Halomonas for efficiently synthesizing nano-selenium as well as method for preparing nano-selenium and application of halomonas

Abstract

The invention discloses halomonas for efficiently synthesizing nano-selenium as well as a method for preparing the nano-selenium and application of the halomonas, and belongs to the technical field of biology. The Halomonas sp. CCTCC NO: M 20242266 disclosed by the invention has strong tolerance to high-concentration selenite, can efficiently convert the selenite into nano-selenium, and is simple in required culture condition, low in cost and easy to operate; the nano-selenium particles produced by utilizing the strain disclosed by the invention are stable in property and high in biological activity. The Halomonas sp. CCTCC NO: M 20242266 or a microbial agent of the Halomonas sp. CCTCC NO: M 20242266 or nano-selenium produced by utilizing the strain has wide development prospects in the fields of functional agriculture including planting industry and breeding industry, functional food processing, health food processing, medical product processing and the like.

Description

Technical Field

[0001] The invention relates to a Halomonas strain capable of efficiently synthesizing nano-selenium and a method and application for preparing the nano-selenium, belonging to the field of biotechnology. Background Art

[0002] Selenium is one of the essential trace elements 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 disease. Daily quantitative selenium supplementation can effectively remove excess free radicals in the human body and improve human immunity, thereby preventing the occurrence of various diseases and delaying aging. However, excessive intake of selenium may lead to selenium poisoning. In some parts of my country, there is excessive selenium in water and soil. These selenium elements mainly exist in the form of highly toxic selenites, which have a potential impact on residents and aquatic organisms 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 be solved.

[0003] There are two main forms of selenium supplementation: inorganic selenium and organic selenium. However, compared with inorganic selenium and organic selenium, nano-selenium has less toxicity and higher bioavailability, and has become the focus of current research. There are generally three methods for obtaining nano-selenium: physical method, chemical method and biological method. The physical method mainly uses laser ablation, microwave radiation, hydrothermal treatment, physical evaporation, electrochemical method and other technical means to prepare it. It has the disadvantages of high energy consumption, high cost, long time, dangerous synthesis environment, and difficult to control particle size distribution. The chemical method usually uses selenium sources such as sodium selenite, selenious acid and selenium dioxide as oxidants, hydrazine and other chemical reagents as reductants, and the oxidant and reductant are combined to undergo redox reactions. The chemical substances added during the preparation process will produce a large amount of pollutants, and the stability, biocompatibility and biological activity of the synthesized nano-selenium are greatly reduced. Therefore, more and more researchers have begun to pay attention to biological methods, especially microbial methods, to synthesize nano-selenium. The microbial method reduces inorganic selenium in the environment into elemental nano-selenium through its own metabolism. The nano-selenium prepared by this method has a 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 food fields.

[0004] At present, many studies have proved that various microorganisms can convert inorganic selenium into nano-selenium, such as Bacillus sp., Lactiplantibacillus plantarum, Pediococcus pentosaceus, Thiocapsa roseopersicina, etc. However, most of these microorganisms have a selenium tolerance of less than 500 mM and a low reduction efficiency, which brings inconvenience to the industrial production of biological nano-selenium. For example, the Chinese invention patent application document with publication number CN110964674A discloses the synthesis of nano-selenium using Thiocapsa roseopersicina, and its selenium tolerance is only 25 g / L (about 316 mM), and the reduction efficiency is unknown; the Chinese invention patent application document with publication number CN118703359A discloses a strain of Pediococcus pentosaceus, which can only convert 5 mmol / L of selenium into selenium in 60 hours. The Se(Ⅳ) reduction rate reached 61.8%, and the reduction rate of 1mmol / L Se(Ⅳ) in 36 hours reached 95.24%, and the selenium tolerance was unknown; the Chinese invention patent document with publication number CN112746086B disclosed a Providencia rettageri, and its highest selenium tolerance was only 445mM; the Chinese invention patent application document with publication number CN118440865A disclosed an Oceanobacillus profundus, although its selenium tolerance was relatively high, its ability to convert and synthesize nano-selenium was only 2-8mM.

[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] In view of the above problems, the present invention screened out a strain of Halomonas sp. SF2000 that can efficiently synthesize nano-selenium, which can grow in 1200mM selenite, can reduce 2mM nano-selenium by 100% within 24 hours, and can 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 so far. Higher synthesis capacity means fewer production times and less related raw materials need to be consumed. It is more convenient, more efficient, more environmentally friendly and more economical, and has a very broad market application value.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The present invention provides a Halomonas sp. SF2000, which is classified and named Halomonas sp. SF2000. The Halomonas sp. SF2000 has been deposited in the China Center for Type Culture Collection on October 21, 2024, with a preservation number of CCTCC NO: M 20242266.

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

[0010] Materials such as high-selenium soil, high-selenium plant rhizosphere soil, salt lake sediment, and selenium-rich organic fertilizers produced in different years (provided by Hefei Zhongke Qiao Biotechnology Co., Ltd.) were collected from Enshi, Hubei Province. 15 grams of each material collected from the environment was added to 200 mL of 100 mM sodium selenite solution, incubated at 37 ° C for 2 days under stirring at 150 rpm, and 100 μL of the supernatant was evenly spread on an LB agar plate containing 500 mM sodium selenite. After incubation at 37 ° C for 2 days, red colonies were observed, which was sodium selenite reduced to nano-selenium. The isolates with Se (IV) reduction activity were selected according to the red nano-selenium secretion, and a single colony was selected. After 3-4 passages on the LB agar plate, it was 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, with flagella, and cell size of (0.2-0.4) × (1-1.5) μm;

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

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

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

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

[0017] The present invention also provides a microbial agent, which contains the Halomonas sp. CCTCCNO: M 20242266 of the present invention.

[0018] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCCNO: M 20242266 as the main microorganism.

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

[0020] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of CCTCC NO: M 20242266 and then expanding the culture.

[0021] In one embodiment of the present invention, the microbial agent contains living cells of the CCTCC NO: M20242266 strain of the present invention, dry cells of the CCTCC NO: M 20242266 strain of the present invention obtained by freeze drying, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, a liquid agent of the CCTCC NO: M 20242266 strain of the present invention, a solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention in any other form.

[0022] In one embodiment of the present invention, the method for preparing the microbial agent is: adding the culture solution 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 biological preparation method for nano-selenium, which comprises the following steps: using the Halomonas sp. CCTCC NO: M 20242266 of the present invention as a biological material and a selenium-containing inorganic salt as a source of selenium, inoculating a seed solution of CCTCC NO: M 20242266 into a selenium-containing culture medium for culturing, purifying the culture solution obtained, and allowing CCTCC NO: M 20242266 to perform biological transformation on the inorganic selenium to prepare nano-selenium.

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

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

[0027] In one embodiment of the present invention, the bacterial concentration of the seed solution is (1-10)×10 9 CFU / mL; the inoculation amount of the 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 selenite.

[0030] In one embodiment of the present invention, the concentration of the 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: centrifuging the culture solution, collecting the precipitate, washing with water, and resuspending; breaking the cells, centrifuging, washing with water, and resuspending; adding sucrose solution, centrifuging, washing with water, and resuspending to obtain particles; freeze-drying the extracted particles to obtain nano-selenium freeze-dried powder.

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

[0034] In one embodiment of the present invention, disrupting the cells is destroying the cells using ultrasound.

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

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

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

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

[0039] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCCNO: M 20242266 as the main microorganism.

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

[0041] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of CCTCC NO: M 20242266 and then expanding the culture.

[0042] In one embodiment of the present invention, the microbial agent contains living cells of the CCTCC NO: M20242266 strain of the present invention, dry cells of the CCTCC NO: M 20242266 strain of the present invention obtained by freeze drying, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, a liquid agent of the CCTCC NO: M 20242266 strain of the present invention, a solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention in any other form.

[0043] In one embodiment of the present invention, the method for preparing the microbial agent is: adding the culture solution 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] The invention also provides application of Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or nano-selenium produced by using CCTCC NO: M 20242266 in planting production.

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

[0047] In one embodiment of the present invention, the field crops are selected from rice, wheat, corn, millet, soybean and the like.

[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 and the like.

[0049] In one embodiment of the present invention, the fruit is selected from strawberry, blueberry, citrus, kiwi, grape, apple, peach, pear, hawthorn, jujube and the like.

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

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

[0052] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCCNO: M 20242266 as the main microorganism.

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

[0054] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of CCTCC NO: M 20242266 and then expanding the culture.

[0055] In one embodiment of the present invention, the microbial agent contains living cells of the CCTCC NO: M20242266 strain of the present invention, dry cells of the CCTCC NO: M 20242266 strain of the present invention obtained by freeze drying, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, a liquid agent of the CCTCC NO: M 20242266 strain of the present invention, a solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention in any other form.

[0056] In one embodiment of the present invention, the method for preparing the microbial agent is: adding the culture solution 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 invention also provides a nano-selenium fertilizer, which contains the Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or the nano-selenium produced by using CCTCC NO: M 20242266.

[0059] In one embodiment of the present invention, the nano-selenium fertilizer is prepared by compounding a nano-selenium suspension and pure water in a volume ratio of 10% to 90%.

[0060] In one embodiment of the present invention, the nano-selenium suspension refers to a nano-selenium suspension prepared by dissolving the nano-selenium freeze-dried powder prepared by the above method using CCTCC NO: M20242266 in water to prepare 5-100 mgSe / L of the nano-selenium suspension.

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

[0062] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCCNO: M 20242266 as the main microorganism.

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

[0064] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of CCTCC NO: M 20242266 and then expanding the culture.

[0065] In one embodiment of the present invention, the microbial agent contains living cells of the CCTCC NO: M20242266 strain of the present invention, dry cells of the CCTCC NO: M 20242266 strain of the present invention obtained by freeze drying, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, a liquid agent of the CCTCC NO: M 20242266 strain of the present invention, a solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention in any other form.

[0066] In one embodiment of the present invention, the method for preparing the microbial agent is: adding the culture solution 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] The invention also provides application of Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or nano-selenium produced by 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 livestock, poultry and other farmed animals or increasing the selenium content in their meat.

[0070] In one embodiment of the present invention, the poultry is selected from chicken, duck, goose and the like.

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

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

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

[0074] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCCNO: M 20242266 as the main microorganism.

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

[0076] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of CCTCC NO: M 20242266 and then expanding the culture.

[0077] In one embodiment of the present invention, the microbial agent contains living cells of the CCTCC NO: M20242266 strain of the present invention, dry cells of the CCTCC NO: M 20242266 strain of the present invention obtained by freeze drying, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, a liquid agent of the CCTCC NO: M 20242266 strain of the present invention, a solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention in any other form.

[0078] In one embodiment of the present invention, the method for preparing the microbial agent is: adding the culture solution 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 invention also provides a nano-selenium feed or a nano-selenium feed additive, wherein the nano-selenium feed or the nano-selenium feed additive contains the Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or the nano-selenium produced by using CCTCC NO: M 20242266.

[0081] In one embodiment of the present invention, the nano-selenium feed is prepared by mixing nano-selenium dry powder and feed raw materials 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 dry powder prepared by mixing the nano-selenium freeze-dried powder prepared by CCTCC NO: M20242266 and corn flour according to the above method, and the nano-selenium content is 1-10 mgSe / kg.

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

[0084] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCCNO: M 20242266 as the main microorganism.

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

[0086] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of CCTCC NO: M 20242266 and then expanding the culture.

[0087] In one embodiment of the present invention, the microbial agent contains living cells of the CCTCC NO: M20242266 strain of the present invention, dry cells of the CCTCC NO: M 20242266 strain of the present invention obtained by freeze drying, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, a liquid agent of the CCTCC NO: M 20242266 strain of the present invention, a solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention in any other form.

[0088] In one embodiment of the present invention, the method for preparing the microbial agent is: adding the culture solution 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 invention also provides application of Halomonas sp. CCTCC NO: M 20242266 or its microbial agent or nano-selenium produced by using CCTCC NO: M 20242266 in preparing selenium-enriched products.

[0091] In one embodiment of the present invention, the selenium-rich products include but are not limited to medicines, functional foods, health products, feeds, and feed additives.

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

[0093] In one embodiment of the present invention, the microbial agent uses Halomonas sp. CCTCCNO: M 20242266 as the main microorganism.

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

[0095] In one embodiment of the present invention, the bacterial agent is prepared by preparing the seed solution of CCTCC NO: M 20242266 and then expanding the culture.

[0096] In one embodiment of the present invention, the microbial agent contains living cells of the CCTCC NO: M20242266 strain of the present invention, dry cells of the CCTCC NO: M 20242266 strain of the present invention obtained by freeze drying, immobilized cells of the CCTCC NO: M 20242266 strain of the present invention, a liquid agent of the CCTCC NO: M 20242266 strain of the present invention, a solid agent of the CCTCC NO: M 20242266 strain of the present invention, or the CCTCC NO: M 20242266 strain of the present invention in any other form.

[0097] In one embodiment of the present invention, the method for preparing the microbial agent is: adding the culture solution 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] Deposit of biological materials:

[0100] Halomonas sp. SF2000, classified and named Halomonas sp. SF2000 Halomonas sp. SF2000, the strain was deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit number CCTCC NO: M 20242266, and the deposit address is Wuhan University, Wuhan, China.

[0101] Beneficial effects:

[0102] The Halomonas SF2000 of the present invention has strong tolerance to high concentration of selenite and can efficiently convert selenite into nano-selenium, and the required culture conditions are simple, low cost and easy to operate. The recovered nano-selenium particles have stable properties and high biological activity, and have obvious advantages in functional agriculture including planting and breeding, functional food processing, health food processing and pharmaceutical product processing.

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

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

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

[0106] 4) The Halomonas SF2000 of the present invention or its microbial agent or the nano-selenium produced by the Halomonas SF2000 of the present invention has a wide range of applications and can be widely used in planting and breeding industries. It can well increase the selenium content of the application objects, coordinate the production of safe, green, efficient and environmentally friendly selenium-rich plants and selenium-rich livestock and poultry, and promote the improvement of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 This is the phylogenetic tree of Halomonas sp. SF2000;

[0108] Figure 2 This is a graph showing the growth of Halomonas sp. SF2000 on plates with different sodium selenite concentrations;

[0109] Figure 3 This is the EDS spectrum analysis diagram of purified nano-selenium;

[0110] Figure 4Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of nano-selenium converted by SF2000;

[0111] Figure 5 is a graph of the nanoparticle size of nano-selenium;

[0112] Figure 6 is the Fourier transform infrared spectrum (FT-IR) of nano-selenium;

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

[0114] Figure 8 The scavenging ability of synthesized nano-selenium on DPPH free radicals;

[0115] Fig. 9 Graph showing the ability of synthesized nano-selenium to scavenge ABTS cationic radicals. DETAILED DESCRIPTION

[0116] In order to further understand the present invention, the present invention is described below in conjunction with embodiments. These descriptions are only to further explain the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0117] The culture medium involved in the following examples is as follows:

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

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

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

[0121] 1. Determination of selenium (IV) content by ascorbic acid reduction method:

[0122] Drawing of standard curve: Using Na2SeO3 as the standard, prepare Na2SeO3 solutions with concentrations of 0, 10, 20, 50, 100, 200, and 250 mg / L respectively. Take 1 mL of Na2SeO3 solution of different concentrations and mix them thoroughly with 0.5 mL of 4 mol / L hydrochloric acid and 1 mL of 1 mol / L ascorbic acid respectively. Let them stand at room temperature for 10 min. Measure the absorbance at 500 nm using UV-vis and draw a standard curve.

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

[0124] 2. Determination of nano-selenium content by spectrophotometry:

[0125] At a wavelength of 490nm, measure the red Se generated by the reduction of 1-10μmol selenite solution with 25μmol NH2OH·HCl. 0 The intensity was used to establish a standard curve.

[0126] The samples were centrifuged at 8000 × g for 20 min to separate the cells, and the absorbance of the supernatant (red solution) was measured spectrophotometrically at 490 nm and 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 such as high-selenium soil from Enshi, Hubei, rhizosphere soil of high-selenium plants, bottom mud of salt lakes, and selenium-rich organic fertilizers produced in different years (provided by Hefei Zhongke Qiao Biotechnology Co., Ltd.) were collected respectively. 15 grams of each material collected from the environment was added to 200 mL of 100 mM sodium selenite solution, and incubated at 37 ° C for 2 days with stirring at 150 rpm. After 2 days, 100 μL of the supernatant was evenly spread on an LB agar plate containing 500 mM sodium selenite. After incubation at 37 ° C for 2 days, red colonies were observed, indicating that sodium selenite was reduced to nano-selenium. The isolates with Se (IV) reduction activity were selected based on the red nano-selenium secretion, and single colonies were selected. After 3-4 passages on the LB agar plate, they were mixed with LB liquid culture medium containing 30% glycerol and stored at -80 ° C for further experiments.

[0130] 2. Identification of strains:

[0131] The strains were subjected to 16S rRNA amplification and sequencing. The bacterial genomic DNA was extracted using a kit, and the universal primers 27F (SEQ ID NO.1: 5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (SEQ ID NO.2: 5′-GGTTACCTTGTTACGACTT-3′) were used. The PCR products were sent to Shanghai Biotech for sequencing. The sequencing results were used to search for the most similar reference sequence of the isolates using the BLAST search method. The BLAST search program (https: / / www.ncbi.nlm.nih.gov) was used to search for nucleotide sequences with significant similarity, and the phylogenetic tree was constructed using the neighbor joining method ( Figure 1 ).

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

[0133] Example 2: Determination of selenium tolerance and reducing ability of strains

[0134] Preparation of Halomonas culture medium: Take out the preserved Halomonas SF2000 glycerol tube from the -80℃ refrigerator, thaw it at room temperature and inoculate it on the LB agar plate. After 24 hours, pick out a single colony and inoculate it into LB liquid medium for 24 hours to obtain Halomonas SF2000 culture medium with an OD600 value of about 1.0 (bacterial concentration is about 2×10 9 CFU / mL);

[0135] 1) Determination of selenium tolerance of strains

[0136] The culture fluid of Halomonas SF2000 cultured for 24 hours was inoculated on LB agar plates containing 600mM, 800mM, 1000mM and 1200mM sodium selenite at an inoculum size of 2% (v / v), and cultured at 37°C for 48 hours. The growth of the strain on the plate was observed, and the plate inoculated with only Halomonas SF2000 without adding sodium selenite was used as a control.

[0137] The results clearly show that Halomonas SF2000 grows well on 600mM, 800mM, and 1000mM sodium selenite plates, and can be seen to reduce sodium selenite to red nano-selenium; although it can also grow on 1200mM sodium selenite plates, its growth is inhibited ( Figure 2 ).

[0138] 2) Determination of the reducing ability of strains

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

[0140] 10 mL of culture was collected from the culture medium every 6 h to determine the bacterial density: the culture was centrifuged at 10,000 g for 10 min to separate the supernatant and the precipitate; the selenium (IV) content of the supernatant was determined by the ascorbic acid reduction method; the nano-selenium content in the precipitate was determined by spectrophotometry using the above-mentioned determination method of "2. Determination of nano-selenium content 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 the reducing ability of Halomonas SF2000 was excellent: it could completely convert 2 mM sodium selenite into nano-selenium within 24 hours, 5 mM sodium selenite into nano-selenium within 36 hours, 10 mM sodium selenite into nano-selenium almost completely within 60 hours (conversion rate was 97.8%), and 20 mM sodium selenite could be converted into about 50% within 60 hours, which is the highest level reported so far; it is comparable to that described in the prior art (Bioprocessing of seleno-oxyanions and tellurite in a novel Bacillus sp. strain STG-83: a solution to removal of toxic oxyanions in presence of nitrate. J Hazard Mater. doi: 10.1016 / j.jhazmat.2008.09.065. Epub 2008 Sep). Compared with Bacillus STG-83 in 26.PMID:18977594.) (which could only reduce 1 mM sodium selenite in 96 h), the reducing ability of Halomonas SF2000 was much higher than that of this strain.

[0145] Example 3: Method for purifying bio-nanoselenium synthesized by strains

[0146] The purification method of bio-nanoselenium is as follows:

[0147] The culture solution of Halomonas SF2000 (bacterial concentration of about 2×10 9 CFU / mL) was added to 200 mL of LB liquid culture medium containing 2 mM sodium selenite at an addition amount of 2% (v / v), and incubated at 37°C with stirring at 150 rpm for 24 h to obtain nano-selenium culture solution.

[0148] Centrifuge 100mL of nano-selenium culture solution at 8000r / min and 4°C for 10 minutes, collect the precipitate, wash it three times with milliq water, and then resuspend the precipitate in 5mL of milliq water, and use ultrasound (3s on / off interval, 200 repetitions in total) to destroy the cells. After the cells are broken, centrifuge at 10000r / min and 4°C for 10 minutes, wash it three times with milliq water, and finally resuspend it with 2mL of milliq water. Then, add 2mL of 80% sucrose solution to the resuspended solution, and take the particles after centrifugation at 10000r / min. Wash the particles three times with milliq water and resuspend them in 2mL of milliq water to obtain a purified nano-selenium solution; freeze-dry the extracted particles to obtain purified biological nano-selenium freeze-dried powder.

[0149] The purified biological nano-selenium freeze-dried powder was sent to Microspectrum Technology Group for testing. EDS energy spectrum analysis showed that its selenium content was 45%. In addition, it also contained C, N, O, Na, P, S, Cl, K and other elements ( 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 dropwise coated on the carbon root, air-dried under the cover for 10-15 minutes, and then observed under a scanning electron microscope (SEM); 100 μL of the nano-selenium sample solution was dropwise coated on a copper mesh plate, air-dried, and 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 nano-selenium was outside the cell and round in shape. Transmission electron microscopy showed that the bacteria had flagella and that there were nanospheres embedded in the extracellular matrix (ECM) outside the cell. 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 show that the average particle size of nano-selenium is about 350nm ( Figure 5 ).

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

[0159] The nanoselenium particles were scanned in the infrared spectrum in attenuated total reflectance (ATR) mode, and the resolution and wave number accuracy of the spectrum were 2 and 0.01 cm, respectively. -1 , a total of 64 scans related to atmospheric CO2 were performed.

[0160] The scan results show:

[0161] At 3100-3500cm -1 A peak (3272.75 cm -1 ), which is the OH stretching vibration of the hydroxyl groups present in the nanoparticles; 2890-3050cm -1 Nearby peaks (2922.66cm -1 ) is attributed to the stretching vibration of the CH bond; amide II (1500 cm -1 ) and amide III (1200-1300cm -1 ) range is related to proteins, while 900-1200cm -1 The bands are related to polysaccharides and nucleic acids; the CO symmetry of the carboxyl group (1300-1450cm -1 ) stretching vibration confirmed that the adsorption area changed significantly, indicating that the carboxyl group plays an important role in the stabilization of nano-Selenium. The protein and polysaccharide signals observed in the FTIR spectrum indicate that the formed nano-Selenium is wrapped by extracellular polymers ( Figure 6 ).

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

[0163] The nanoselenium particle samples were dispersed on a low background noise sample holder and analyzed on an X-ray diffractometer (Rigaku, Japan). The X-ray diffraction patterns were recorded at 45 kV voltage and 112 mA current. The scans were performed in a continuous mode in the 2θ range of 10°–90° with a scan speed of 0.02° min -1 .

[0164] The scan results show:

[0165] The XRD spectrum of nano-Se shows a diffuse mode without any sharp Bragg reflection ( Figure 7 ), indicating the amorphous nature of nano-Selenium.

[0166] Example 5: Study on the antioxidant effect of purified bio-nanoselenium

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

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

[0169] 100 μL of sample solution with gradient concentrations (0.1, 0.2, 0.5, 1, 2 mM) was mixed with 100 μL of anhydrous ethanol as a control sample.

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

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

[0172]

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

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

[0175] 7.4 mM ABTS solution was mixed with 2.6 mM potassium persulfate solution to prepare ABTS+ solution, and the mixture was allowed to stand in the dark at room temperature for 12-16 hours until a stable oxidation state was achieved. ABTS+ solution was adjusted with sodium phosphate buffer (pH 7.4) to 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 different concentrations (so that the final concentrations of the samples were 0.1, 0.2, 0.5, 1, and 2 mM, respectively), wherein SeNPs was prepared by mixing the purified biological nano-selenium freeze-dried powder obtained in Example 3 with water. The mixture was allowed to stand for 6 minutes in the dark, and the absorbance was measured at 734 nm.

[0177] The ability to remove ABTS+ is calculated according to the following formula:

[0178]

[0179] Wherein: A0 is the absorbance of the sample without adding the ABTS+ solution, A1 and A2 are the absorbance of the sample with and without adding the ABTS+ solution, respectively.

[0180] The results of the antioxidant experiment showed that the scavenging activity trends of the SeNPs synthesized in this study for DPPH and ABTS were basically the same. In the concentration range of 0.1-2mM, the scavenging ability of SeNPs for both increased with the increase of concentration ( Figure 8 , Fig. 9 ), among which, the scavenging ability of SeNPs for DPPH was the highest at 50.98%, which was lower than vitamin C (100%), but higher than Na2SeO3 (26.13%); for the scavenging of ABTS, 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 Agent

[0182] (1) Preparation of bacterial suspension

[0183] The culture solution of Halomonas SF2000 (with a bacterial concentration of about 2×10 9 CFU / mL) was added to LB liquid culture medium containing 2 mM sodium selenite at an addition amount of 2% (v / v), and incubated at 37°C and 150 rpm for 24 h to obtain a culture solution of Halomonas SF2000 containing nano-selenium;

[0184] (2) Adding freeze-drying protective agent

[0185] Trehalose is selected as a protective agent, and a trehalose solution of 10 times the required concentration is first prepared, sterilized at 100° C. for 20 min, and then mixed with an equal volume of the culture solution of Halomonas SF2000 containing nano-selenium obtained in step (1);

[0186] (3) Freeze-drying

[0187] The culture solution of Halomonas SF2000 containing nano-selenium and to which trehalose protective agent has been added in step (2) is mixed evenly and poured into a plate. The plate is sealed with plastic wrap and quickly placed in a -80°C ultra-low temperature refrigerator for pre-freezing for 24 hours. The pre-frozen plate is quickly transferred to a freeze dryer for freeze drying. The freeze-dried plate is quickly transferred to an ultra-clean workbench and sealed with plastic wrap and sealing film. The freeze-dried powder is stored at -20°C. The number of live bacteria of Halomonas SF2000 in the freeze-dried powder is not less than 2×10 9 CFU / g.

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

[0189] The culture solution of Halomonas SF2000 (bacterial concentration of about 2×10 9 CFU / mL) was added to 200 mL of LB liquid culture medium containing 2 mM sodium selenite at an addition amount of 2% (v / v), and incubated at 37°C with stirring at 150 rpm for 24 h to obtain nano-selenium culture solution.

[0190] Centrifuge 100mL of nano-selenium culture solution at 8000r / min and 4°C for 10 minutes, collect the precipitate, wash it three times with milliq water, and then resuspend the precipitate in 5mL of milliq water, and use ultrasound (3s on / off interval, 200 repetitions in total) to destroy the cells. After the cells are broken, centrifuge at 10000r / min and 4°C for 10 minutes, wash it three times with milliq water, and finally resuspend it with 2mL of milliq water. Then, add 2mL of 80% sucrose solution to the resuspended solution, and take the particles after centrifugation at 10000r / min. Wash the particles three times with milliq water and resuspend them in 2mL of milliq water to obtain a purified nano-selenium solution; freeze-dry the extracted particles to obtain purified biological nano-selenium freeze-dried powder.

[0191] 1) Application of Halomonas SF2000 in rice

[0192] The purified bio-nanoselenium freeze-dried powder obtained above was prepared into 5 mg / kg, 10 mg / kg and 20 mg / kg solutions (the solvent was water), and sprayed on the leaves when the rice entered the filling stage, with a spraying amount of 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 (the measurement method was determined in accordance with “GB5009.93-2017 Determination of Selenium in Food”, the same below) and the yield were determined. The results are shown in Table 2.

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

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

[0196] As can be seen from Table 2, the selenium content of rice treated with nano-selenium in the 5ppm and 10ppm groups reached the selenium content standard specified in "GB / T 22499-2008 Selenium-enriched Rice", while the selenium content of rice treated with nano-selenium in the 20ppm group exceeded the standard; secondly, the rice yield treated with nano-selenium in the 5ppm and 10ppm groups increased by about 5% compared with the control, and the rice yield treated with nano-selenium in the 20ppm group decreased by about 5%. Therefore, it is recommended that the concentration of foliar spraying of nano-selenium should not exceed 20ppm in the specific application process of selenium-enriched rice production.

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

[0198] The purified bio-nanoselenium freeze-dried powder obtained above was prepared into solutions of 3 mg / kg, 5 mg / kg and 10 mg / kg (the solvent was water), and sprayed on the leaves 4 weeks before Suzhou Green was put on the market, 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 in the aboveground part and the average plant weight of Suzhou Green were measured. The results are shown in Table 3.

[0200] Table 3 Effect of nano-selenium treatment on Suzhou green selenium content and yield

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

[0202] As shown in Table 3, the selenium content of the aboveground part of Suzhou green rice treated with nano-selenium in the 3ppm and 5ppm groups reached the standard of selenium-rich vegetables specified in "GHT1135-2017 Selenium-rich Agricultural Products", while the selenium content of the aboveground part of Suzhou green rice treated with nano-selenium in the 10ppm group exceeded the upper limit of the standard; secondly, the average weight of Suzhou green rice treated with nano-selenium in the 3ppm and 5ppm groups increased by about 6 and 11% respectively compared with the control, and the average weight of Suzhou green rice treated with nano-selenium in the 10ppm group was no different from the control. Therefore, it is recommended that the maximum concentration of foliar spraying of nano-selenium be controlled below 10ppm in the specific application process of selenium-rich rice production.

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

[0204] The culture solution of Halomonas SF2000 (bacterial concentration of about 2×10 9CFU / mL) was added to 200 mL of LB liquid culture medium containing 2 mM sodium selenite at an addition amount of 2% (v / v), and incubated at 37°C with stirring at 150 rpm for 24 h to obtain nano-selenium culture solution.

[0205] Centrifuge 100mL of nano-selenium culture solution at 8000r / min and 4°C for 10 minutes, collect the precipitate, wash it three times with milliq water, and then resuspend the precipitate in 5mL of milliq water, and use ultrasound (3s on / off interval, 200 repetitions in total) to destroy the cells. After the cells are broken, centrifuge at 10000r / min and 4°C for 10 minutes, wash it three times with milliq water, and finally resuspend it with 2mL of milliq water. Then, add 2mL of 80% sucrose solution to the resuspended solution, and take the particles after centrifugation at 10000r / min. Wash the particles three times with milliq water and resuspend them in 2mL of milliq water to obtain a purified nano-selenium solution; freeze-dry the extracted particles to obtain purified biological nano-selenium freeze-dried powder.

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

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

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

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

[0210] Nano-selenium concentration 0ppm(control) 2ppm 4ppm 6ppm Selenium content in chicken 34 μg / kg 216 μg / kg 367 μg / kg 527 μg / kg Selenium content in eggs 46 μg / kg 259 μg / kg 403 μg / kg 561 μg / kg Average weight 1358g / pc 1372g / pc 1364g / pc 1347g / pc

[0211] As can be seen from Table 4, the selenium content of chicken and eggs treated with nano-selenium in the 2ppm and 4ppm groups reached the standard for selenium-rich meat specified in "GH T 1135-2017 Selenium-rich 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 body 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 bio-nanoselenium freeze-dried powder obtained above was fully mixed with corn flour as a carrier, and prepared into 2 mg / kg, 4 mg / kg and 6 mg / kg nano-selenium feeds respectively, and fed to each sheep (male) at a standard of 150 grams per day. After 30 consecutive days of feeding, the selenium content and average body weight of the mutton were measured, and compared with sheep fed with an equal 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 0ppm(control) 2ppm 4ppm 6ppm Selenium content in lamb 29 μg / kg 186 μg / kg 284 μg / kg 394 μg / kg Average weight 53.4 kg / pc 54.2 kg / pc 52.9 kg / pc 53.7 kg / pc

[0216] As can be seen from Table 5, the absorption rate of nano-selenium in sheep is lower than that in chickens. The selenium content in mutton of the three treatment groups all reached the standard for selenium-rich meat specified in "GH T1135-2017 Selenium-rich Agricultural Products" and did not exceed the standard. Secondly, there was no significant difference in the average body weight of each treatment group compared with the control, indicating that nano-selenium had no significant effect on the body weight of sheep.

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

[0218] The culture solution of Halomonas SF2000 (bacterial concentration of about 2×10 9 CFU / mL) was added to 200 mL of LB liquid culture medium containing 2 mM sodium selenite at an addition amount of 2% (v / v), and incubated at 37°C with stirring at 150 rpm for 24 h to obtain nano-selenium culture solution.

[0219] Centrifuge 100mL of nano-selenium culture solution at 8000r / min and 4°C for 10 minutes, collect the precipitate, wash it three times with milliq water, and then resuspend the precipitate in 5mL of milliq water, and use ultrasound (3s on / off interval, 200 repetitions in total) to destroy the cells. After the cells are broken, centrifuge at 10000r / min and 4°C for 10 minutes, wash it three times with milliq water, and finally resuspend it with 2mL of milliq water. Then, add 2mL of 80% sucrose solution to the resuspended solution, and take the particles after centrifugation at 10000r / min. Wash the particles three times with milliq water and resuspend them in 2mL of milliq water to obtain a purified nano-selenium solution; freeze-dry the extracted particles to obtain purified biological nano-selenium freeze-dried powder.

[0220] The purified biological nano-selenium freeze-dried powder is added to starch in a blender at a ratio of 1:2000 (v / v), and mixed thoroughly to form a premix. The premix and starch are then mixed thoroughly at a ratio of 1:2000 (v / v), a wetting agent is added, and the raw materials are made into microparticles in a granulator. Dry in a drying oven at 60°C to control the moisture content of the microparticles to about 8%, pass the microparticles through an 80-mesh sieve, and mix the qualified microparticles with the lubricant evenly. The mixer speed is 200rpm and the mixing time is 30min. Fill the raw material microparticles into the capsule shell in an automatic sampler, and control the weight of each capsule to 1g. After formal production, the average tablet weight is sampled and sampled for content determination. The semi-finished capsules are bottled, 100 capsules per bottle, sealed and packaged for storage, and nano-selenium health products are obtained.

[0221] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Halomonas sp. SF2000, characterized in that: The Halomonas SF2000 was deposited in the China Center for Type Culture Collection on October 21, 2024, with the deposit number being CCTCC NO: M 20242266.

2. A microbial agent, characterized in that: The microbial agent contains the Halomonas SF2000 described in claim 1; Preferably, the microbial agent uses the Halomonas SF2000 described in claim 1 as the main microorganism; Preferably, the microbial agent contains living cells of the Halomonas SF2000 described in claim 1, dry cells of the Halomonas SF2000 described in claim 1 obtained by freeze-drying, immobilized cells of the Halomonas SF2000 described in claim 1, a liquid agent of the Halomonas SF2000 described in claim 1, a solid agent of the Halomonas SF2000 described in claim 1, or the Halomonas SF2000 described in claim 1 in any other form.

3. A biological preparation method of nano-selenium, characterized in that: The method comprises: inoculating the seed liquid of the Halomonas SF2000 described in claim 1 or the microbial agent described in claim 2 into a culture medium containing selenium salts for culturing to prepare nano-selenium.

4. Use of the Halomonas SF2000 described in claim 1 or the microbial agent described in claim 2 or the preparation method described in claim 3 in agricultural production and processing; Preferably, the agricultural production and processing include but are not limited to the preparation of selenium-rich fertilizers, selenium-rich feeds, selenium-rich health foods and nano-selenium drugs.

5. Use of the Halomonas SF2000 described in claim 1 or the microbial agent described in claim 2 or the preparation method described in claim 3 in plant production; Preferably, the application includes promoting the growth of field crops, vegetables or fruits or increasing their yield or increasing their selenium content; Preferably, the application is to prepare the Halomonas SF2000 described in claim 1, the microbial agent described in claim 2 or the nano-selenium obtained by the preparation method described in claim 3 into a solution and spray it on the leaves of plants.

6. A nano-selenium fertilizer, characterized in that: The nano-selenium fertilizer contains the Halomonas SF2000 described in claim 1, the microbial agent described in claim 2 or the nano-selenium obtained by the preparation method described in claim 3.

7. Application of the Halomonas SF2000 described in claim 1 or the microbial agent described in claim 2 or the preparation method described in claim 3 in aquaculture production; Preferably, the aquaculture production includes but is not limited to promoting the growth of livestock, poultry and other farmed animals or increasing the selenium content in their meat; Preferably, the application is feeding feed containing the Halomonas SF2000 described in claim 1, the microbial agent described in claim 2 or the nano-selenium obtained by the preparation method described in claim 3 to farmed animals.

8. A nano-selenium feed or a nano-selenium feed additive, characterized in that: The nano-selenium feed or nano-selenium feed additive contains the Halomonas SF2000 described in claim 1, the microbial agent described in claim 2 or the nano-selenium obtained by the preparation method described in claim 3.

9. Use of the Halomonas SF2000 described in claim 1 or the microbial agent described in claim 2 or the preparation method described in claim 3 in the preparation of selenium-enriched products.

10. The use according to claim 9, characterized in that: The selenium-rich products include but are not limited to medicines, functional foods, health products, feed, feed additives, and fertilizers.

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