bidirectional coating of EPS endows the composite nanoparticles with excellent anti-agglomeration ability and environmental tolerance. The polysaccharide and protein components in EPS effectively prevent the aggregation and sedimentation of nanoparticles through steric hindrance and electrostatic repulsion effects, enabling the product to maintain a good dispersion state under different storage and use conditions.

[0089] Comparative experiment Comparative Experiment 1: The Method of This Invention vs. the Traditional "Conversion Then Mixing" Method Experimental Objective The method of "sequential conversion of the same fermentation system" in this invention is compared with the method of "separate conversion and then mixing" in the prior art to verify the synergistic effect of the method of this invention.

[0090] Experimental Design The present invention group: carried out zinc-enriched pre-culture + selenium-enriched fermentation according to the method of Example 1 (the same fermentation system was converted sequentially).

[0091] Control group 1 (converted and then mixed separately): Bacillus subtilis was fermented independently for 72 h in selenium-containing medium (3.4 mmol / L sodium selenite, without zinc) and zinc-containing medium (0.5 g / L zinc ions, without selenium) to prepare nano-selenium fermentation broth and nano-zinc fermentation broth, respectively, and then mixed at a volume ratio of 1:1.

[0092] Control group 2 (simultaneous addition): The activated bacterial solution was simultaneously inoculated into a culture medium containing sodium selenite (3.4 mmol / L) and zinc sulfate (0.5 g / L zinc ions), without stepwise pre-culture, and directly fermented for 72 h.

[0093] Each group had 3 replicates, and the remaining fermentation conditions were the same as in Example 1.

[0094] Determination methods The particle size and yield of nano-selenium and nano-zinc were determined in the same manner as in Experiment 2. Composite ratio determination: EDS energy dispersive spectroscopy analysis was used to statistically determine the proportion of composite nanoparticles containing both Se and Zn elements among all nanoparticles.

[0095] Experimental results ; Results Analysis The yield of nano-selenium in this invention group (2.38 mmol / L) was significantly higher than that in control group 1 (1.52 mmol / L) and control group 2 (1.35 mmol / L), representing increases of 56.6% and 76.3%, respectively. The zinc conversion rate (71.2%) was also significantly better than that in control group 1 (48.5%) and control group 2 (42.3%). Regarding particle size, both the nano-selenium and nano-zinc in this invention group had the smallest particle sizes (186 nm and 85 nm), indicating that sequential conversion within the same system is more conducive to generating smaller and more uniform nanoparticles.

[0096] Of particular importance is that the proportion of composite nanoparticles in the present invention group is as high as 72.3%, meaning that most of the nanoparticles are composite particles containing both Se and Zn elements, while the proportion of composite particles in control group 1 is 0 (because they cannot form true composite particles after separate conversion and mixing), and the proportion of composite particles in control group 2 is only 28.6%. In terms of stability, the sedimentation rate of the present invention group after standing for 7 days is only 8.5%, while that of control group 1 and control group 2 is as high as 35.6% and 42.8%, respectively.

[0097] The above results indicate that the "sequential conversion within the same fermentation system" method of this invention achieves true selenium-zinc composite nanoparticle formation through a cascade mechanism involving selenium reduction-induced GSH and EPS secretion, GSH-driven zinc nanoparticle formation, and bidirectional EPS coating. Compared with existing technologies that involve "separate conversion followed by mixing" or "simultaneous addition," the method of this invention has significant advantages in terms of nanoparticle yield, particle size uniformity, composite ratio, and stability, demonstrating synergistic effects.

[0098] Comparative Experiment 2: The Growth-Promoting Effect of the Invention's Microbial Fertilizer vs. Conventional Selenium-Zinc Fertilizer on Rice Experimental Objective The effects of the bio-nano selenium-zinc microbial fertilizer prepared in this invention on the promotion of rice growth and the influence of conventional selenium-zinc fertilizer (direct addition of inorganic salts) on the selenium-zinc content of the grains were compared.

[0099] Experimental Design A pot experiment was conducted using the rice variety "Xiangliangyou 900". Three treatment groups were set up, with 5 pots in each group and 3 plants per pot. Treatment A (the microbial fertilizer group of this invention): The biological nano-selenium zinc microbial fertilizer liquid product prepared according to the method of Example 1 was diluted 50 times and applied to the roots by irrigation. 50 mL was applied to each pot once during the tillering stage and once during the booting stage of rice.

[0100] Treatment B (conventional selenium-zinc fertilizer group): Apply equal amounts of inorganic salt solution of selenium and zinc (Na2SeO3 3.4mmol / L + ZnSO4·7H2O 0.5g / L zinc ions), in the same manner and at the same time as treatment A.

[0101] Treatment C (blank control group): Apply an equal volume of water.

[0102] Determination methods After rice matures, it is harvested, and plant height, effective panicle number, thousand-grain weight, and yield per plant are measured. Selenium content in the grains is determined using hydride generation-atomic fluorescence spectrometry (HG-AFS); zinc content in the grains is determined using ICP-OES.

[0103] Experimental results ; Results Analysis Compared with the blank control group, the rice plant height of the microbial fertilizer group of this invention increased by 17.7%, the number of effective panicles increased by 39.1%, the thousand-grain weight increased by 14.0%, and the yield per plant increased by 44.4%. Compared with the conventional selenium-zinc fertilizer group, the yield per plant of the microbial fertilizer group of this invention increased by 28.3%, indicating that the growth-promoting effect of the microbial fertilizer of this invention is significantly better than that of conventional inorganic selenium-zinc fertilizer.

[0104] Regarding the nutritional quality of the seeds, the selenium content (0.38 mg / kg) of the seeds in the microbial fertilizer group of this invention is 1.81 times that of the conventional selenium-zinc fertilizer group (0.21 mg / kg) and 6.33 times that of the blank control group (0.06 mg / kg); the zinc content (28.5 mg / kg) of the seeds is 1.53 times that of the conventional selenium-zinc fertilizer group (18.6 mg / kg) and 1.88 times that of the blank control group (15.2 mg / kg).

[0105] The above results indicate that the bio-nano selenium-zinc microbial fertilizer prepared in this invention, due to the presence of selenium and zinc in a bio-nano form and their protection by EPS coating, exhibits higher bioavailability and crop absorption and utilization rates. Compared with direct application of inorganic salts, the microbial fertilizer of this invention can more effectively transport selenium and zinc to crop grains, while simultaneously promoting crop growth through the growth-promoting effects of Bacillus subtilis (secreting plant growth hormones, solubilizing phosphorus and potassium, etc.).

[0106] Comparative Experiment 3: The Effects of the Invention's Microbial Fertilizer on Tomato Yield and Quality Experimental Objective The application effect of the microbial fertilizer of this invention on vegetable crops (tomatoes) was verified, proving its universality on different crops.

[0107] Experimental Design A greenhouse pot experiment was conducted using the tomato variety "Pink Crown". Three treatment groups were set up, with 5 pots in each group and 2 plants per pot. Treatment A (the microbial fertilizer group of this invention): Same as treatment A in comparative experiment 2, applied once each during the flowering period and fruit enlargement period of tomatoes.

[0108] Treatment B (conventional selenium-zinc fertilizer group): Same as treatment B in control experiment 2.

[0109] Treatment C (blank control group): Apply an equal volume of water.

[0110] Determination methods Tomatoes were harvested in batches after ripening, and the yield per plant was recorded. Selenium content in the fruit was determined using HG-AFS; zinc content using ICP-OES; vitamin C content using the 2,6-dichlorophenolindophenol titration method; and soluble sugar content using the anthrone colorimetric method.

[0111] Experimental results ; Results Analysis Compared with the blank control group, the tomato yield per plant in the microbial fertilizer group of this invention increased by 39.0%, the selenium content of the fruit increased by 540%, the zinc content of the fruit increased by 82.4%, the vitamin C content increased by 43.9%, and the soluble sugar content increased by 28.8%. Compared with the conventional selenium-zinc fertilizer group, the yield per plant in the microbial fertilizer group of this invention increased by 23.4%, the selenium content of the fruit increased by 77.8%, the zinc content of the fruit increased by 46.2%, the vitamin C content increased by 27.8%, and the soluble sugar content increased by 17.3%.

[0112] The above results indicate that the microbial fertilizer of this invention has the same effect on tomatoes as it does on rice, both showing significant growth-promoting, yield-increasing, and quality-improving effects. This microbial fertilizer not only effectively increases the selenium and zinc content in fruits but also improves the nutritional quality of fruits (vitamin C and soluble sugar content), demonstrating its universal applicability across crops.

[0113] Summary of comparative experiments Based on the above three sets of comparative experiments, the following conclusions can be drawn: Process advantages: The method of "sequential conversion in the same fermentation system" of this invention, through the stepwise strategy of "zinc-enriched pre-culture and selenium-enriched fermentation" and the cascade linkage mechanism mediated by GSH / EPS, realizes the synchronous and efficient synthesis and compounding of nano-selenium and nano-zinc. It is significantly better than the control schemes of "separate conversion and then mixing" and "simultaneous addition" in terms of yield, particle size, compounding ratio and stability (comparative experiment 1).

[0114] Fertilizer efficacy advantages: The bio-nano selenium-zinc microbial fertilizer prepared by this invention is significantly superior to conventional inorganic selenium-zinc fertilizer in promoting crop growth (plant height, number of effective panicles / fruits, yield) and improving the nutritional quality of crop grains / fruits (selenium content, zinc content) (comparative experiment 2 and comparative experiment 3).

[0115] Universality: The microbial fertilizer of this invention showed significant growth-promoting and selenium- and zinc-enriching effects on both rice (monocotyledonous grass) and tomato (dicotyledonous solanaceous crop), indicating that it has good cross-crop universality.

[0116] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A zinc- and selenium-enriched microbial fertilizer, characterized in that, The invention comprises bio-selenium nanoparticles and bio-zinc nanoparticles formed by microbial strains capable of reducing selenite to nano-elemental selenium and converting zinc ions into nano-zinc particles in the same fermentation system through sequential biotransformation of inorganic selenium and inorganic zinc sources; the surface of the bio-selenium nanoparticles is coated with extracellular polysaccharides and proteins produced by microbial metabolism, and the surface of the bio-zinc nanoparticles is coated with glutathione produced by microbial metabolism.

2. The bio-nano selenium-zinc microbial fertilizer according to claim 1, characterized in that, The microbial strain is Bacillus subtilis; the particle size of the bio-nano selenium is 70-500 nm, and the particle size of the bio-nano zinc is 20-200 nm.

3. The zinc-selenium-enriched microbial fertilizer according to claim 1, characterized in that, The bio-nano selenium, bio-nano zinc, and extracellular polysaccharides together form composite nanoparticles, the average particle size of which is 50-300 nm.

4. A method for preparing zinc-selenium-enriched microbial fertilizer as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Strain activation: The microbial strain is inoculated into the activation medium and cultured at a temperature of 25-40℃ and a rotation speed of 100-250r / min for 12-48h to obtain the activated bacterial solution; S2: Zinc-enriched pre-culture: The activated bacterial solution obtained in step S1 is inoculated into a fermentation medium containing an inorganic zinc source at an inoculation rate of 1% to 15%, and pre-cultured for 12 to 48 hours at a temperature of 25 to 40°C, a pH of 5.5 to 8.0, and a rotation speed of 100 to 250 r / min; the concentration of the inorganic zinc source in the fermentation medium is 0.1 to 5 g / L. S3: Selenium-enriched fermentation culture: An inorganic selenium source is added to the culture system obtained in step S2 to make the concentration of the inorganic selenium source in the fermentation medium 0.5-10 mmol / L. Fermentation culture is continued for 24-96 h at a temperature of 25-40℃, pH 5.5-8.0, and a rotation speed of 100-250 r / min. The inorganic selenium source is sodium selenite and / or sodium selenate, and the inorganic zinc source is zinc sulfate, zinc chloride, and / or zinc acetate. During fermentation, the microorganisms reduce selenite to nano-selenium and secrete glutathione and extracellular polysaccharides. The glutathione reduces inorganic zinc to nano-zinc, and the extracellular polysaccharides simultaneously coat the surface of nano-selenium and nano-zinc. S4: Product collection: Collect the fermentation broth obtained in step S3 to obtain the liquid product of bio-nano selenium zinc microbial fertilizer; or after drying, obtain the solid product of bio-nano selenium zinc microbial fertilizer.

5. The preparation method according to claim 4, characterized in that, The fermentation medium in step S2 comprises the following components by weight: 10-50 parts carbon source, 5-30 parts nitrogen source, 1-10 parts inorganic salt, 0.01-10 parts inorganic zinc source, and 50-90 parts water; the carbon source is selected from one or more of glucose, sucrose, starch, and molasses; the nitrogen source is selected from one or more of yeast extract, peptone, soybean meal powder, ammonium sulfate, and urea.

6. The preparation method according to claim 4, characterized in that, During the fermentation process described in step S3, the concentration of selenite in the fermentation broth is monitored in real time. When the reduction rate of selenite is lower than 60% of the initial reduction rate, an inorganic zinc source is added to the fermentation system to maintain the total concentration of zinc ions in the fermentation system at 0.1–1.0 g / L. At the same time, the free zinc ions in the generated nano-selenium complex system maintain the concentration of free zinc ions in the system at 0.05–0.5 mmol / L.

7. The preparation method according to claim 4, characterized in that, The fermentation culture described in step S3 is a stepwise fed-batch fermentation: first, an inorganic selenium source is added to make the initial concentration of the selenium source in the fermentation medium 0.5-2 mmol / L. When the selenite reduction rate reaches more than 80%, the inorganic selenium source is added to the final concentration of 5-10 mmol / L.

8. The preparation method according to claim 4, characterized in that, The microbial strain has a reduction rate of over 65% for selenite and a conversion rate of over 70% for zinc ions.

9. The preparation method according to claim 4, characterized in that, The drying process described in step S4 is spray drying, freeze drying, or vacuum drying. Before the drying process, a protective agent is added to the fermentation broth. The protective agent is selected from one or more of trehalose, skim milk powder, and glycerol. The amount of protective agent added is 1% to 10% of the mass of the fermentation broth.

10. The application of a zinc-selenium-enriched microbial fertilizer as described in any one of claims 1 to 3, or a zinc-selenium-enriched microbial fertilizer prepared by the method according to any one of claims 4 to 9, in promoting crop growth and increasing the selenium and zinc content in crop grains; wherein the crop is rice, wheat, corn, soybean, tomato, pepper, strawberry, or apple.

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