A strain of Bacillus licheniformis for efficient biosynthesis of nano-selenium and its application and green preparation method of nano-selenium

Abstract

The present invention belongs to the field of functional microbial application technology, specifically a strain of Bacillus licheniformis for efficient biosynthesis of nano-selenium, its application and a green method for preparing nano-selenium. Bacillus licheniformis B1-Se for efficient biosynthesis of nano-selenium has been deposited in the Guangdong Provincial Microbial Culture Collection Center on June 27, 2024, with a deposit number of GDMCC NO: 64806. The present invention uses a method of specific screening and directional domestication by gradually increasing the concentration of sodium selenite, from wild strains to efficient engineered strains, so that it can stably tolerate a sodium selenite concentration environment of up to 6600 mg / L, and the sodium selenite conversion rate is increased to 92.19%, and the nano-selenium production is increased to 2778.05 mg / L, reaching the international leading level.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganism application, and specifically relates to a Bacillus licheniformis strain for efficient biosynthesis of nano-selenium and its application and a green method for preparing nano-selenium. Background Art

[0002] Selenium is one of the essential trace elements for animals. It has biological functions such as improving the body's immunity, anti-virus, anti-tumor, anti-oxidation, and improving intestinal flora. However, selenium in nature mainly exists in the form of inorganic selenium and organic selenium. Inorganic selenium is mainly selenite and selenate, which are obtained from the by-products of metal mines. Organic selenium includes natural organic selenium, such as selenoproteins in animals and plants; artificial biological transformation of organic selenium, such as selenium-enriched yeast, selenium-enriched edible fungi, etc.; and artificially synthesized organic selenium, such as selenized carrageenan, etc. Common ones include selenoproteins, selenium-containing amino acids, selenols, selenoethers, selenols, etc. and their derivatives.

[0003] At present, there are several methods for synthesizing nano-selenium:

[0004] (1) Physical synthesis method: Nano-selenium is prepared by laser ablation, microwave radiation, hydrothermal treatment, physical evaporation, electrochemical method and other technical means. This method has fast preparation speed, good stability and high purity, but it has disadvantages such as high equipment cost, dangerous synthesis environment and difficult to control particle size distribution.

[0005] (2) Chemical synthesis method: Usually, selenium sources such as sodium selenite, selenious acid and selenium dioxide are used as oxidants, and chemical reagents such as Vc and hydrazine are used as reducing agents to prepare nano-selenium through redox reactions. This method is simple to operate and can be assembled at the atomic or molecular level to achieve control of particle size, crystal form and shape. However, the stability, biocompatibility and biological activity of the prepared nano-selenium may be reduced, and additional stabilizers or modifiers need to be added. In addition to the above methods, there are other chemical principle methods, such as sol-gel method, hydrothermal reaction method, solution evaporation thermal decomposition method, photochemical synthesis method, electrochemical method, etc. These methods have their own advantages and disadvantages. For example, the sol-gel method can be uniformly doped at the molecular level and the synthesis temperature is low, but the raw materials are expensive, time-consuming, and easy to agglomerate after drying. The hydrothermal reaction method can obtain tiny particles without high-temperature calcination, with good dispersibility and crystallinity, but the reaction process requires high temperature and high pressure, and has high dependence and strict requirements on equipment.

[0006] (3) Biosynthesis: The conversion of selenium sources into nano-selenium through microorganisms or plants is an emerging field that is environmentally friendly and low-cost. The biosynthesis method has mild reaction conditions and strong stability. It is a new trend in the preparation of nano-selenium and has broad application prospects.

[0007] However, there is no green and effective method among the current biosynthesis methods. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention aims to provide a strain of Bacillus licheniformis that can efficiently biosynthesize nano-selenium. The strain has a high sodium selenite conversion rate and nano-selenium yield. The strain is used to prepare nano-selenium, which can achieve green production. The prepared nano-selenium has good antibacterial activity, which provides a possible way to develop new antibacterial materials.

[0009] In order to achieve the above invention objectives, the present invention is implemented by the following technical solutions:

[0010] One of the purposes of the invention of this application is to protect a strain of Bacillus licheniformis B1-Se that can efficiently biosynthesize nano-selenium. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024, with the collection number GDMCC NO: 64806; the address of the collection unit is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0011] Furthermore, the present application also protects the use of the above-mentioned Bacillus licheniformis B1-Se in tolerance to sodium selenite.

[0012] Since Bacillus licheniformis B1-Se has good tolerance to sodium selenite, Bacillus licheniformis B1-Se in the present application can also be used to prepare sodium selenite conversion products.

[0013] Furthermore, the present application also protects the use of the above-mentioned Bacillus licheniformis B1-Se in the efficient biosynthesis of nano-selenium (such as green production of nano-selenium products).

[0014] Preferably, the Bacillus licheniformis B1-Se described above can stably tolerate a sodium selenite concentration environment of up to 6600 mg / L.

[0015] Preferably, in the above application, Bacillus licheniformis B1-Se increases the conversion rate of sodium selenite to 92.19%.

[0016] Preferably, in the above applications, Bacillus licheniformis B1-Se can increase the nano-selenium production to 2778.05 mg / L in the application of efficient biosynthesis of nano-selenium.

[0017] The present application also protects a green method for preparing nano-selenium, which uses the Bacillus licheniformis B1-Se described above.

[0018] Preferably, the green method for preparing nano-selenium comprises the following steps:

[0019] (1) Seed solution preparation:

[0020] Bacillus licheniformis B1-Se was used as a bacterial strain and inoculated into a beef extract peptone medium for cultivation to prepare a seed solution;

[0021] (2) preparing a transformation medium containing nano-selenium;

[0022] (3) Fermentation broth inoculation and culture:

[0023] The activated Bacillus licheniformis B1-Se seed solution is inoculated into the above-prepared transformation medium containing nano-selenium to obtain a fermentation solution;

[0024] (4) Separation and purification of nano-selenium:

[0025] The fermentation liquid is centrifuged to obtain a precipitate, which is resuspended and then subjected to ultrasonic oscillation; the solution after ultrasonic treatment is centrifuged to obtain a supernatant, which is then centrifuged again, the obtained precipitate is washed with distilled water and resuspended, extracted in a n-hexane solution, filtered through a microporous filter membrane, and nitrogen-dried or freeze-dried to prepare SeNPs red powder.

[0026] Preferably, in step (1) of the green method for preparing nano-selenium, the culture conditions are: culture in a constant temperature incubator at 37° C. for 24 hours.

[0027] Preferably, in step (2) of the green method for preparing nano-selenium, the steps of preparing the transformation medium containing nano-selenium are:

[0028] Each 1000 mL of culture medium contained the following mass of basal medium components: peptone 10.0 g, yeast extract 5.0 g, NaCl 5.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, CaCl2 0.1 g;

[0029] The trace elements in each 1000mL culture medium include: FeSO4·7H2O 5.0mg, ZnSO4·7H2O2.8mg, MnSO4·H2O 0.8mg, CuSO4·5H2O 0.25mg, Na2MoO4·2H2O 0.06mg, H3BO30.3mg;

[0030] Vitamins in each 1000mL culture medium include: 20mg thiamine, 20mg riboflavin, 20mg nicotinamide, 20mg D-biotin, 20mg D-calcium pantothenate;

[0031] a. Weigh the basic culture medium components according to the proportion, make up to 997mL with deionized water, sterilize at high temperature and high pressure, and then cool to room temperature for use;

[0032] b. Prepare 2 mL of 500-fold concentrated trace element mother solution and 1 mL of 400-fold concentrated vitamin mother solution, sterilize with a 0.22 μm sterile filter membrane and set aside;

[0033] c. Under sterile conditions, add the trace element concentrated mother solution and the vitamin concentrated mother solution to the basal culture medium respectively and mix well;

[0034] d. Adjust the pH of the culture medium to 7.2;

[0035] e. Add sodium selenite precursor at a final concentration of 6600 mg / L to obtain.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (I) High efficiency: The selected Bacillus licheniformis B1-Se has a higher sodium selenite conversion rate and nano-selenium yield, which improves production efficiency.

[0038] (ii) Environmentally friendly: This method uses biosynthesis to prepare nano-selenium, avoiding the harmful chemicals used in traditional chemical synthesis methods and reducing environmental pollution.

[0039] (III) Innovative domestication strategy: Through targeted domestication strategy, the strain’s tolerance to high concentrations of sodium selenite was improved, further increasing the yield and conversion rate of nano-selenium.

[0040] (iv) Easy for large-scale production: The proposed culture and fermentation conditions are easy to control, which is conducive to industrial production and large-scale application.

[0041] (V) Application potential: The prepared nano-selenium has good antibacterial activity, which provides the possibility for the development of new antibacterial materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the standard curve for SeNPs detection in the embodiment of the present invention;

[0043] Figure 2 This is the colony morphology of strain B1-Se in the embodiment of the present invention;

[0044] Figure 3 This is the growth curve of strain B1-Se in the embodiment of the present invention at a concentration of 3500 mg / L sodium selenite;

[0045] Figure 4 This is the fermentation process of strain B1-Se in the embodiment of the present invention at a concentration of 3500 mg / L sodium selenite;

[0046] Figure 5 This is the phylogenetic evolutionary tree of strain B1-Se in the embodiment of the present invention;

[0047] Figure 6 It is the fifth trait improvement strain of strain B1-Se tolerant to sodium selenite in the embodiment of the present invention;

[0048] Figure 7 The high-purity nano-selenium powder is bioconverted by the strain B1-Se in the embodiment of the present invention;

[0049] Figure 8 This is a scanning electron microscope image of the strain B1-Se nano-selenium sample in the example of the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with embodiments, which are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in the field without creative work are all within the scope of protection of the present invention.

[0051] In the present application, % not specifically indicated indicates the mass percentage, i.e., wt%; reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially.

[0052] The following examples involve reagent preparation standards and index testing methods as follows:

[0053] (1) Preparation of culture medium and reagents:

[0054] Beef extract peptone medium (NA): 2g yeast powder, 5g beef extract, 10g peptone, 5g sodium chloride, pH 7.0, add water to 1000mL, sterilization conditions: sterilize at 121℃ for 20min.

[0055] Lactic acid bacteria medium (MRS): yeast powder 5g, peptone 10g, beef extract 8g, glucose 20g, sodium acetate 5g, diammonium citrate 2g, Tween 80 1mL, K2HPO4 2g, MgSO4·7H2O0.2g, CaCO3 16g, MnSO4·H2O 0.05g, pH 6.3-6.8, add water to 1000mL, sterilization conditions: sterilize at 115℃ for 30min.

[0056] Preparation of PBS solution: potassium dihydrogen phosphate (KH2PO4): 0.27g, disodium hydrogen phosphate (Na2HPO4): 1.42g, sodium chloride (NaCl): 8g, potassium chloride (KCl) 0.2g, add about 800mL of deionized water and stir thoroughly to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally adjust the volume to 1L.

[0057] 1 mol / L Na2S solution: Accurately weigh 7.804 g of Na2S solid, completely dissolve it in ultrapure water, dilute to 100 ml and store for later use.

[0058] 20mmol / L hydroxylamine solution: Accurately weigh 0.1341g of hydroxylamine solid, completely dissolve it in ultrapure water, dilute to 100ml and store for later use.

[0059] 1 mol / L NaOH solution: Accurately weigh 6 g of NaOH solid, completely dissolve it in ultrapure water, dilute to 150 ml and store for later use.

[0060] 0.1% SDS (w / v) (1 mol / L NaOH as solvent): Accurately weigh 0.1 g of SDS solid and completely dissolve it in 1 mol / L NaOH solution. Make up to 100 ml with 1 mol / L NaOH solution and store for later use.

[0061] (2) Preparation of SeNPs (selenium nanoparticles) detection standard curve:

[0062] As shown in Table 1 below, a series of gradient solutions of 0-600 mg / L sodium selenite were prepared using 1 g / L sodium selenite mother solution. Take 10 mL of each solution, add 2 mL of 1 mol / L Na2S solution, and mix slowly. After 1 hour, the solution is reddish brown, and the absorbance is measured at a wavelength of 500 nm. With the sodium selenite content (mg / L) as the horizontal coordinate and the corresponding absorbance value as the vertical coordinate, a standard curve is established to calculate the concentration of sodium selenite after conversion. The obtained standard curve is as follows: Figure 1 As shown, R 2 =0.9993, good linear relationship.

[0063] Table 1 Preparation of SeNPs content gradient standard solution

[0064]

[0065] (3) Determination of SeNPs content in samples:

[0066] Forward determination: Take 10mL of bacterial solution, centrifuge at 10000r / min for 10min, and resuspend in SDS solution. Ultrasonicate at 240W and 40kHz for 30min, then centrifuge at 10000r / min for 10min. Wash the bacterial precipitate by repeated centrifugation 3 times with 10ml ultrapure water, add 2mL of 1mol / LNa2S solution, react at room temperature for 1h, and shake the solution intermittently during the reaction. Take the supernatant after centrifugation and measure the absorbance at 500nm. Substitute the absorbance value into the standard curve to calculate the corresponding sodium selenite concentration and the converted SeNPs content.

[0067] Nano-selenium concentration (mg / L) = measured sodium selenite concentration (mg / L) × nano-selenium molar mass / sodium selenite molar mass = measured sodium selenite concentration (mg / L) × 78.96 / 172.94

[0068] Reverse determination: Take 10mL of bacterial solution, centrifuge at 10000r / min for 10min, take the supernatant and add 2mL of 1mol / L Na2S solution, react at room temperature for 1h, and shake the solution intermittently during this period. Take the supernatant after centrifugation and measure the absorbance at 500nm. Substitute the absorbance value into the standard curve to calculate the corresponding residual sodium selenite concentration and the converted SeNPs content.

[0069] Nano-selenium concentration (mg / L) = (amount of sodium selenite added - measured sodium selenite concentration) × nano-selenium molar mass / sodium selenite molar mass = (amount of sodium selenite added - measured sodium selenite concentration) × 78.96 / 172.94

[0070] (4) Calculation of sample selenium conversion rate:

[0071] Forward determination: Selenium conversion rate = measured sodium selenite concentration (mg / L) / amount of sodium selenite added to the culture medium (mg / L) × 100%

[0072] Reverse determination: Selenium conversion rate = 1-measured sodium selenite concentration (mg / L) / amount of sodium selenite added to the culture medium (mg / L) × 100%

[0073] (4) Evaluation of bacterial proliferation level (OD 600 Determination)

[0074] The sample was shaken and diluted 5 times with pure water, and the absorbance at a wavelength of 600 nm was detected by UV spectrophotometer in parallel 3 times.

[0075] Example 1: Screening and identification of efficient bioconversion strains

[0076] Strain collection: Collect samples from selenium-rich environments such as soil, water or plant rhizosphere.

[0077] Pre-culture: The collected samples were inoculated into NA medium containing 1000 mg / L sodium selenite for aerobic culture or MRS medium for anaerobic culture for pre-culture.

[0078] Screening: The sample solution after pre-culture was spread on a solid medium containing 1000 mg / L sodium selenite by dilution and plate method, and the strains with red colonies were selected as candidate strains; the colony morphology and 1000x microscopic images obtained by the initial screening are as follows Figure 2 As shown, the strain was named B1.

[0079] Tolerance test: The selected B1 original strain was subjected to sodium selenite tolerance test, and its growth curve at the maximum sodium selenite tolerance concentration of 3500 mg / L that did not cause a decrease in OD value was recorded as follows: Figure 3 As shown, the color changes during the whole fermentation process are as follows Figure 4 As shown, at this time, after 48 hours of cultivation, the conversion rate of sodium selenite reached 70.25% and the output of nano-selenium reached 1122.7 mg / L.

[0080] Strain identification: The selected high-tolerance strains were identified using molecular biological methods such as 16S rRNA gene sequencing. The extracted total DNA was used as a template to perform PCR amplification of the bacterial 16S rDNA V4 region. The 16S amplification primers were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAATCGC-3′). The PCR reaction system was 25 μL: 12.5 μL Mix premix, 9.5 μL ddH2O, 1.0 μL each of forward and reverse primers, and 1.0 μL DNA template. PCR reaction conditions: 94°C pre-denaturation for 1 min; 94°C denaturation for 20 s, 54°C annealing for 30 s, 72°C extension for 30 s, a total of 30 cycles; 72°C extension for 5 min. After the PCR amplification reaction is completed, 3% agarose gel electrophoresis is used to detect the target DNA concentration. When the detection concentration reaches the requirement, it is sent to Beijing Qingke Bioengineering Co., Ltd. (Chengdu Testing Center) for amplicon first-generation sequencing.

[0081] The sequencing results were analyzed by MEGA 7.0 software to construct a phylogenetic tree, which confirmed that the biological species of strain B1 was Bacillus licheniformis.

[0082] Example 2: Directed domestication of strains tolerant to high concentrations of sodium selenite and preservation of dominant strains

[0083] Initial concentration: Start inoculation and culture at a sodium selenite concentration of 3500 mg / L.

[0084] Concentration gradient increase: The amount of each increase can be adjusted according to the response of the strain, and the initial plan is to increase by 100 mg / L per generation.

[0085] Incremental frequency: After each generation, observe the growth of the strain and adjust the increment frequency as needed.

[0086] Adaptation time: After each increase in concentration, give the strain enough time (2-3 days) to adapt to the new environment.

[0087] Observation records: After each increase in the concentration of sodium selenite, the NA medium without selenium was used as the control group. The growth of the two groups of strains in the same period of time was observed and recorded, including colony morphology, growth rate, nano-selenium bioconversion rate and other data.

[0088] Fitness assessment: After each incremental concentration increase, the fitness of the strain should be assessed. If the strain fails to grow at the new concentration, it may be necessary to reduce the incremental step size or increase the adaptation time.

[0089] Verification of trait stability: Each time a group with significantly improved tolerance to sodium selenite appears in a generation, the original strain must be preserved and removed from the sodium selenite stress environment. After continuous subculture in the basic culture medium, its tolerance should be re-evaluated. If the excellent trait can be stably inherited, the next step of directed domestication can be carried out.

[0090] The results of directed domestication are as follows Figure 6 As shown in the figure, strain B1-Se was adapted to the sodium selenite concentration step by step for 4 months, with a total of 34 generations. Among them, there were 5 generations showing tolerance to higher sodium selenite concentrations and higher nano-selenium bioconversion rates. After 5 times of trait improvement, the domesticated new strain B1-Se can stably tolerate the highest sodium selenite concentration of 6600mg / L. After 48h of cultivation, the sodium selenite conversion rate increased to 92.19% and the nano-selenium production increased to 2778.05mg / L.

[0091] The above-mentioned Bacillus licheniformis B1-Se, which has been specifically screened and functionally domesticated, is deposited in the Guangdong Provincial Microbiological Culture Collection Center. The preservation date is June 27, 2024, and the preservation number is GDMCCNO:64806.

[0092] Example 3: Green preparation of nano-selenium by strain B1-Se and characterization of its products

[0093] (1) Seed solution preparation:

[0094] Bacillus licheniformis B1-Se was used as a bacterial strain, inoculated into a beef extract peptone medium, and cultured in a constant temperature incubator at 37° C. for 24 hours to obtain a seed solution.

[0095] (2) Preparation of culture medium:

[0096] Prepare the transformation medium containing nano-selenium as follows:

[0097] Basic medium (per 1000 mL): peptone 10.0 g, yeast extract 5.0 g, NaCl 5.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, CaCl2 0.1 g;

[0098] Trace element solution (per 1000 mL): FeSO4·7H2O 5.0 mg, ZnSO4·7H2O 2.8 mg, MnSO4·H2O0.8 mg, CuSO4·5H2O 0.25 mg, Na2MoO4·2H2O 0.06 mg, H3BO3 0.3 mg;

[0099] Vitamin solution (per 1000 mL): thiamine (VB1) 20 mg, riboflavin 20 mg, niacinamide 20 mg, D-biotin 20 mg, D-calcium pantothenate 20 mg;

[0100] Hybrid Approach:

[0101] 1. Weigh the above basic culture medium components, dilute to 1000 mL with deionized water, sterilize at high temperature and high pressure, and cool to room temperature for use.

[0102] 2. Prepare trace element solution and vitamin solution separately, sterilize them with 0.22μm sterile filter membrane and set aside.

[0103] 3. Under sterile conditions, add 2 mL of trace element solution and 1 mL of vitamin solution to the basal culture medium respectively and mix well.

[0104] 4. Adjust the pH of the culture medium to 7.2.

[0105] 5. Add sodium selenite precursor (Na2SeO3) at a final concentration of 6600 mg / L.

[0106] (3) Fermentation broth inoculation and culture:

[0107] The activated Bacillus licheniformis B1-Se seed solution was inoculated into the selenium-containing medium prepared above at an inoculation amount of 5% (v / v). The culture conditions were 37° C., 220 rpm shaking culture on a shaker, and the fermentation time was 48 hours.

[0108] (4) Separation and purification of nano-selenium:

[0109] Take 240ml of fermentation liquid and centrifuge it at 10000r / min for 10min. After the precipitate is obtained, it is resuspended with 0.1% SDS (w / v) (1mol / LNaOH as solvent) and then treated with ultrasonic oscillation at a power of 240W and a frequency of 40kHz for 2h. The solution after ultrasonic treatment is centrifuged at 3000r / min for 10min, and the supernatant is centrifuged at 12000r / min for 10min. The precipitate is washed 3 times with distilled water and resuspended, extracted 2-3 times with n-hexane solution, filtered through a 0.45μm microporous membrane, and dried by nitrogen blowing or freeze drying to obtain the following Figure 7 SeNPs are shown as red powder.

[0110] (5) Characterization of Nano-Selenium Products:

[0111] Scanning electron microscopy (SEM) characterization: Take a small amount of prepared nano-selenium sample and observe it using a scanning electron microscope. The results are as follows Figure 8 It is shown that nano-selenium is spherical or quasi-spherical, with a particle size of about 300-370nm.

[0112] (6) Evaluation of antibacterial activity of nano-selenium:

[0113] The antibacterial activity of nano-selenium on Escherichia coli, Staphylococcus aureus and Clostridium perfringens was evaluated by paper diffusion method. The prepared nano-selenium was dispersed in deionized water at a concentration of 100 μg / mL. The bacterial suspension was evenly spread on a nutrient agar plate, and then a sterile filter paper soaked with nano-selenium solution was placed on the plate. The culture condition was 37°C, and the diameter of the inhibition zone was measured after culturing for 24 hours. The results are shown in Table 2 below. Nano-selenium has a significant antibacterial effect on Escherichia coli, Staphylococcus aureus and Clostridium perfringens.

[0114] Table 2 Evaluation of antibacterial activity of bionanoselenium

[0115]

[0116] The above basic examples of the present invention and their further selected examples can be freely combined to form multiple embodiments, all of which are embodiments that can be adopted and claimed by the present invention. In the scheme of the present invention, each selected example can be arbitrarily combined with any other basic example and selected example. Those skilled in the art will know that there are many combinations.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus licheniformis B1-Se that efficiently biosynthesizes nano-selenium was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024, with the collection number GDMCCNO:64806.

2. Use of Bacillus licheniformis B1-Se as claimed in claim 1 in sodium selenite tolerance.

3. Use of Bacillus licheniformis B1-Se as claimed in claim 1 in sodium selenite conversion products.

4. Use of Bacillus licheniformis B1-Se as claimed in claim 1 in green and efficient biosynthesis of nano-selenium.

5. The use according to claim 2, characterized in that: Bacillus licheniformis B1-Se can stably tolerate an environment with a sodium selenite concentration of up to 6600 mg / L.

6. The use according to claim 3, characterized in that: Bacillus licheniformis B1-Se increased the conversion rate of sodium selenite to 92.19%.

7. The use according to claim 4, characterized in that: The application of Bacillus licheniformis B1-Se in the green and efficient biosynthesis of nano-selenium has increased the nano-selenium production to 2778.05 mg / L.

8. A green method for preparing nano-selenium, characterized in that The Bacillus licheniformis B1-Se as claimed in claim 1 is used.

9. The green method for preparing nano-selenium according to claim 8, characterized in that The following steps are involved: (1) Seed solution preparation: Bacillus licheniformis B1-Se was used as a bacterial strain and inoculated into a beef extract peptone medium for cultivation to prepare a seed solution; (2) preparing a transformation medium containing nano-selenium; (3) Fermentation broth inoculation and culture: The activated Bacillus licheniformis B1-Se seed solution is inoculated into the above-prepared transformation medium containing nano-selenium to obtain a fermentation solution; (4) Separation and purification of nano-selenium: The fermentation liquid is centrifuged to obtain a precipitate, which is resuspended and then subjected to ultrasonic oscillation; the solution after ultrasonic treatment is centrifuged to obtain a supernatant, which is then centrifuged again, the obtained precipitate is washed with distilled water and resuspended, extracted in a n-hexane solution, filtered through a microporous filter membrane, and nitrogen-dried or freeze-dried to prepare SeNPs red powder.

10. The green method for preparing nano-selenium according to claim 8, characterized in that: In step (1), the culture conditions are: culture in a constant temperature incubator at 37° C. for 24 hours; In step (2), the method steps for preparing the transformation medium containing nano-selenium are as follows: Each 1000 mL of culture medium contained the following mass of basal medium components: peptone 10.0 g, yeast extract 5.0 g, NaCl 5.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, CaCl2 0.1 g; Each 1000mL culture medium contains trace elements: FeSO4·7H2O 5.0mg, ZnSO4·7H2O 2.8mg, MnSO4·H2O0.8mg, CuSO4·5H2O 0.25mg, Na2MoO4·2H2O 0.06mg, H3BO3 0.3mg; Vitamins in each 1000mL culture medium include: 20mg thiamine, 20mg riboflavin, 20mg nicotinamide, 20mg D-biotin, 20mg D-calcium pantothenate; a. Weigh the basic culture medium components according to the proportion, make up to 997mL with deionized water, sterilize at high temperature and high pressure, and then cool to room temperature for use; b. Prepare 2 mL of 500-fold concentrated trace element stock solution and 1 mL of 400-fold concentrated vitamin stock solution, sterilize them with a 0.22 μm sterile filter membrane and set aside; c. Under sterile conditions, add the trace element concentrated mother solution and the vitamin concentrated mother solution to the basal culture medium respectively and mix well; d. Adjust the pH of the culture medium to 7.2; e. Add sodium selenite precursor at a final concentration of 6600 mg / L to obtain.

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