A highly efficient halomonas bacterium for synthesizing nano-selenium and its preparation method and application

Abstract

This invention discloses a highly efficient halomonas strain for synthesizing selenium nanoparticles, along with its preparation method and applications, belonging to the field of biotechnology. The halomonas strain CCTCC NO: M 20242266 of this invention exhibits strong tolerance to high concentrations of selenite and can efficiently convert selenite into selenium nanoparticles. The required culture conditions are simple, low-cost, and easy to operate. The selenium nanoparticles produced using this strain are stable and have high bioactivity. The halomonas strain CCTCC NO: M 20242266 or its microbial agents, or the selenium nanoparticles produced using this strain, have broad development prospects in functional agriculture, including crop and animal husbandry, functional food processing, health food processing, and pharmaceutical product processing.

Description

Technical Field

[0001] This invention relates to a highly efficient halomonas bacterium for synthesizing nano-selenium, as well as the method for preparing nano-selenium and its applications, belonging to the field of biotechnology. Background Technology

[0002] Selenium is an essential trace element for the human body and one of the 15 dietary nutrients that must be supplemented daily. Studies have shown that severe selenium deficiency can lead to more than 40 diseases, such as cancer and cardiovascular diseases. Consistent daily selenium supplementation can effectively remove excess free radicals from the body, improve immunity, and thus prevent various diseases and delay aging. However, excessive selenium intake can lead to selenium poisoning. In some parts of my country, selenium levels in water bodies and soil exceed standards, primarily in the form of highly toxic selenite. This poses a potential threat to residents and aquatic life living around polluted water bodies. How to remove or reduce the toxic effects of selenium in these water bodies has become an urgent problem to solve.

[0003] There are two main forms of selenium supplementation: inorganic selenium and organic selenium. However, compared with inorganic and organic selenium, nano-selenium has lower toxicity and higher bioavailability, making it a current research focus. There are generally three methods for obtaining nano-selenium: physical, chemical, and biological methods. Physical methods mainly utilize techniques such as laser ablation, microwave radiation, hydrothermal treatment, physical evaporation, and electrochemical methods. These methods have drawbacks such as high energy consumption, high cost, long processing time, hazardous synthesis environment, and difficulty in controlling particle size distribution. Chemical methods typically use selenium sources such as sodium selenite, selenite acid, and selenium dioxide as oxidants and chemical reagents such as hydrazine as reducing agents. The oxidant and reducing agent combine to undergo a redox reaction. The chemical substances added during the preparation process generate a large amount of pollutants, and the stability, biocompatibility, and bioactivity of the synthesized nano-selenium are greatly reduced. Therefore, more and more researchers are beginning to focus on biological methods, especially microbial methods, to synthesize nano-selenium. Microbial methods reduce inorganic selenium in the environment to elemental nano-selenium through their own metabolism. The nano-selenium produced by this method has small particle size, uniform and stable structure, high temperature resistance, safety, and environmental friendliness. It has become the mainstream method for preparing nano-selenium and has broad application prospects in environmental remediation, agriculture, and functional foods.

[0004] Numerous studies have demonstrated that various microorganisms can convert inorganic selenium into nano-selenium, such as Bacillus sp., Lactiplantibacillus plantarum, Pediococcus pentosaceus, and Thiocapsa roseopersicina. However, most of these microorganisms exhibit selenium tolerance below 500 mM and low reduction efficiency, hindering the industrial production of bio-nano-selenium. For instance, Chinese invention patent application CN110964674A discloses the synthesis of nano-selenium using Thiocapsa roseopersicina, but its selenium tolerance is only 25 g / L (approximately 316 mM), and the reduction efficiency is unknown. Chinese invention patent application CN118703359A discloses a strain of Pediococcus pentosaceus that can only tolerate 5 mmol / L selenium for 60 hours. The reduction rate of Se(Ⅳ) reached 61.8%, and the reduction rate of 1 mmol / L Se(Ⅳ) reached 95.24% after 36 hours. The selenium tolerance is unknown. Chinese invention patent document CN112746086B discloses a *Providencia rettgeri*, whose highest selenium tolerance is only 445 mM. Chinese invention patent application document CN118440865A discloses an *Oceanobacillus profundus*, which has a high selenium tolerance, but its ability to synthesize nano-selenium is only 2-8 mM.

[0005] Therefore, there is an urgent need to find a strain with higher selenium tolerance and stronger ability to reduce inorganic selenium in order to improve the industrial production efficiency of nano-selenium. Summary of the Invention

[0006] To address the aforementioned issues, this invention has screened out a Halomonas SF2000 strain capable of efficiently synthesizing nano-selenium. This strain can grow in 1200 mM selenite, achieving 100% reduction of 2 mM nano-selenium within 24 hours, and reducing 5 mM and 10 mM selenite by over 90% within 60 hours, as well as approximately 50% of 20 mM selenite. This represents the highest level reported to date. Higher synthesis capacity means fewer production cycles, requiring less raw material, making it more convenient, efficient, environmentally friendly, and economical, thus possessing significant market application value.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] This invention provides a strain of Halomonas sp. SF2000, classified and named Halomonas sp. SF2000. The Halomonas sp. was deposited at the China Center for Type Culture Collection on October 21, 2024, with accession number CCTCC NO: M 20242266.

[0009] The screening process for Halomonas sp. SF2000 is as follows:

[0010] Materials were collected from high-selenium soil, rhizosphere soil of high-selenium plants, salt lake sediment, and selenium-enriched organic fertilizer produced in different years (provided by Hefei Zhongke Qiao Biotechnology Co., Ltd.). 15 grams of each collected material were added to 200 mL of a 100 mM sodium selenite solution and incubated at 37°C with stirring at 150 rpm for 2 days. After 2 days, 100 μL of the supernatant was evenly spread onto LB agar plates containing 500 mM sodium selenite. After incubation at 37°C for 2 days, red colonies were observed, indicating that sodium selenite had been reduced to nano-selenium. Isolates with Se(IV) reduction activity were selected based on the red nano-selenium secretion. Single colonies were selected and passaged 3-4 times on LB agar plates before being mixed with LB liquid medium containing 30% glycerol and stored at -80°C. The strain was identified as *Halomonas* sp. by 16S rRNA sequencing and named SF2000.

[0011] In one embodiment of the present invention, the Halomonas sp. CCTCC NO: M20242266 has the following characteristics:

[0012] (1) Morphological characteristics: Gram-negative bacteria, non-spore-forming, rod-shaped, flagellated, cell size (0.2-0.4)×(1-1.5)μm;

[0013] (2) Colony characteristics: The colonies are yellow, raised, with intact edges, smooth surface, and opaque;

[0014] (3) Growth characteristics: aerobic, chemoheterotrophic, growth temperature is 4-40℃;

[0015] (4) Strong selenium tolerance: It can grow in 1200mM selenite;

[0016] (5) Strong ability to produce nano selenium: It has a high selenium reducing power, and can reduce 2mM selenite to nano selenium by 100% within 24 hours, and reduce 5mM and 10mM selenite by more than 90% within 60 hours, and reduce 20mM selenite by about 50%; and the purity of the purified nano selenium produced can reach up to 45%.

[0017] The present invention also provides a microbial inoculant containing the Halomonas sp. of the present invention, CCTCC NO: M 20242266.

[0018] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCC NO: M 20242266 as the main microorganism.

[0019] In one embodiment of the present invention, the number of CCTCC NO: M 20242266 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0020] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of CCTCC NO: M 20242266 and then expanding it.

[0021] In one embodiment of the present invention, the microbial agent contains live cells of the CCTCC NO: M20242266 strain of the present invention, freeze-dried dried cells of the CCTCC NO: M 20242266 strain of the present invention, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, liquid agent of the CCTCC NO: M 20242266 strain of the present invention, solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention existing in any other form.

[0022] In one embodiment of the present invention, the method for preparing the microbial agent is as follows: adding the culture medium of Halomonas SF2000 to a culture medium containing sodium selenite and incubating to obtain the agent.

[0023] In one embodiment of the present invention, the culture medium containing sodium selenite is a culture medium containing 2 mM sodium selenite.

[0024] The present invention also provides a method for the biological preparation of nano-selenium, wherein the method comprises: using the Halomonas sp. CCTCC NO: M 20242266 of the present invention as biological material, using selenium-containing inorganic salts as the source of selenium, inoculating the seed culture of CCTCC NO: M20242266 into a culture medium containing selenium salts for cultivation, purifying the culture medium, and allowing CCTCC NO: M 20242266 to perform biotransformation on inorganic selenium to prepare nano-selenium.

[0025] In one embodiment of the present invention, the method specifically includes the following steps:

[0026] In one embodiment of the present invention, the method for preparing the seed culture of CCTCC NO: M 20242266 includes the following steps: taking out the preserved SF2000 glycerol tube of Halomonas, thawing it at room temperature and inoculating it onto a plate, picking out a single colony after 24 hours and inoculating it into a liquid culture medium to obtain the seed culture of CCTCC NO: M 20242266.

[0027] In one embodiment of the present invention, the bacterial concentration of the seed solution is (1-10)×10⁻⁶. 9 CFU / mL; the inoculation amount of seed solution is 1% to 3%; preferably, the inoculation amount is 2%.

[0028] In one embodiment of the present invention, the culture conditions are 30-37°C and 150-250 rpm.

[0029] In one embodiment of the present invention, the selenium-containing inorganic salt is a selenite.

[0030] In one embodiment of the present invention, the concentration of selenium-containing inorganic salt in the culture medium is 2-20 mM.

[0031] In one embodiment of the present invention, the culture time is 24 to 60 hours.

[0032] In one embodiment of the present invention, the purification method is as follows: centrifuge the culture medium, collect the precipitate, wash and resuspend it with water; rupture the cells, centrifuge, wash and resuspend them with water; add sucrose solution, centrifuge, wash and resuspend them with water to obtain particles; freeze-dry the extracted particles to obtain nano-selenium freeze-dried powder.

[0033] In one embodiment of the present invention, the centrifugation conditions are 8000-10000 r / min, 4°C, and the centrifugation time is 10-15 min.

[0034] In one embodiment of the invention, cell rupture is achieved by using ultrasound to destroy cells.

[0035] In one embodiment of the present invention, the concentration of the sucrose solution is 80% (w / v).

[0036] This invention also provides the application of Halomonas sp. CCTCC NO: M 20242266 or its microbial agents or nano-selenium produced using CCTCC NO: M 20242266 in agricultural production and processing.

[0037] In one embodiment of the present invention, the agricultural production and processing includes, but is not limited to, the preparation of selenium-enriched fertilizers, selenium-enriched feeds, selenium-enriched health foods, and nano-selenium drugs.

[0038] In one embodiment of the present invention, the microbial agent contains Halomonas asp. CCTCC NO: M 20242266 of the present invention.

[0039] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCC NO: M 20242266 as the main microorganism.

[0040] In one embodiment of the present invention, the number of CCTCC NO: M 20242266 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0041] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of CCTCC NO: M 20242266 and then expanding it.

[0042] In one embodiment of the present invention, the microbial agent contains live cells of the CCTCC NO: M20242266 strain of the present invention, freeze-dried dried cells of the CCTCC NO: M 20242266 strain of the present invention, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, liquid agent of the CCTCC NO: M 20242266 strain of the present invention, solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention existing in any other form.

[0043] In one embodiment of the present invention, the method for preparing the microbial agent is as follows: adding the culture medium of Halomonas SF2000 to a culture medium containing sodium selenite and incubating to obtain the agent.

[0044] In one embodiment of the present invention, the culture medium containing sodium selenite is a culture medium containing 2 mM sodium selenite.

[0045] This invention also provides the application of Halomonas sp. CCTCC NO: M 20242266 or its microbial inoculants or nano-selenium produced using CCTCC NO: M 20242266 in agricultural production.

[0046] In one embodiment of the invention, the application includes promoting the growth of field crops, vegetables, or fruits, increasing their yield, or increasing their selenium content.

[0047] In one embodiment of the present invention, the field crop is selected from rice, wheat, corn, millet, soybean, etc.

[0048] In one embodiment of the present invention, the vegetables are selected from sweet potatoes, garlic, cabbage, lettuce, kale, okra, celery, zucchini, pumpkin, tomato, cucumber, eggplant, pepper, etc.

[0049] In one embodiment of the present invention, the fruit is selected from strawberries, blueberries, citrus fruits, kiwifruit, grapes, apples, peaches, pears, hawthorns, dates, etc.

[0050] In one embodiment of the present invention, the application is to spray a solution of Halomonas sp. CCTCC NO: M20242266 or its microbial agent or nano-selenium produced using CCTCC NO: M 20242266 onto the leaves of plants.

[0051] In one embodiment of the present invention, the microbial agent contains Halomonas asp. CCTCC NO: M 20242266 of the present invention.

[0052] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCC NO: M 20242266 as the main microorganism.

[0053] In one embodiment of the present invention, the number of CCTCC NO: M 20242266 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0054] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of CCTCC NO: M 20242266 and then expanding it.

[0055] In one embodiment of the present invention, the microbial agent contains live cells of the CCTCC NO: M20242266 strain of the present invention, freeze-dried dried cells of the CCTCC NO: M 20242266 strain of the present invention, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, liquid agent of the CCTCC NO: M 20242266 strain of the present invention, solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention existing in any other form.

[0056] In one embodiment of the present invention, the method for preparing the microbial agent is as follows: adding the culture medium of Halomonas SF2000 to a culture medium containing sodium selenite and incubating to obtain the agent.

[0057] In one embodiment of the present invention, the culture medium containing sodium selenite is a culture medium containing 2 mM sodium selenite.

[0058] The present invention also provides a nano-selenium fertilizer, wherein the nano-selenium fertilizer contains Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or nano-selenium produced using CCTCC NO: M 20242266.

[0059] In one embodiment of the present invention, the nano-selenium fertilizer is a mixture of nano-selenium suspension and purified water at a volume ratio of 10% to 90%.

[0060] In one embodiment of the present invention, the nano-selenium suspension refers to the nano-selenium lyophilized powder prepared by the above method using CCTCC NO: M20242266, dissolved in water to prepare a nano-selenium suspension with a concentration of 5-100 mg Se / L.

[0061] In one embodiment of the present invention, the microbial agent contains Halomonas asp. CCTCC NO: M 20242266 of the present invention.

[0062] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCC NO: M 20242266 as the main microorganism.

[0063] In one embodiment of the present invention, the number of CCTCC NO: M 20242266 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0064] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of CCTCC NO: M 20242266 and then expanding it.

[0065] In one embodiment of the present invention, the microbial agent contains live cells of the CCTCC NO: M20242266 strain of the present invention, freeze-dried dried cells of the CCTCC NO: M 20242266 strain of the present invention, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, liquid agent of the CCTCC NO: M 20242266 strain of the present invention, solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention existing in any other form.

[0066] In one embodiment of the present invention, the method for preparing the microbial agent is as follows: adding the culture medium of Halomonas SF2000 to a culture medium containing sodium selenite and incubating to obtain the agent.

[0067] In one embodiment of the present invention, the culture medium containing sodium selenite is a culture medium containing 2 mM sodium selenite.

[0068] This invention also provides the application of Halomonas sp. CCTCC NO: M 20242266 or its microbial agents or nano-selenium produced using CCTCC NO: M 20242266 in aquaculture production.

[0069] In one embodiment of the present invention, the aquaculture production includes, but is not limited to, promoting the growth of farmed animals such as livestock or poultry or increasing the selenium content in their meat.

[0070] In one embodiment of the present invention, the poultry is selected from chickens, ducks, geese, etc.

[0071] In one embodiment of the present invention, the livestock is selected from pigs, cattle, sheep, etc.

[0072] In one embodiment of the present invention, the application is to feed farmed animals with feed containing Halomonas sp. CCTCC NO: M 20242266 or its microbial agent, or nano-selenium produced using CCTCC NO: M 20242266.

[0073] In one embodiment of the present invention, the microbial agent contains Halomonas asp. CCTCC NO: M 20242266 of the present invention.

[0074] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCC NO: M 20242266 as the main microorganism.

[0075] In one embodiment of the present invention, the number of CCTCC NO: M 20242266 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0076] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of CCTCC NO: M 20242266 and then expanding it.

[0077] In one embodiment of the present invention, the microbial agent contains live cells of the CCTCC NO: M20242266 strain of the present invention, freeze-dried dried cells of the CCTCC NO: M 20242266 strain of the present invention, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, liquid agent of the CCTCC NO: M 20242266 strain of the present invention, solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention existing in any other form.

[0078] In one embodiment of the present invention, the method for preparing the microbial agent is as follows: adding the culture medium of Halomonas SF2000 to a culture medium containing sodium selenite and incubating to obtain the agent.

[0079] In one embodiment of the present invention, the culture medium containing sodium selenite is a culture medium containing 2 mM sodium selenite.

[0080] The present invention also provides a nano-selenium feed or nano-selenium feed additive, wherein the nano-selenium feed or nano-selenium feed additive contains Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or nano-selenium produced using CCTCC NO: M 20242266.

[0081] In one embodiment of the present invention, the nano-selenium feed is made by mixing nano-selenium dry powder and feed ingredients in a mass ratio of 1:(10-100).

[0082] In one embodiment of the present invention, the nano selenium dry powder refers to the nano selenium freeze-dried powder prepared by the above method using CCTCC NO: M20242266 and mixed with corn flour to form nano selenium dry powder, with a nano selenium content of 1-10 mgSe / kg.

[0083] In one embodiment of the present invention, the microbial agent contains Halomonas asp. CCTCC NO: M 20242266 of the present invention.

[0084] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCC NO: M 20242266 as the main microorganism.

[0085] In one embodiment of the present invention, the number of CCTCC NO: M 20242266 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0086] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of CCTCC NO: M 20242266 and then expanding it.

[0087] In one embodiment of the present invention, the microbial agent contains live cells of the CCTCC NO: M20242266 strain of the present invention, freeze-dried dried cells of the CCTCC NO: M 20242266 strain of the present invention, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, liquid agent of the CCTCC NO: M 20242266 strain of the present invention, solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention existing in any other form.

[0088] In one embodiment of the present invention, the method for preparing the microbial agent is as follows: adding the culture medium of Halomonas SF2000 to a culture medium containing sodium selenite and incubating to obtain the agent.

[0089] In one embodiment of the present invention, the culture medium containing sodium selenite is a culture medium containing 2 mM sodium selenite.

[0090] The present invention also provides the application of Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or nano-selenium produced using CCTCC NO: M 20242266 in the preparation of selenium-enriched products.

[0091] In one embodiment of the present invention, the selenium-enriched product includes, but is not limited to, drugs, functional foods, health products, feed, and feed additives.

[0092] In one embodiment of the present invention, the microbial agent contains Halomonas asp. CCTCC NO: M 20242266 of the present invention.

[0093] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCC NO: M 20242266 as the main microorganism.

[0094] In one embodiment of the present invention, the number of CCTCC NO: M 20242266 cells in the bacterial agent is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0095] In one embodiment of the present invention, the microbial agent is obtained by preparing a seed culture of CCTCC NO: M 20242266 and then expanding it.

[0096] In one embodiment of the present invention, the microbial agent contains live cells of the CCTCC NO: M20242266 strain of the present invention, freeze-dried dried cells of the CCTCC NO: M 20242266 strain of the present invention, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, liquid agent of the CCTCC NO: M 20242266 strain of the present invention, solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention existing in any other form.

[0097] In one embodiment of the present invention, the method for preparing the microbial agent is as follows: adding the culture medium of Halomonas SF2000 to a culture medium containing sodium selenite and incubating to obtain the agent.

[0098] In one embodiment of the present invention, the culture medium containing sodium selenite is a culture medium containing 2 mM sodium selenite.

[0099] Preservation of biological materials:

[0100] Halomonas sp. SF2000, classified as Halomonas sp. SF2000, was deposited on October 21, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242266, located at Wuhan University, Wuhan, China.

[0101] Beneficial effects:

[0102] The *Haloxylon ammodendron* SF2000 strain of this invention exhibits strong tolerance to high concentrations of selenite and can efficiently convert selenite into nano-selenium. The required culture conditions are simple, low-cost, and easy to operate. The recovered nano-selenium particles are stable and have high biological activity, offering significant advantages in functional agriculture, including crop and animal husbandry, functional food processing, health food processing, and pharmaceutical product processing.

[0103] 1) The Halomonas SF2000 of the present invention has extremely high selenium tolerance and can grow in 1200mM selenite;

[0104] 2) The Halomonas SF2000 of the present invention has extremely strong inorganic selenium reducing power. It can reduce 2mM nano selenium by 100% within 24 hours, and reduce 5mM and 10mM selenite by more than 90% within 60 hours, and reduce 20mM selenite by about 50%, which is the highest level reported to date.

[0105] 3) The purified nano-selenium produced by the Halomonas SF2000 of this invention has a purity of up to 45% and exhibits good antioxidant activity;

[0106] 4) The Halomonas SF2000 of the present invention or its microbial agent, or the nano-selenium produced by Halomonas SF2000 of the present invention, has a wide range of applications. It can be widely used in planting and breeding industries, and can effectively increase the selenium content of the target organisms, synergistically produce safe, green, efficient and environmentally friendly selenium-enriched plants and selenium-enriched livestock and poultry, and promote the improvement of product quality. Attached Figure Description

[0107] Figure 1 Phylogenetic tree of Halomonas sp. SF2000;

[0108] Figure 2 Figure 1 shows the growth of Halomonas sp. SF2000 on plates with different sodium selenite concentrations.

[0109] Figure 3 The image shows the EDS energy dispersive spectroscopy (EDS) spectrum of purified selenium nanoparticles.

[0110] Figure 4Images of SF2000 converted selenium nanoparticles obtained by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0111] Figure 5 This is a nanoparticle size diagram of nano-selenium;

[0112] Figure 6 The image shows the Fourier transform infrared (FT-IR) spectrum of nano-selenium.

[0113] Figure 7 X-ray diffraction (XRD) pattern of nano-selenium;

[0114] Figure 8 A graph showing the DPPH free radical scavenging ability of synthesized selenium nanoparticles;

[0115] Figure 9 A graph showing the ability of synthesized selenium nanoparticles to scavenge ABTS cation free radicals. Detailed Implementation

[0116] To further understand the present invention, the present invention will be described below with reference to embodiments. These descriptions are only for further explaining the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0117] The culture media involved in the following examples are as follows:

[0118] LB agar plate: 10g peptone, 5g yeast extract, 10g sodium chloride, 20g agar powder, dissolved in 1L deionized water.

[0119] LB liquid medium: 10g peptone, 5g yeast extract, and 10g sodium chloride dissolved in 1L of deionized water.

[0120] The measurement methods involved in the following examples are as follows:

[0121] 1. The content of selenium (IV) was determined by the ascorbic acid reduction method:

[0122] Standard curve plotting: Using Na2SeO3 as a standard, Na2SeO3 solutions with concentrations of 0, 10, 20, 50, 100, 200, and 250 mg / L were prepared. 1 mL of each Na2SeO3 solution was thoroughly mixed with 0.5 mL of 4 mol / L hydrochloric acid and 1 mL of 1 mol / L ascorbic acid, and allowed to stand at room temperature for 10 min. The absorbance was measured at 500 nm using UV-vis, and a standard curve was plotted.

[0123] Sample measurement: Take 1 mL of sample and mix thoroughly with 0.5 mL of 4 mol / L hydrochloric acid and 1 mL of 1 mol / L ascorbic acid. Let stand for 10 min and measure the absorbance at a wavelength of 500 nm. Calculate the selenium (IV) content in the sample according to the selenium (IV) standard curve.

[0124] 2. Spectrophotometric determination of nano-selenium content:

[0125] At a wavelength of 490 nm, the reduction of 1–10 μmol selenite solution to 25 μmol NH₂OH·HCl to produce red Se was measured. 0 To determine the intensity, a standard curve is established.

[0126] The sample was centrifuged at 8000×g for 20 min to separate cells, and the absorbance of the supernatant (red solution) was measured spectrophotometrically at 490 nm. Se was calculated based on the standard curve. 0 concentration.

[0127] Example 1: Isolation and Identification of Halomonas sp. SF2000

[0128] 1. Strain screening and isolation:

[0129] Materials were collected from high-selenium soil, rhizosphere soil of high-selenium plants, bottom sediment of salt lake, and selenium-enriched organic fertilizer produced in different years (provided by Hefei Zhongke Qiao Biotechnology Co., Ltd.). 15 grams of each material collected from the environment were added to 200 mL of 100 mM sodium selenite solution and incubated at 37°C with stirring at 150 rpm for 2 days. After 2 days, 100 μL of the supernatant was evenly spread onto LB agar plates containing 500 mM sodium selenite. After incubation at 37°C for 2 days, red colonies were observed, indicating that sodium selenite had been reduced to nano-selenium. Based on the red nano-selenium secretion, isolates with Se(IV) reduction activity were selected. Single colonies were selected and passaged 3-4 times on LB agar plates. The colonies were then mixed with LB liquid medium containing 30% glycerol and stored at -80°C for further experiments.

[0130] 2. Identification of the strain:

[0131] 16S rRNA amplification and sequencing were performed on the bacterial strain. Bacterial genomic DNA was extracted using a kit, and PCR products were sent to Shanghai Sangon Biotech for sequencing using universal primers 27F (SEQ ID NO.1: 5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (SEQ ID NO.2: 5′-GGTTACCTTGTTACGACTT-3′). The sequencing results were used to find the most similar reference sequence for the isolate using the BLAST search method. The BLAST search program (https: / / www.ncbi.nlm.nih.gov) was used to search for significantly similar nucleotide sequences, and a phylogenetic tree was constructed using the neighborhood linking method. Figure 1 ).

[0132] BLAST comparison analysis results showed that the strain is Halomonas sp. SF2000, and was deposited at the China Center for Type Culture Collection on October 21, 2024, with accession number CCTCC NO: M20242266, and deposit address is Wuhan University, Wuhan, China.

[0133] Example 2: Determination of the selenium tolerance and reducing power of the strain

[0134] Preparation of *Haloxylon ammodendron* culture medium: *Haloxylon ammodendron* SF2000 glycerol tubes stored at -80℃ were removed from the freezer, thawed at room temperature, and inoculated onto LB agar plates. After 24 hours, single colonies were picked and inoculated into LB liquid medium and cultured for 24 hours to obtain *Haloxylon ammodendron* SF2000 culture medium with an OD600 value of approximately 1.0 (bacterial concentration approximately 2 × 10⁻⁶). 9 CFU / mL);

[0135] 1) Selenium tolerance test of the strain

[0136] The SF2000 culture medium of Halomonas bacteria, which had been cultured for 24 hours, was inoculated at a rate of 2% (v / v) onto LB agar plates containing 600mM, 800mM, 1000mM, and 1200mM sodium selenite, respectively. The plates were incubated at 37°C for 48 hours, and the growth of the bacteria on the plates was observed. Plates inoculated with only Halomonas SF2000 without sodium selenite were used as controls.

[0137] The results clearly show that *Haloxylon ammodendron* SF2000 grew well on 600mM, 800mM, and 1000mM sodium selenite plates, and the reduction of sodium selenite to red nano-selenium was visible; although it could also grow on 1200mM sodium selenite plates, its growth was inhibited. Figure 2 ).

[0138] 2) Determination of the reducing power of the strain

[0139] The above-mentioned SF2000 culture medium of Halomonas bacteria cultured for 24 hours was added to 200 mL of LB liquid medium containing 2 mM, 5 mM, 10 mM and 20 mM sodium selenite, respectively, at an addition rate of 2% (v / v), and incubated at 37°C with stirring at 150 rpm.

[0140] Every 6 hours, 10 mL of culture was collected from the culture medium to determine the bacterial density. The culture was centrifuged at 10,000 g for 10 minutes to separate the supernatant and precipitate. The content of selenium (IV) in the supernatant was determined by ascorbic acid reduction. Using the method described in "2. Spectrophotometric determination of nano selenium content" above, the content of nano selenium in the precipitate was determined by spectrophotometry. The reduction efficiency of sodium selenite in each group over time is shown in Table 1.

[0141] The conversion efficiency is calculated as follows: (nano-selenium content in the precipitate / total selenium content of added selenite) × 100%.

[0142] Table 1. Transformation efficiency of strains to different concentrations of selenite over time.

[0143]

[0144] The results showed that *Haloxymonas SF2000* has excellent reducing ability: it can completely convert 2 mM sodium selenite into nano-selenium within 24 hours, 5 mM sodium selenite into nano-selenium within 36 hours, and 10 mM sodium selenite into almost complete nano-selenium within 60 hours (conversion rate of 97.8%). Furthermore, it can convert approximately 50% of 20 mM sodium selenite within 60 hours, which is the highest level reported to date. This is consistent with existing technologies (Bioprocessing of seleno-oxyanions and tellurite in a novel *Bacillus sp. strain* STG-83: solution to removal of toxic oxyanions in presence of nitrate. *J HazardMater*. doi:10.1016 / j.jhazmat.2008.09.065. *Epub* 2008 Sep). Compared to Bacillus STG-83 (which can only reduce 1 mM sodium selenite in 96 h) in 26.PMID:18977594., the reducing ability of Halomonas SF2000 is much higher.

[0145] Example 3: Method for purifying bio-nano selenium synthesized by bacterial strains

[0146] The purification method for biological nano-selenium is as follows:

[0147] The above-mentioned Halomonas SF2000 culture medium (bacterial concentration approximately 2×10⁻⁶) cultured for 24 hours in Example 2 was used. 9 Add CFU / mL to 200 mL of LB liquid medium containing 2 mM sodium selenite at a dosage of 2% (v / v), and incubate at 37°C and 150 rpm for 24 h to obtain nano-selenium culture medium.

[0148] Centrifuge 100 mL of selenium nanoparticle culture medium at 8000 rpm for 10 minutes at 4°C, collect the precipitate, wash three times with milliq water, and then resuspend the precipitate in 5 mL of milliq water. Disrupt the cells using ultrasound (3-second on / off intervals, 200 repetitions). After cell lysis, centrifuge at 10000 rpm for 10 minutes at 4°C, wash three times with milliq water, and finally resuspend in 2 mL of milliq water. Then, add 2 mL of 80% sucrose solution to the resuspended solution, centrifuge at 10000 rpm, and collect the particles. Wash the particles three times with milliq water and resuspend in 2 mL of milliq water to obtain a purified selenium nanoparticle solution. Freeze-dry the extracted particles to obtain purified biological selenium nanoparticle lyophilized powder.

[0149] The purified bio-nano selenium freeze-dried powder was sent to Microspection Technology Group for testing. EDS energy dispersive spectroscopy analysis showed that its selenium content was 45%, and it also contained elements such as C, N, O, Na, P, S, Cl, and K. Figure 3 ).

[0150] Example 4: Characterization of bio-nano selenium

[0151] 1) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observation of nano-selenium

[0152] The nano-selenium culture solution obtained in Example 3 was drop-coated onto carbon atoms, air-dried under a cover for 10-15 minutes, and then observed under a scanning electron microscope (SEM). 100 μL of nano-selenium sample solution was drop-coated onto a copper grid plate, air-dried, and then the morphology and size of the nano-selenium were measured under a transmission electron microscope (TEM).

[0153] The results showed that the Halomonas sp SF2000 strain was rod-shaped with a cell size of (0.2-0.4) × (1-1.5) μm. The synthesized selenium nanoparticles were extracellular and spherical. Transmission electron microscopy revealed that the bacterial cells had flagella and extracellular nanospheres embedded in the extracellular matrix (ECM). Figure 4 ).

[0154] 2) Nanoparticle size analysis of nano-selenium

[0155] The particle size of nano-selenium was analyzed by dynamic light scattering (DLS) using a particle size analyzer.

[0156] The purified nano-selenium solution (1 mL) obtained in Example 3 was transferred to a polystyrene test tube and measured at 25°C.

[0157] The results showed that the average particle size of the nano-selenium was approximately 350 nm. Figure 5 ).

[0158] 3) Fourier transform infrared (FT-IR) spectroscopy analysis of nano-selenium

[0159] The selenium nanoparticles were scanned in attenuated total reflectance (ATR) mode in infrared spectroscopy, with a spectral resolution of 2 and a wavenumber accuracy of 0.01 cm⁻¹. -1 A total of 64 scans related to atmospheric CO2 were conducted.

[0160] The scan results show:

[0161] In the 3100-3500cm range -1 A peak was observed at 3272.75 cm. -1 This refers to the OH stretching vibration of hydroxyl groups present in the nanoparticles; 2890-3050 cm⁻¹ -1 Nearby peak (2922.66cm) -1 This is attributed to the stretching vibration of the CH bond; amide II (1500 cm⁻¹) -1 ) and amide III (1200-1300cm) -1 The range is related to protein, while 900-1200cm -1 The wavelengths are associated with polysaccharides and nucleic acids; the CO symmetry of the carboxyl group (1300-1450 cm⁻¹) -1 Stretch vibrations confirmed significant changes in the adsorption region, indicating that the carboxyl groups play a crucial role in the stabilization of selenium nanoparticles. Protein and polysaccharide signals observed in FTIR spectroscopy suggest that the formed selenium nanoparticles are encapsulated by extracellular polymers. Figure 6 ).

[0162] 4) X-ray diffraction (XRD) analysis of nano-selenium

[0163] The selenium nanoparticle sample was dispersed on a low-background-noise sample holder and analyzed using an X-ray diffractometer (Rigaku, Japan). X-ray diffraction patterns were recorded at 45 kV and 112 mA. Scans were performed continuously in the 2θ range of 10°–90° at a scan rate of 0.02° / min. -1 .

[0164] The scan results show:

[0165] The XRD spectrum of nano-selenium showed a diffusion mode without any sharp Bragg reflections. Figure 7 This indicates the amorphous nature of nano-selenium.

[0166] Example 5: Study on the antioxidant properties of purified bio-nano selenium

[0167] 1) Determination of DPPH free radical scavenging ability

[0168] 100 μL of nano-selenium sample solutions, sodium selenite sample solutions, or vitamin C sample solutions (Vc) at gradient concentrations (0.1, 0.2, 0.5, 1, 2 mM) were mixed with 100 μL of freshly prepared 0.5 mM DPPH-alcohol solution in a 96-well culture plate. The nano-selenium sample solutions (SeNPs) were prepared by mixing the purified bio-selenium nano-lyophilized powder obtained in Example 3 with water.

[0169] 100 μL of sample solutions with gradient concentrations (0.1, 0.2, 0.5, 1, 2 mM) were mixed with 100 μL of anhydrous ethanol to serve as control samples.

[0170] After stirring evenly, incubate in the dark at room temperature for 30 minutes, and then measure the absorbance at 517 nm.

[0171] The DPPH free radical scavenging rate is calculated by the following formula:

[0172]

[0173] In the formula: A0 is the absorbance of the blank control without the sample (a mixture of 100 μL anhydrous ethanol and 100 μL DPPH-alcohol solution), A1 is the absorbance of a mixture of 100 μL nano selenium sample, sodium selenite sample solution, or vitamin C solution and 100 μL DPPH-alcohol solution, and A2 is the absorbance of a mixture of 100 μL sample solution and 100 μL anhydrous ethanol.

[0174] 2) Determination of ABTS free radical cation scavenging ability

[0175] An ABTS+ solution was prepared by mixing a 7.4 mM ABTS solution with a 2.6 mM potassium persulfate solution, and the mixture was allowed to stand in the dark at room temperature for 12–16 hours until a stable oxidation state was reached. The ABTS+ solution was adjusted with sodium phosphate buffer (pH 7.4) to achieve an absorbance of 0.700 ± 0.01 at 734 nm.

[0176] 1 mL of ABTS·+ solution was added to 4 mL of different samples (Na2SeO3, SeNPs, Vc) of varying concentrations (resulting in final concentrations of 0.1, 0.2, 0.5, 1, and 2 mM, respectively). The SeNPs were prepared by mixing the purified bio-nano selenium lyophilized powder obtained in Example 3 with water. The mixture was allowed to stand in the dark for 6 min, and the absorbance was measured at 734 nm.

[0177] The ability to clear ABTS+ is calculated using the following formula:

[0178]

[0179] In the formula: A0 is the absorbance of the sample with only ABTS+ solution added and no sample added; A1 and A2 are the absorbance of the sample with ABTS+ solution added and the sample without ABTS+ solution added, respectively.

[0180] Antioxidant experiments showed that the scavenging activity of the synthesized SeNPs against DPPH and ABTS exhibited similar trends. Within the concentration range of 0.1-2 mM, the scavenging ability of SeNPs against both increased with increasing concentration. Figure 8 , Figure 9 Among them, SeNPs had the highest DPPH scavenging ability at 50.98%, which was lower than vitamin C (100%) but higher than Na2SeO3 (26.13%). For ABTS scavenging, the maximum antioxidant activity of SeNPs (58.92%) was lower than vitamin C (100%) but much higher than Na2SeO3 (11.65%).

[0181] Example 6: Preparation of Halomonas SF2000-nano selenium microbial inoculant

[0182] (1) Preparation of bacterial suspension

[0183] The Halomonas SF2000 culture broth (bacterial concentration approximately 2 × 10⁻⁶) cultured for 24 hours in Example 2 above was used. 9 The CFU / mL solution was added to LB liquid medium containing 2 mM sodium selenite at a dosage of 2% (v / v) and incubated at 37°C and 150 rpm for 24 h to obtain the SF2000 culture medium of Halomonas containing nano-selenium.

[0184] (2) Add freeze-drying protectant

[0185] Trehalose was selected as a protective agent. A 10-fold trehalose solution of the required concentration was prepared and sterilized at 100°C for 20 min. Then it was mixed with an equal volume of the SF2000 culture medium containing nano-selenium obtained in step (1).

[0186] (3) Freeze-drying

[0187] After thoroughly mixing the SF2000 culture medium containing nano-selenium containing trehalose protectant (as in step (2)), pour it into agar plates. Seal the dispensed plates with plastic wrap and quickly place them in an ultra-low temperature freezer at -80℃ for 24 hours for pre-freezing. Then, quickly transfer the pre-frozen plates to a freeze dryer for freeze-drying. After freeze-drying, quickly transfer the plates to a clean bench and seal them with plastic wrap and sealing film. Store the freeze-dried powder at -20℃, ensuring the viable count of SF2000 halometabolites in the freeze-dried powder is not less than 2 × 10⁻⁶. 9 CFU / g.

[0188] Example 7: Application of Halomonas SF2000 in agricultural planting

[0189] The SF2000 culture medium of Halomonas spp. cultured for 24 hours in Example 2 above (bacterial concentration approximately 2×10⁻⁶) was used to prepare the culture medium. 9 Add CFU / mL to 200 mL of LB liquid medium containing 2 mM sodium selenite at a dosage of 2% (v / v), and incubate at 37°C and 150 rpm for 24 h to obtain nano-selenium culture medium.

[0190] Centrifuge 100 mL of selenium nanoparticle culture medium at 8000 rpm for 10 minutes at 4°C, collect the precipitate, wash three times with milliq water, and then resuspend the precipitate in 5 mL of milliq water. Disrupt the cells using ultrasound (3-second on / off intervals, 200 repetitions). After cell lysis, centrifuge at 10000 rpm for 10 minutes at 4°C, wash three times with milliq water, and finally resuspend in 2 mL of milliq water. Then, add 2 mL of 80% sucrose solution to the resuspended solution, centrifuge at 10000 rpm, and collect the particles. Wash the particles three times with milliq water and resuspend in 2 mL of milliq water to obtain a purified selenium nanoparticle solution. Freeze-dry the extracted particles to obtain purified biological selenium nanoparticle lyophilized powder.

[0191] 1) Application of Halomonas SF2000 in rice

[0192] The purified biological nano-selenium freeze-dried powder obtained above was prepared into solutions of 5 mg / kg, 10 mg / kg and 20 mg / kg (solvent is water). The solutions were sprayed on the leaves of rice when the rice entered the grain-filling stage, and the spraying amount was 150 kg / mu. The control group was sprayed with an equal amount of water on the leaves.

[0193] After the rice matured, the selenium content in the rice grains (measured according to GB5009.93-2017 Determination of Selenium in Food, the same below) and yield were determined, and the results are shown in Table 2.

[0194] Table 2. Effects of nano-selenium treatment on selenium content and yield in rice.

[0195] Nano Selenium Concentration 0 ppm (control) 5ppm 10ppm 20ppm rice selenium content 12μg / kg 186μg / kg 284μg / kg 495μg / kg Rice yield 513 kg / mu 538 kg / mu 544 kg / mu 487 kg / mu

[0196] As shown in Table 2, the selenium content of rice treated with nano-selenium at 5 ppm and 10 ppm both met the selenium content standard specified in GB / T 22499-2008 Selenium-Enriched Rice, while the selenium content of rice treated with nano-selenium at 20 ppm exceeded this standard. Secondly, the yield of rice treated with nano-selenium at 5 ppm and 10 ppm increased by about 5% compared with the control, while the yield of rice treated with nano-selenium at 20 ppm decreased by about 5%. Therefore, it is recommended that the concentration of nano-selenium applied by foliar spray should not exceed 20 ppm in the specific application process of selenium-enriched rice production.

[0197] 2) Application of Halomonas SF2000 in vegetables (Suzhou Bok Choy)

[0198] The purified bio-nano selenium freeze-dried powder obtained above was prepared into solutions of 3 mg / kg, 5 mg / kg and 10 mg / kg (solvent was water). The solutions were sprayed on the leaves of Suzhou Qing 4 weeks before the market launch, with a spraying amount of 150 kg / mu. The control group was sprayed with an equal amount of water on the leaves.

[0199] After harvesting, the selenium content and average plant weight of the above-ground parts of Suzhou green were measured, and the results are shown in Table 3.

[0200] Table 3. Effects of nano-selenium treatment on selenium content and yield in Suzhou green tea.

[0201] Nano Selenium Concentration 0 ppm (control) 3ppm 5ppm 10ppm Selenium content in aboveground parts (dry weight) 24μg / kg 343μg / kg 586μg / kg 938μg / kg Average plant weight (fresh weight) 236 grams / plant 251 grams / plant 263 grams / plant 239 grams / plant

[0202] Table 3 shows that the selenium content of the aboveground parts of *Clerodendrum thomsoniae* treated with 3 ppm and 5 ppm nano-selenium both met the standards for selenium-enriched vegetables specified in *GHT1135-2017 Selenium-Enriched Agricultural Products*, while the selenium content of the aboveground parts of *Clerodendrum thomsoniae* treated with 10 ppm nano-selenium exceeded the upper limit of this standard. Furthermore, the average weight per plant of *Clerodendrum thomsoniae* treated with 3 ppm and 5 ppm nano-selenium increased by approximately 6% and 11% respectively compared to the control, while the average weight per plant of *Clerodendrum thomsoniae* treated with 10 ppm nano-selenium was not different from the control. Therefore, it is recommended that the maximum concentration of nano-selenium for foliar spraying be controlled below 10 ppm in the specific application of selenium-enriched rice production.

[0203] Example 8: Application of Halomonas SF2000 in Aquaculture

[0204] The SF2000 culture medium of Halomonas spp. cultured for 24 hours in Example 2 above (bacterial concentration approximately 2×10⁻⁶) was used to prepare the culture medium. 9Add CFU / mL to 200 mL of LB liquid medium containing 2 mM sodium selenite at a dosage of 2% (v / v), and incubate at 37°C and 150 rpm for 24 h to obtain nano-selenium culture medium.

[0205] Centrifuge 100 mL of selenium nanoparticle culture medium at 8000 rpm for 10 minutes at 4°C, collect the precipitate, wash three times with milliq water, and then resuspend the precipitate in 5 mL of milliq water. Disrupt the cells using ultrasound (3-second on / off intervals, 200 repetitions). After cell lysis, centrifuge at 10000 rpm for 10 minutes at 4°C, wash three times with milliq water, and finally resuspend in 2 mL of milliq water. Then, add 2 mL of 80% sucrose solution to the resuspended solution, centrifuge at 10000 rpm, and collect the particles. Wash the particles three times with milliq water and resuspend in 2 mL of milliq water to obtain a purified selenium nanoparticle solution. Freeze-dry the extracted particles to obtain purified biological selenium nanoparticle lyophilized powder.

[0206] 1) Application of Halomonas SF2000 in chickens (eggs)

[0207] The purified biological nano-selenium freeze-dried powder obtained above was thoroughly mixed with soybean meal and corn flour as carriers to prepare nano-selenium feeds of 2 mg / kg, 4 mg / kg and 6 mg / kg respectively.

[0208] The chickens were fed 10 grams of selenium per day for 30 consecutive days. The selenium content in the eggs and chicken meat was measured (measured according to GB5009.93-2017 Determination of Selenium in Food, the same below) and the average weight were compared with chickens fed the same amount of soybean meal and corn flour as a control. The results are shown in Table 4.

[0209] Table 4. Effects of nano-selenium treatment on selenium content and body weight in chickens (eggs).

[0210] Nano Selenium Concentration 0 ppm (control) 2ppm 4ppm 6ppm Chicken selenium content 34μg / kg 216μg / kg 367μg / kg 527μg / kg Egg selenium content 46μg / kg 259μg / kg 403μg / kg 561μg / kg average weight 1358 grams / each 1372 grams / each 1364 grams / each 1347 grams / each

[0211] As shown in Table 4, the selenium content of chicken and eggs treated with nano-selenium in the 2ppm and 4ppm groups both met the standard for selenium-enriched meat specified in "GH T 1135-2017 Selenium-Enriched Agricultural Products", while the selenium content of chicken and eggs treated with nano-selenium in the 6ppm group exceeded the upper limit of the standard. Secondly, there was no significant difference in the average weight of each treatment group compared with the control, indicating that nano-selenium has little effect on the weight of chickens.

[0212] 2) Application of Halomonas SF2000 in sheep

[0213] The purified biological nano-selenium freeze-dried powder obtained above was thoroughly mixed with corn flour as a carrier and formulated into nano-selenium feeds of 2 mg / kg, 4 mg / kg and 6 mg / kg respectively. Each male sheep was fed 150 grams per day. After 30 days of continuous feeding, the selenium content of the mutton and the average weight were measured and compared with sheep fed the same amount of ordinary corn flour as a control. The results are shown in Table 5.

[0214] Table 5. Effects of nano-selenium treatment on selenium content and body weight in sheep.

[0215] Nano Selenium Concentration 0 ppm (control) 2ppm 4ppm 6ppm Selenium content in mutton 29μg / kg 186μg / kg 284μg / kg 394μg / kg average weight 53.4 kg / animal 54.2 kg / animal 52.9 kg / animal 53.7 kg / animal

[0216] As shown in Table 5, sheep absorbed nano-selenium at a lower rate than chickens. The selenium content of the mutton in all three treatment groups met the standard for selenium-enriched meat specified in "GH T1135-2017 Selenium-Enriched Agricultural Products" and did not exceed the standard. Secondly, the average weight of each treatment group was not significantly different from that of the control, indicating that nano-selenium had no significant effect on the weight of sheep.

[0217] Example 9: Application of Halomonas SF2000 in the preparation of nano-selenium health products

[0218] The SF2000 culture medium of Halomonas spp. cultured for 24 hours in Example 2 above (bacterial concentration approximately 2×10⁻⁶) was used to prepare the culture medium. 9 Add CFU / mL to 200 mL of LB liquid medium containing 2 mM sodium selenite at a dosage of 2% (v / v), and incubate at 37°C and 150 rpm for 24 h to obtain nano-selenium culture medium.

[0219] Centrifuge 100 mL of selenium nanoparticle culture medium at 8000 rpm for 10 minutes at 4°C, collect the precipitate, wash three times with milliq water, and then resuspend the precipitate in 5 mL of milliq water. Disrupt the cells using ultrasound (3-second on / off intervals, 200 repetitions). After cell lysis, centrifuge at 10000 rpm for 10 minutes at 4°C, wash three times with milliq water, and finally resuspend in 2 mL of milliq water. Then, add 2 mL of 80% sucrose solution to the resuspended solution, centrifuge at 10000 rpm, and collect the particles. Wash the particles three times with milliq water and resuspend in 2 mL of milliq water to obtain a purified selenium nanoparticle solution. Freeze-dry the extracted particles to obtain purified biological selenium nanoparticle lyophilized powder.

[0220] The purified bio-nano selenium freeze-dried powder was mixed with starch at a ratio of 1:2000 (v / v) in a mixer to form a premix. The premix and starch were then mixed thoroughly at the same ratio, and a wetting agent was added. The raw material was then granulated in a granulator. The granules were dried in a 60℃ drying oven until the moisture content was controlled at approximately 8%. The granules were then passed through an 80-mesh sieve. The qualified granules were mixed evenly with a lubricant at a mixer speed of 200 rpm for 30 minutes. The granules were then automatically added to capsule shells, with each capsule weighing 1g. After formal production, the average tablet weight was randomly sampled and samples were taken for content determination. The semi-finished capsules were bottled, 100 capsules per bottle, sealed, and stored to obtain the nano selenium health product.

[0221] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Halomonas sp. SF2000, characterized in that, The salt bacterium SF2000 is preserved in China Center for Type Culture Collection on October 21, 2024, and the preservation number is CCTCC NO: M 20242266.

2. A microbial inoculant, characterized in that, The microbial agent contains the salt bacterium SF2000 of claim 1.

3. A microbial inoculant, characterized in that, The microbial agent contains the viable cells of the salt bacterium SF2000 of claim 1, the dry cells of the salt bacterium SF2000 of claim 1 obtained by freeze-drying, the cells of the salt bacterium SF2000 of claim 1 immobilized, or the liquid microbial agent of the salt bacterium SF2000 of claim 1.

4. A method for biologically producing nano-selenium, characterized in that, The method is to inoculate the seed liquid of the salt bacterium SF2000 of claim 1 or the microbial agent of claim 2 into a culture medium containing selenium salt, and then to prepare nano-selenium after cultivation.

5. The use of the salt bacterium SF2000 of claim 1 or the microbial agent of claim 2 in the preparation of selenium-rich fertilizer or selenium-rich feed.

6. The use of the salt bacterium SF2000 of claim 1 or the microbial agent of claim 2 in promoting the growth of vegetables or rice, increasing the yield or selenium content of vegetables or rice; the use is to prepare a solution of the salt bacterium SF2000 of claim 1 or the microbial agent of claim 2 and spray it on the leaves of vegetables or rice.

7. A nano-selenium fertilizer, characterized in that, The nano-selenium fertilizer contains the salt bacterium SF2000 of claim 1, the microbial agent of claim 2, or the nano-selenium prepared by the preparation method of claim 4.

8. The use of the salt bacterium SF2000 of claim 1 or the microbial agent of claim 2 in the preparation of feed for promoting the growth of chickens or sheep or increasing the selenium content in the meat of chickens or sheep. The use is to feed chickens or sheep with feed containing the salt bacterium SF2000 of claim 1 or the microbial agent of claim 2.

9. A nano-selenium feed or nano-selenium feed additive, characterized in that, The nano-selenium feed or nano-selenium feed additive contains the salt bacterium SF2000 of claim 1, the microbial agent of claim 2, or the nano-selenium prepared by the preparation method of claim 4.

10. The use of the salt bacterium SF2000 of claim 1 or the microbial agent of claim 2 in the preparation of selenium-rich products, which are feed additives.

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