Method for biosynthesizing zero-valent nano-selenium by using microorganisms and application of zero-valent nano-selenium

Abstract

The invention relates to a method for biosynthesizing zero-valent nano-selenium by utilizing pantoea, the method comprises the following steps of: adding selenite into a strain culture medium, culturing pantoea, and separating and purifying from a synthetic product to obtain the zero-valent nano-selenium, the strain name of the pantoea is XZ-1, the classification name is pantoea Pantoeasp., the preservation number is CGMCC No.32330, the collection number is CGMCC No.32330, the collection number is CGMCC No.32330, the collection number is CGMCC No.32330, the collection number is CGMCC No.32330, the collection number is CGMCC No.32330, and the collection number is CGMCC No.32330. The preservation date is October 24, 2024, the strain XZ-1 is applied to synthesize zero-valent nano-selenium, biological nano-selenium is obtained from the synthesized product after multiple times of separation and purification, and the purified biological nano-selenium is mixed with a protective agent and a dispersing agent according to a certain proportion to prepare a nano-selenium fertilizer raw material, so that the problems that the nano-selenium is agglomerated and is easy to oxidize and lose efficacy are solved; the bioavailability of selenium is obviously improved, and the selenium element loss is reduced. The prepared nano-selenium can be used for original ingredients of fertilizers and feeds.

Description

Technical Field

[0001] The invention belongs to the technical field of preparing nano-selenium by microorganisms, and in particular to a method for biosynthesizing zero-valent nano-selenium by microorganisms and its application. Background Art

[0002] Selenium is one of the indispensable trace elements in all life activities. It plays a vital role in protecting cell membrane structure and biological functions, enhancing immunity and preventing various diseases. Selenium exists in three main forms in nature: organic selenium, inorganic selenium and elemental selenium. These forms are widely distributed in fossil fuels, rock sediments and marine environments. Different forms of selenium have great differences in its absorption efficiency and toxicity, among which: elemental nano-selenium > organic selenium > inorganic selenium. In terms of biological activity, elemental nano-selenium is nearly 5 times that of inorganic selenium and about 2.5 times that of organic selenium. Nano-selenium has the characteristics of low or non-toxicity, high biological activity and easy absorption. Research experiments have shown that elemental nano-selenium particles can comprehensively improve the antioxidant response ability of livestock and poultry bodies, enhance animal immunity, inhibit intestinal tumors, and improve the quality of livestock and poultry meat. It has the characteristics of low-dose biological toxicity and high bioavailability compared to sodium selenite.

[0003] Common methods for preparing nano-selenium include physical, chemical and biological methods. Among them, the physical method has strict requirements on experimental conditions, the synthesis cost is expensive, time-consuming, and it is not easy to achieve large-scale production. The chemical preparation process may use toxic chemicals, and the synthesized nano-selenium may not be conducive to human absorption, causing impacts on the environment and human health. Compared with physical and chemical methods, biological methods have many advantages. It is usually carried out under mild experimental conditions, does not require high equipment investment and complex operating procedures, and is conducive to reducing production costs and achieving large-scale production. More importantly, the biological method uses biological systems (such as microorganisms, plants or enzyme systems) to synthesize nano-selenium. This process avoids the use of toxic chemicals, thereby significantly reducing pollution to the environment. In addition, the use of microorganisms to prepare nano-selenium can not only improve the shortcomings of physical and chemical methods in terms of preparation methods, but also the synthesized nanoparticles have uniform particle size, high stability, and good biocompatibility, which greatly expands the application of nano-selenium.

[0004] Therefore, with the increasingly active research on the preparation of nanomaterials, the method of biosynthesis has gradually attracted the attention of researchers. In 1999, PNAS first reported the reduction of silver nanoparticles by Pseudomonas stutzeri, opening up a new method for the preparation of metal nanoparticles - in-situ reduction using microorganisms. The in-situ synthesis of metal nanoparticles using microorganisms refers to the reduction of metal ions to zero-valent metal nanoparticles by the action of cell surface functional groups or intracellular reductases. Currently, the research on the mechanism is divided into two aspects according to different methods. One is that carboxyl, amide, keto, and aldehyde groups on the cell membrane or cell wall surface can act as electron donors for metal ions. These active organic functional groups on the cell surface can adsorb, complex, and chelate metal ions, and in-situ reduce them into metal particles on the cell wall. At the same time, the cell wall surface of the bacteria interacts with the metal particles, preventing their migration and reducing aggregation, thereby obtaining metal nanoparticles. The other is the enzyme-catalyzed reduction process. The enzymes produced by microorganisms act as catalysts in the process of reducing and generating nanoparticles. They use electron carriers to transfer the electrons of reducing substances to metal ions and reduce them, thereby synthesizing metal nanoparticles. At the same time, these help to provide a natural coating film for the synthesized nanoparticles, prevent the aggregation of nanoparticles, and help the nanoparticles maintain long-term stability. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies in the prior art and provide a method for biosynthesizing zero-valent nano-selenium using strain XZ-1 and its application.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] A method for biosynthesizing zero-valent nano-selenium using Pantoea, the method uses Pantoea to biosynthesize nano-selenium: by adding sodium selenite to the strain culture medium, culturing Pantoea, and separating and purifying zero-valent nano-selenium from the synthesis product;

[0008] The strain name of the Pantoea is XZ-1, and its taxonomic name is: Pantoea sp., the preservation number is: CGMCC No. 32330, the preservation date is: October 24, 2024, and the preservation unit is: China General Microbiological Culture Collection Center, and the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0009] Moreover, the inorganic salts include sodium salt and potassium salt of selenous acid.

[0010] Moreover, the particle size of the zero-valent nano-selenium is 80 - 100 nm.

[0011] Moreover, the concentration of the sodium salt and potassium salt of selenous acid is 0.1 - 100 mM, preferably 5 mM.

[0012] Moreover, the obtained synthetic products include cell lysates, cell suspensions, and the cell precipitate obtained by centrifuging the culture medium.

[0013] A method for biosynthesizing zero-valent nano-selenium using Pantoea includes the following steps:

[0014] (1) Strain activation, including the preparation and activation process of the culture medium:

[0015] The culture medium formula is as follows: peptone: yeast extract powder: sodium chloride: potassium chloride, lactose are prepared according to the following mass ratio: 1:0.5:1:0.05:0.05. When preparing the culture medium, the above materials are mixed and deionized water is added, and the mass-volume ratio is 1:25, with the unit of g:mL;

[0016] The activation process is as follows: Place the prepared culture medium in an autoclave, sterilize it at 120 °C for 20 min, and then cool it at room temperature; use an inoculation loop to inoculate strain XZ-1 into the sterilized activated culture medium solution, place the solution in a shaker, and shake and culture it at 30 °C and 120 rpm for 14 h;

[0017] (2) Strain culture, including the preparation and culture process of the inoculation medium:

[0018] Prepare the inoculation medium: The mass ratio of peptone: yeast extract powder: sodium chloride: potassium chloride: magnesium chloride: calcium chloride is: 5:3:2:0.5:0.3:0.2; when preparing the culture medium, the mass-volume ratio of the above mixed materials to deionized water is 1:50, with the unit of g:mL;

[0019] The culture process is as follows: Place the prepared culture medium in an autoclave, sterilize it at 120 °C for 20 min, and then cool it and store it at room temperature; aspirate 1 mL of the activated cell bacterial solution and transfer it to the inoculation medium solution, with the inoculation amount being 1%, place the solution in a shaker, and shake and culture it at 30 °C and 120 rpm for 8 - 10 h until the OD600 reaches 1 - 1.2;

[0020] (3) Nano-selenium preparation, including the formula of the selenium reduction medium and the nano-selenium preparation process:

[0021] The formula of the selenium reduction medium is: peptone: yeast extract powder: sodium chloride: potassium chloride: Na2HPO4: KH2PO4: sodium selenite mass ratio is 15:10:5:0.2:1.44:0.24:0.005;

[0022] The nano-selenium preparation process is: After the culture medium is sterilized at 120 °C for 20 min, add 10 mM sodium lactate filtered through a 0.45 μm filter membrane, shake well and set aside;

[0023] Centrifuge the cell lysate obtained in step (2) at 8000 rpm for 10 min to collect the bacterial cells, inoculate the bacterial cells into the selenium reduction medium, and shake culture at 30 °C and 120 rpm for 8 - 12 h;

[0024] (4) Separation and purification of biological nano - selenium: For the reaction product obtained after selenium reduction in step (3), centrifuge at 8000 rpm for 25 min to collect the cell precipitate, wash it 2 - 3 times with sterile water by centrifuging at 10000 rpm for 10 - 20 min each time. After collecting the precipitate, resuspend it in 10 - 20 mL of sterile water, and then ultrasonically disrupt the cells on ice at a power of 800 W, with a start - stop interval of 30 s for 30 min to fully lyse the bacterial cells; centrifuge the lysate at 10000 rpm for 20 min, add sterile water with a mass twice that of the solid precipitate after collecting the precipitate to obtain a biological nano - selenium suspension; directly freeze the obtained biological nano - selenium suspension in liquid nitrogen for 10 min, and then put it into a freeze - dryer for freeze - drying. The freeze - drying time is 24 - 48 hours to obtain pure biological nano - selenium dry powder;

[0025] (5) The storage condition of the pure biological nano - selenium dry powder is: stored at room temperature in vacuum.

[0026] A nano - selenium matrix fertilizer containing zero - valent nano - selenium prepared by the above method.

[0027] Moreover, in the method, zero - valent nano - selenium is compounded with a dispersant and a protective agent to form a nano - selenium matrix fertilizer.

[0028] Moreover, the dispersant is: sodium carboxymethyl cellulose, gum arabic powder, 2 - naphthalenesulfonic acid, disodium EDTA, or a combination of any two or three of them; when the dispersant is compounded with zero - valent nano - selenium, its added mass is 0.5‰ - 1‰ of the mass of zero - valent nano - selenium;

[0029] The protective agent is: ferrous chloride, nano - zero - valent iron, nano - zinc oxide, manganese sulfate, zinc sulfate, or a combination of any two or three of them; when the protective agent is compounded with zero - valent nano - selenium, its added mass is 5‰ - 10‰ of the mass of zero - valent nano - selenium.

[0030] A method for using a nano - selenium matrix fertilizer, comprising the following steps:

[0031] Spray the nano - selenium matrix fertilizer as a foliar selenium fertilizer. The dosage per mu in vegetable fields is 1 - 3 g, and the water consumption per mu is 50 L. The dosage for wheat and rice is 2 - 5 g, and the water consumption per mu is 100 L. Spray it in two times;

[0032] Or, use the nano - selenium matrix fertilizer as a feed matrix: the dosage per ton is 0.2 - 1 g.

[0033] The advantages and positive effects obtained by the present invention are:

[0034] 1. The method of the present invention uses strain XZ-1 to prepare biological nano-selenium and carry out separation and purification, and at the same time applies it to fertilizers and feeds. The biological fermentation process is used to prepare nano-selenium, which has the characteristics of environmental friendliness, high stability, uniform particles, safety and high efficiency. The obtained biological nano-selenium is used in selenium-rich fertilizers and selenium-rich feeds.

[0035] 2. The present invention applies strain XZ-1 to synthesize zero-valent nano-selenium, and obtains biological nano-selenium from the synthesis product through multiple separation and purification steps. After mixing the purified biological nano-selenium with a protective agent and a dispersant in a certain proportion, the raw material of nano-selenium fertilizer is prepared, which solves the problems of nano-selenium agglomeration and easy oxidation and inactivation, significantly improves the biological utilization rate of selenium, and reduces the loss of selenium element. The prepared nano-selenium can be used as the original ingredient of fertilizers and feeds.

[0036] 3. The present invention synthesizes zero-valent nano-selenium through strain XZ-1, and uses the biosynthesized elemental nano-selenium as the raw material of selenium fertilizer. Compared with the selenium forms of inorganic selenium and organic selenium used in conventional selenium fertilizers, the elemental nano-selenium has uniform particles, high biological activity, and high crop absorption efficiency; at the same time, compared with traditional physical and chemical synthesis of nano-selenium, the experimental conditions are mild (no high temperature and high pressure) and no secondary pollution is generated. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the cell growth curve diagram of strain XZ-1 in the present invention under different concentrations of sodium selenite;

[0038] Figure 2 It is the nano-selenium yield diagram of strain XZ-1 in the present invention under different treatments of sodium selenite concentrations;

[0039] Figure 3 It is the scanning electron microscope (SEM) image and energy spectrum (EDX) diagram of bacterial cells and nano-selenium of strain XZ-1 in the present invention under 5 mM sodium selenite;

[0040] Figure 4 It is the transmission electron microscope (TEM) photo of the selenium nanoparticles generated by strain XZ-1 in the present invention under 5 mM sodium selenite treatment;

[0041] Figure 5 It is the X-ray absorption near-edge structure (XANES) spectral data diagram of the selenium nanoparticles generated in the present invention.

[0042] Figure 6 It is the comparison diagram of the influence of the selenium fertilizer made from the selenium nanoparticles generated in the present invention on the growth of plants (Epipremnum aureum). DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention will be further described below in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0044] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically noted in this article, those of ordinary skill in the art can refer to various common reference books, scientific and technological literature, or relevant specifications, manuals, etc. before the filing date of this invention application for implementation.

[0045] The strain XZ-1 used in the present invention was collected from highly arsenic-contaminated soil in Tibet. That is, 1-2 g of soil was added to 20 mL of tryptone, yeast extract, and glucose (TYEG) medium with a pH of 7.0, 5-10 ml / L of sodium arsenate was added, and incubated at 28 °C for 48 h. Then, 1 mL of the culture was collected every 24 h and added to a new culture medium one by one. After repeating the above steps 2 times, 1 mL of the culture was taken and serially diluted to 10 -6 , and then inoculated onto a TYEG medium plate containing the same sodium arsenate. The plate was further cultured at 28 °C for 24 h. The isolate was purified by the streak plate method and stored at -80 °C in 30% sterile glycerol, and stored after purification.

[0046] A method for biosynthesizing zero-valent nano-selenium using Pantoea, the method uses Pantoea to biosynthesize nano-selenium: by adding selenite to the strain medium, culturing Pantoea, and separating and purifying zero-valent nano-selenium from the synthesis product; the strain name of the Pantoea is XZ-1, the taxonomic name is: Pantoea sp., the preservation number is: CGMCC No. 32330, the preservation date is: October 24, 2024, and the preservation unit is: China General Microbiological Culture Collection Center, the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0047] The method for biosynthesizing zero-valent nano-selenium provided by the present invention includes the following steps:

[0048] (1) Strain activation, including the preparation and activation process of the medium:

[0049] The medium formula is as follows: peptone: yeast extract powder: sodium chloride: potassium chloride, lactose are prepared according to the following mass ratio: 1:0.5:1:0.05:0.05. When preparing the medium, the above materials are mixed and deionized water is added, and the mass-volume ratio is 1:25, with the unit of g:mL;

[0050] The activation process is as follows: Place the prepared culture medium in an autoclave and sterilize it at 120°C for 20 minutes, then cool it at room temperature; Use an inoculation loop to inoculate strain XZ-1 into the sterilized activated culture medium solution, place the solution in a shaker, and culture it with shaking at 30°C and 120 rpm for 14 hours;

[0051] (2) Strain culture, including the preparation of inoculation medium and the culture process:

[0052] Prepare the inoculation medium: The mass ratio of peptone: yeast extract powder: sodium chloride: potassium chloride: magnesium chloride: calcium chloride is: 5:3:2:0.5:0.3:0.2; When preparing the medium, the mass-volume ratio of the above mixed materials to deionized water is 1:50, with the unit of g:mL;

[0053] The culture process is as follows: Place the prepared culture medium in an autoclave and sterilize it at 120°C for 20 minutes, then cool it and store it at room temperature; Pipette 1 mL of the activated cell suspension and transfer it to the inoculation medium solution, with the inoculation amount being 1%, place the solution in a shaker, and culture it with shaking at 30°C and 120 rpm for 8 - 10 hours until the OD600 reaches 1 - 1.2;

[0054] (3) Preparation of nano-selenium, including the formula of selenium reduction medium and the process of nano-selenium preparation:

[0055] The formula of the selenium reduction medium is: The mass ratio of peptone: yeast extract powder: sodium chloride: potassium chloride: Na2HPO4: KH2PO4: sodium selenite is 15:10:5:0.2:1.44:0.24:0.005;

[0056] The process of nano-selenium preparation is: After the medium is sterilized at 120°C for 20 minutes, add 10 mM sodium lactate filtered through a 0.45 μm filter membrane, shake well and set aside;

[0057] Centrifuge the cell solution obtained in step (2) at 8000 rpm for 10 minutes to collect the bacterial cells, inoculate the bacterial cells into the selenium reduction medium, and culture it with shaking at 30°C and 120 rpm for 8 - 12 hours;

[0058] (4) Separation and purification of biological nano-selenium: For the reaction product obtained after selenium reduction in step (3), centrifuge at 8000 rpm for 25 min to collect the cell precipitate, wash it 2 - 3 times by centrifuging at 10000 rpm for 10 - 20 min with sterilized water. After collecting the precipitate, resuspend it in 10 - 20 mL of sterilized water, and then ultrasonically disrupt the cells on ice with a power of 800 W, with a start-stop interval of 30 s for 30 min to fully lyse the bacterial cells. Centrifuge the lysate at 10000 rpm for 20 min, and after collecting the precipitate, add sterilized water with a mass twice that of the solid precipitate to obtain a biological nano-selenium suspension. Directly freeze the obtained biological nano-selenium suspension in liquid nitrogen for 10 min, and then place it in a freeze dryer for freeze drying. The freeze drying time is 24 - 48 hours to obtain pure biological nano-selenium dry powder;

[0059] (5) The storage condition of the pure biological nano-selenium dry powder is: storage under normal temperature and vacuum.

[0060] The steps of the selenium reduction preparation experiment of the present invention can also be carried out in a fermenter. The medium formula remains unchanged, and the relevant medium volume needs to be changed according to the volume of the fermenter. Preferably, it is 60% of the fermenter volume, and the fermentation parameters are set according to the shaking table culture parameters, with a fermentation time of 12 - 24 h.

[0061] The present invention also provides selenium-rich feed and selenium-rich fertilizer prepared from the nano-selenium matrix fertilizer, wherein the content of biological nano-selenium is 200 - 1000 μg / kg and 5 - 10 g / kg respectively.

[0062] In a specific embodiment of the present invention, the nano-selenium matrix fertilizer is mixed evenly with feed raw materials at a ratio of 200 - 1000 μg / kg to prepare selenium-rich feed. After feeding the feed to livestock and poultry such as laying hens, broilers, and cattle, selenium-rich eggs, selenium-rich chicken meat, etc. that can be reprocessed are obtained.

[0063] In another specific embodiment of the present invention, the nano-selenium matrix fertilizer is formulated into selenium-rich fertilizer at a ratio of 5 - 10 g / kg. The fertilizer is used for the cultivation of vegetables such as tomatoes and cucumbers, for the cultivation of fruits such as apples and kiwifruits, for the application in the cultivation of edible fungi such as Flammulina velutipes, Lentinula edodes, and Auricularia auricula, for the cultivation of food crops such as wheat, rice, and corn, for the cultivation of miscellaneous grains such as soybeans, peanuts, millet, and sweet potatoes, and for tea cultivation to obtain selenium-rich vegetables, selenium-rich fruits, selenium-rich edible fungi, selenium-rich crops, and selenium-rich tea that can be reprocessed. The dosage of nano-selenium per mu in vegetable fields is 1 - 3 g, and the water consumption per mu is 50 L. The dosage of nano-selenium for wheat and rice is 2 - 5 g, and the water consumption per mu is 100 L, which is sprayed in two times.

[0064] Specifically, the relevant preparation and detection are as follows:

[0065] Example 1

[0066] Biological Preparation of Selenium Nanoparticles

[0067] Based on the liquid LB medium, an activation medium, an inoculation medium, and a selenium reduction medium were prepared. The specific operations were as follows: The activation medium for strain XZ-1 had the following formula: Weigh 1 g of peptone, 0.5 g of yeast extract powder, 1 g of sodium chloride, 0.02 g of potassium chloride, and 10 mM of lactose and dissolve them in 100 mL of deionized water. Adjust the pH value to about 7. After autoclaving at 121 °C for 20 min, store it.

[0068] Use an inoculation loop to inoculate strain XZ-1 into the activation medium solution. Place the solution in a shaker and shake-culture it at 30 °C and 120 rpm for 14 h. The inoculation medium formula was: Weigh 5 g of peptone, 3 g of yeast extract powder, 2 g of sodium chloride, 0.5 g of potassium chloride, 0.3 g of magnesium chloride, and 0.2 g of calcium chloride and dissolve them in 500 mL of deionized water. Stir for 5 minutes to form a uniform solution, and adjust the pH value to about 7. After autoclaving at 121 °C for 20 min, store it. Pipette the activated cell suspension and transfer it to the inoculation medium solution with an inoculation amount of 1%. Place the solution in a shaker and shake-culture it at 30 °C and 120 rpm for 8 - 10 h, OD 600 Reach 1.2.

[0069] The selenium reduction medium formula was: 15 g / L of peptone, 10 g / L of yeast extract powder, 5 g / L of sodium chloride, 0.2 g / L of potassium chloride, 1.44 g / L of Na2HPO4, 0.24 g / L of KH2PO4, 5 mM of sodium selenite, and adjust the pH value to about 7. After autoclaving at 121 °C for 20 min, add 10 mM of sodium lactate filtered through a 0.45 μm filter membrane, shake well and set aside.

[0070] Centrifuge the above-obtained cell culture solution at 8000 rpm for 10 min to collect the bacterial cells. Disperse the collected bacterial cells into 500 mL of the selenium reduction medium and shake-culture them at 30 °C and 120 rpm for 8 - 12 h, then centrifuge to collect the elemental selenium nanoparticles.

[0071] Through comparison, the present invention found that the addition of lactose increased the bacterial cell yield by 10 - 15%. The addition of sodium lactate to the selenium reduction medium increased the elemental selenium yield by about 20%.

[0072] Example 2

[0073] Growth Curves of Strain XZ-1 Synthesizing Nano Selenium under Different Concentrations of Sodium Selenite

[0074] The strain XZ-1 was cultured by shaking at 30 °C and 120 rpm using the inoculation medium shown in Example 1, and the inoculation amount was 1%. Before inoculation, sodium selenite solutions with concentrations of 0, 1, 2, 5, 7, and 10 mM were added to the medium respectively. Under different concentrations of sodium selenite, the growth curve of the strain XZ-1 synthesizing nano-selenium is as Figure 1 shown. It can be seen from the figure that the strain XZ-1 grows well in the presence of 0-5 mM sodium selenite; it can still grow in the presence of 10 mM sodium selenite.

[0075] Example 3

[0076] Yield of nano-selenium synthesized by the strain XZ-1 under different sodium selenite concentration treatments

[0077] The strain XZ-1 was cultured using the culture method in Example 2, and the concentrations of sodium selenite in the culture medium were 0, 1, 2, 5, 7, and 10 mM respectively. After culturing at 30 °C for 12 h, the supernatant was collected by centrifugation at 4000 g for 15 min, and the nano-selenium content in the solution was measured. The results are as Figure 2 shown. The strain XZ-1 can almost completely convert sodium selenite into nano-selenium at low concentrations. In the case of 1 mM sodium selenite, 0.93 mM nano-selenium can be generated; in the presence of 2 mM sodium selenite, 1.45 mM nano-selenium is generated; in the presence of 5 mM sodium selenite, 1.68 mM nano-selenium is generated, reaching the highest yield of synthesized nano-selenium.

[0078] Example 4

[0079] Scanning electron microscope (SEM) images and energy-dispersive X-ray spectroscopy (EDX) images of the strain XZ-1 cells and synthesized nano-selenium

[0080] Take the mixed bacterial liquid of the strain XZ-1 treated with 5 mM sodium selenite in Example 1 and centrifuge it at 8000 r / min for 25 min to collect the synthesized nano-selenium particles and bacterial cells. Observe the microscopic morphology and surface elements of nano-selenium and the strain XZ-1 cells through SEM images and EDX images. The results are as Figure 3 shown. The SEM images and EDX results show that a large number of nano-selenium particles are generated in the cell culture environment.

[0081] Example 5: Transmission electron microscope (TEM) images of selenium nanoparticles synthesized by the strain XZ-1

[0082] Select the elemental selenium nanoparticles obtained by centrifugation from the strain XZ-1 described in Example 1 treated with 5 mM sodium selenite for transmission electron microscopy characterization. The results are as Figure 4 shown. The selenium nanoparticles synthesized by the strain XZ-1 are uniformly sized circular particles with a particle size of about 80 nm;

[0083] Example 6

[0084] Near-edge X-ray absorption fine structure spectrum (XANES) of selenium nanoparticles synthesized by strain XZ-1

[0085] XANES characterization and analysis were carried out on selenium standards with different valence states and nano-selenium generated by strain XZ-1 through synchrotron radiation experiments. The results are as Figure 5 shown. By comparison, it can be known that the valence state of nano-selenium generated by strain XZ-1 cells is zero valence, which proves that the method of the present invention synthesizes elemental nano-selenium.

[0086] Example 7

[0087] Comparison chart of the effect of selenium fertilizer prepared from selenium nanoparticles synthesized by strain XZ-1 on the growth of plants (Epipremnum aureum)

[0088] The nano-selenium matrix fertilizer prepared from the generated selenium nanoparticles was used as a foliar selenium fertilizer. After spraying on plants (Epipremnum aureum) for one week, the growth of Epipremnum aureum was observed. The results are as Figure 6 shown. The left 1 (CK) is the blank control, that is, spraying the matrix fertilizer without zero-valent nano-selenium. The growth of Epipremnum aureum after spraying nano-selenium matrix fertilizers with different dilution multiples is shown from the left 2 to the left 4. By comparison, it can be seen that the Epipremnum aureum after spraying nano-selenium matrix fertilizer has more leaves than that after spraying the matrix fertilizer without zero-valent nano-selenium. In addition, the nano-selenium matrix fertilizer diluted 100 times has a greater promoting effect on the growth of Epipremnum aureum, and this formula can be referred to in the actual use process. This example proves that the nano-selenium matrix fertilizer based on biosynthesized selenium nanoparticles has a promoting effect on the growth of plants (Epipremnum aureum).

[0089] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that: without departing from the spirit and scope of the present invention and the appended claims, various substitutions, changes and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A method for synthesizing zero-valent nano-selenium using Pantoea, characterized in that: The method uses Pantoea to biosynthesize nano-selenium: by adding selenite to the strain culture medium, cultivating Pantoea, and separating and purifying the synthesized product to obtain zero-valent nano-selenium; The strain name of Pantoea is XZ-1, and the classification name is: Pantoea sp., the preservation number is: CGMCC No. 32330, the preservation date is: October 24, 2024, the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The method for biosynthesizing zero-valent nano-selenium using Pantoea according to claim 1, characterized in that: The inorganic salts include sodium salt and potassium salt of selenious acid.

3. The method for biosynthesizing zero-valent nano-selenium using Pantoea according to claim 1, characterized in that: The particle size of the zero-valent nano-selenium is 80-100 nm.

4. The method for biosynthesizing zero-valent nano-selenium using Pantoea according to claim 1, characterized in that: The concentration of the sodium salt and potassium salt of selenious acid is 0.1-100mM, preferably 5mM.

5. The method for biosynthesizing zero-valent nano-selenium using Pantoea according to claim 1, characterized in that: The obtained synthetic products include cell lysate, cell suspension, and bacterial cell sediment obtained by centrifuging the culture fluid.

6. The method for biosynthesizing zero-valent nano-selenium using Pantoea according to claim 1, characterized in that: The steps include: (1) Strain activation, including culture medium preparation and activation process: The culture medium formula is as follows: peptone: yeast extract powder: sodium chloride: potassium chloride, lactose are prepared according to the following mass ratio: 1:0.5:1:0.05:0.

05. When preparing the culture medium, the above materials are mixed and deionized water is added. The mass volume ratio is 1:25, and the unit is g:mL; The activation process is as follows: the prepared culture medium is placed in an autoclave, sterilized at 120°C for 20 minutes, and then cooled at room temperature; the strain XZ-1 is inoculated into the sterilized activation culture medium solution using an inoculation loop, and the solution is placed in a shaker, and cultured at 30°C and 120 rpm for 14 hours; (2) Strain culture, including preparation of inoculation medium and culture process: Prepare inoculation medium: the mass ratio of peptone: yeast extract powder: sodium chloride: potassium chloride: magnesium chloride: calcium chloride is 5:3:2:0.5:0.3:0.2; when preparing the medium, the mass volume ratio of the above mixed materials to deionized water is 1:50, the unit is g:mL; The culture process is as follows: the prepared culture medium is placed in an autoclave, sterilized at 120°C for 20 minutes, and then cooled and stored at room temperature; 1 mL of activated cell culture solution is transferred to the inoculation culture medium solution, with the inoculation volume being 1%, and the solution is placed in a shaker, shaken and cultured at 30°C and 120 rpm for 8-10 hours, until the OD600 reaches 1-1.2; (3) Preparation of nano-selenium, including the formula of selenium reduction medium and the process of nano-selenium preparation: The formula of selenium reduction medium is: the mass ratio of peptone: yeast extract powder: sodium chloride: potassium chloride: Na2HPO4: KH2PO4: sodium selenite is 15:10:5:0.2:1.44:0.24:0.005; The preparation process of nano-selenium is as follows: after the culture medium is sterilized at 120°C for 20 minutes, 10 mM sodium lactate filtered through a 0.45 um filter membrane is added, and the mixture is shaken and set aside for use; The cell solution obtained in step (2) was centrifuged at 8000 rpm for 10 min to collect the bacterial cells, and the bacterial cells were inoculated into a selenium-reducing medium and cultured at 30° C. and 120 rpm for 8-12 h; (4) Separation and purification of biological nano-selenium: The reaction product obtained after selenium reduction in step (3) is centrifuged at 8000 rpm for 25 min to collect the cell precipitate, which is washed 2-3 times with sterile water at 10000 rpm for 10-20 min, and the precipitate is collected and resuspended in 10-20 mL of sterile water, and then the cells are ultrasonically disrupted on ice at a power of 800 W, with a start-stop interval of 30 s, and the disruption is performed for 30 min to fully lyse the bacteria; the lysate is centrifuged at 10000 rpm for 20 min, and the precipitate is collected and sterile water of 2 times the mass of the solid precipitate is added to obtain a biological nano-selenium suspension; the obtained biological nano-selenium suspension is directly frozen with liquid nitrogen for 10 min, and placed in a freeze dryer for freeze drying, with the cold drying time being 24 to 48 hours to obtain pure biological nano-selenium dry powder; (5) The storage conditions of pure biological nano-selenium dry powder are: vacuum storage at room temperature.

7. A nano-selenium matrix fertilizer comprising zero-valent nano-selenium prepared by the method according to any one of claims 1 to 5.

8. The nano-selenium matrix fertilizer according to claim 7, characterized in that: The method comprises compounding zero-valent nano-selenium with a dispersant and a protective agent to form a nano-selenium matrix fertilizer.

9. The nano-selenium matrix fertilizer according to claim 7, characterized in that: The dispersant is: sodium carboxymethyl cellulose, araba gum powder, 2-naphthalenesulfonic acid, EDTA disodium salt or a combination of any two or three thereof; when the dispersant is compounded with zero-valent nano-selenium, the added mass thereof is 0.5‰ to 1‰ of the mass of the zero-valent nano-selenium; The protective agent is: ferrous chloride, nano zero-valent iron, nano zinc oxide, manganese sulfate, zinc sulfate or any combination of two or three thereof; when the protective agent is compounded with zero-valent nano selenium, the added mass thereof is 5‰ to 10‰ of the mass of zero-valent nano selenium.

10. A method for using the nano-selenium matrix fertilizer according to claim 7, characterized in that: The steps include: Spray the nano-selenium matrix fertilizer as foliar selenium fertilizer. The dosage for vegetable fields is 1-3g per mu, and the water consumption per mu is 50L. The dosage for wheat and rice is 2-5g, and the water consumption per mu is 100L. Spray it twice. Alternatively, the nano-selenium matrix fertilizer can be used as a feed matrix: the dosage is 0.2 to 1 g per ton.

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