Ochrobactrum ochrobactrum strain capable of efficiently tolerating selenate and biologically synthesizing nano-selenium and application of ochrobactrum ochrobactrum strain

Abstract

The invention discloses an ochrobactrum strain capable of efficiently tolerating selenate and biologically synthesizing nano-selenium and application of the ochrobactrum strain, and belongs to the technical field of microbial fermentation and nano-biology. The screened ochrobactrum strain which is efficient, selenium-resistant and capable of biologically synthesizing nano-selenium is classified and named as Ochrobactrum sp. RS1 and is preserved in the China Center for Type Culture Collection on December 15, 2025, the preservation date is December 15, 2025, and the preservation number is CCTCC NO: M20252876. After the strain is subjected to selenium-resistant domestication, the highest tolerance concentration of the strain to sodium selenite can reach 300 nM, the sodium selenite can be reduced into uniform spherical nano-selenium particles in a liquid culture medium, and the average particle size is 190.0-282.5 nm. By optimizing fermentation conditions (PH, temperature and culture time), the conversion rate of the domesticated ochrobactrum anthropi RS1 to sodium selenite reaches 70.20% + / -0.39%. Compared with a traditional chemical and physical nano-selenium synthesis method, the production efficiency of nano-selenium is improved by fermenting sodium selenite with ochrobactrum anthropi RS1, the production cost is reduced, and the method has the advantages of being environmentally friendly, easy and convenient to operate and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial fermentation and nanobiotechnology, and particularly relates to a high-efficiency selenium-resistant Ochrobactrum strain capable of biosynthesizing nano-selenium and application thereof. BACKGROUND

[0002] Selenium is one of the essential trace elements for humans and animals, and has multiple biological activities such as anti-oxidation, immune regulation and anti-cancer. Compared with inorganic selenium and organic selenium, nano-selenium has lower toxicity, higher bioavailability and unique nanometer characteristics, and has broad application prospects in the fields of medicine, functional food, environmental remediation and the like. At present, the synthesis methods of nano-selenium mainly include physical method, chemical method and biological method. The physical method such as laser ablation and microwave irradiation has the disadvantages of high energy consumption, high cost and dangerous synthesis environment; the chemical method usually needs to use chemical reducing agents, which will produce a large amount of pollutants, and the synthesized nano-selenium has low stability, biocompatibility and biological activity.

[0003] Nano-selenium biosynthesis is generally the synthesis of nano-selenium in and out of cells by microorganisms (such as bacteria, fungi and yeasts) through their own metabolic action or plant and animal extracts as reducing agents and stabilizers. This method has the advantages of low cost, good safety, high efficiency, no season limitation for raw material source and production, and environmental friendliness. More importantly, the nano-selenium prepared by microorganisms has good stability because its surface is often wrapped with an organic macromolecular layer such as protein and polysaccharide, which can effectively prevent aggregation. Direct artificial synthesis of organic selenium has the problems of high technical difficulty and high cost. In comparison, microbial fermentation biosynthesis of nano-selenium has the characteristics of fast reproduction, easy operation and strong transformation ability. However, the relatively low selenite tolerance and slow biosynthesis limit the application of microbial fermentation for preparing nano-selenium, and therefore, finding excellent selenium-resistant strains is the core problem of microbial fermentation of nano-selenium.

[0004] In nature, many microorganisms play an important role in the morphological transformation of selenium. The morphology and transformation process of selenium in soil are very complex, and the mechanism of soil selenium activation is not clear at present. However, there should be selenium-resistant microorganisms and specific microbial community structure in the environment of high-selenium areas. Therefore, starting from the perspective of soil microorganisms, it is necessary to screen selenium-resistant microorganisms with high efficiency and stability and selenium metabolic transformation ability, utilize soil microbial resources, efficiently transform selenate to obtain nano-selenium fertilizer, promote plant growth and resistance, and promote the development and utilization of selenium-rich products.

[0005] Although a variety of microorganisms have been reported to be used for synthesizing nano-selenium in the prior art, such as Pseudomonas putida (Wang et al., 2017), Alcaligenes sp. (Wang et al., 2017), Providencia rettgeri (Wang et al., 2017) and Bacillus megaterium (Wang et al., 2017), the selenium tolerance of these microorganisms is still relatively low, and the biosynthesis efficiency of nano-selenium is low. Pseudomonas putida KT2440 Alcaligenes sp. YBY Providencia sp. DCX Bacillus megaterium ATCC 55000 ​​​) and so on, the tolerance concentration of sodium selenite is limited to within 100 mM, which seriously restricts the improvement of the industrialized biosynthesis efficiency and yield of nano-selenium. Although a few strains such as Proteus mirabilis YC801 increase the tolerance to 100 mM, it still needs to be further improved. In addition, the existing researches are mostly focused on strain screening and nano-selenium characterization, and the systematic optimization of high-efficiency synthesis conditions is still insufficient, and the conversion rate of sodium selenite of most strains is less than 60%. For example, the conversion rate of Providencia sp. is about 50% at a concentration of 20 mM, or a high conversion can only be achieved at an extremely low substrate concentration, resulting in low nano-selenium yield and difficulty in meeting the needs of large-scale production. Providencia rettgeri HF16 ) and so on, the tolerance concentration of sodium selenite is limited to within 100 mM, which seriously restricts the improvement of the industrialized biosynthesis efficiency and yield of nano-selenium. Although a few strains such as Proteus mirabilis YC801 increase the tolerance to 100 mM, it still needs to be further improved. In addition, the existing researches are mostly focused on strain screening and nano-selenium characterization, and the systematic optimization of high-efficiency synthesis conditions is still insufficient, and the conversion rate of sodium selenite of most strains is less than 60%. For example, the conversion rate of Providencia sp. is about 50% at a concentration of 20 mM, or a high conversion can only be achieved at an extremely low substrate concentration, resulting in low nano-selenium yield and difficulty in meeting the needs of large-scale production. SUMMARY

[0006] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a high-selenium-resistant and nano-selenium-biosynthetic Proteus strain and its application, so as to solve the technical problems of limited sodium selenite tolerance, low conversion efficiency and the need for systematic optimization of high-efficiency synthesis conditions.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The first aspect of the present application discloses a high-selenium-resistant and nano-selenium-biosynthetic Proteus strain, which is classified and named as Proteus sp. RS1, and is preserved in the China Center for Type Culture Collection on December 15, 2025, with the preservation number CCTCC NO: M20252876. Ochrobactrum The first aspect of the present application discloses a high-selenium-resistant and nano-selenium-biosynthetic Proteus strain, which is classified and named as Proteus sp. RS1, and is preserved in the China Center for Type Culture Collection on December 15, 2025, with the preservation number CCTCC NO: M20252876.

[0008] The second aspect of the present application discloses the application of the high-selenium-resistant and nano-selenium-biosynthetic Proteus strain in nano-selenium synthesis.

[0009] The third aspect of the present application discloses the application of the high-selenium-resistant and nano-selenium-biosynthetic Proteus strain in promoting plant growth.

[0010] Preferably, the promotion of plant growth is the promotion of the growth of root length, root weight and root thickness of plants.

[0011] The fourth aspect of the present application discloses a method for synthesizing nano-selenium, wherein the above-mentioned high-selenium-resistant and nano-selenium-biosynthetic Proteus strain is inoculated into LB liquid medium containing sodium selenite, and when the culture solution turns red, the bacterial liquid is centrifuged to remove supernatant, washed, fixed, gradient dehydrated, and replaced with acetone to obtain nano-selenium.

[0012] Preferably, the culture temperature is 25-35℃, and the culture time is 36-72 h.

[0013] Preferably, the pH value of the LB liquid medium containing sodium selenite is 6-8.

[0014] Preferably, the concentration of sodium selenite added in the LB liquid medium containing sodium selenite is 1-10 mM.

[0015] In a fifth aspect, the present application discloses the nano selenium synthesized by the method.

[0016] Preferably, the average particle size of the nano selenium is 190.0-282.5 nm.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application provides a high-efficiency selenium-resistant and nano-selenium-biosynthetic Ochrobactrum strain, which is classified and named as Ochrobactrum Ochrobactrum sp. RS1, and is preserved in the China Center for Type Culture Collection on December 15, 2025, with a preservation number of CCTCC NO: M20252876. After being domesticated for selenium resistance, the strain can resist a sodium selenite concentration of 300 mM, and can reduce the sodium selenite in a liquid medium into uniform spherical nano selenium particles (RS1-SeNPs) with a uniform morphology and an average particle size of 190.0-282.5 nm. By optimizing the fermentation conditions (pH, temperature, and culture time), it is proved that the conversion rate of the domesticated Ochrobactrum sp. RS1 to sodium selenite reaches 70.20%±0.39%, which significantly improves the production efficiency of nano selenium and reduces the production cost compared with traditional chemical and physical methods, and has the advantages of environmental friendliness and simple operation. With the increase of the sodium selenite concentration, the particle size of the nano selenium decreases and the particle size range becomes narrower, which makes the absorption and utilization of the nano selenium in the organism more efficient. In addition, the organic matters such as proteins, lipids and sugars attached to the surface of the RS1-SeNPs further enhance the stability of the RS1-SeNPs and reduce the agglomeration phenomenon. In addition, experiments prove that the RS1-SeNPs have a significant growth-promoting effect on Salvia miltiorrhiza seedlings, and can significantly increase the root length, root weight and root thickness of Salvia miltiorrhiza by 30.3%, 81.8% and 36.5% respectively. This growth-promoting effect not only improves the growth rate of plants, but also enhances the stress resistance of plants, providing a new idea for agricultural production and the quality improvement of medicinal plants.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] Preservation instructions The strain of the pale bacillus with the ability of synthesizing nano selenium is preserved as follows: Preservation date: December 15, 2025; Preservation place: China Center for Type Culture Collection; Preservation number: CCTCC NO: M20252876. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a cell morphology diagram of the strain RS1 of the present application under different concentrations of sodium selenite; Figure 2 is a colony morphology diagram and phylogenetic analysis diagram of the strain RS1 of the present application; wherein A is a colony morphology diagram of RS1, and B is a phylogenetic analysis diagram; Figure 3 is a growth curve diagram of the strain RS1 of the present application under different concentrations of sodium selenite; Figure 4 is a scanning electron microscope diagram of the strain RS1 and RS1-SeNPs of the present application under different concentrations of sodium selenite; wherein A and B are SEM diagrams under different magnifications; Figure 5 is a transmission electron microscope image and particle size distribution diagram of RS1-SeNPs under different concentrations of sodium selenite of the present application; wherein A and B are transmission electron microscope images under different magnifications, and C is a particle size distribution diagram; Figure 6 is a Fourier transform infrared spectrogram of nano selenium of the present application; Figure 7 is an influence of temperature (A), initial pH (B), culture time (C) and sodium selenite concentration (D) on the growth of RS1 and the conversion rate of sodium selenite of the present application; Figure 8 is a response surface optimization diagram of the present application; wherein (A) is an influence diagram of the interaction of pH and temperature on the conversion rate of sodium selenite, (B) is an influence diagram of the interaction of pH and culture time on the conversion rate of sodium selenite, and (C) is an influence diagram of the interaction of temperature and culture time on the conversion rate of sodium selenite; the left diagram is a three-dimensional response surface diagram, and the right diagram is a two-dimensional contour diagram; Figure 9 is an influence of the pale bacillus RS1, purified RS1-SeNPs and the combined action of the pale bacillus RS1 and RS1-SeNPs on the growth indexes of salvia miltiorrhiza, i.e. plant height (A), total fresh weight (B), root length (C), root weight (D) and root thickness (E); difference analysis adopts LSD test (p<0.05), and different letters represent significant differences; Figure 10 is a comparison diagram of the pale bacillus RS1 and RS1-SeNPs treating salvia miltiorrhiza plants for 30 days of the present application. DETAILED DESCRIPTION

[0021] To enable persons skilled in the art to understand the features and effects of the present application, the following only generally describes and defines the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used herein are in the ordinary meaning understood by those skilled in the art of the present application, and in the event of conflict, the definitions in the present specification shall prevail.

[0022] Theories or mechanisms described and disclosed herein, whether correct or not, should not be considered limiting on the scope of the present application, i.e., the present application can be practiced without regard to any particular theory or mechanism.

[0023] Herein, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have encompassed and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0024] Herein, unless otherwise specified, "comprise", "include", "contain", "have", or similar words encompass the meaning of "consist of" and "consist essentially of", for example, "A comprises a" encompasses the meaning of "A comprises a and other" and "A comprises only a".

[0025] Herein, for the sake of brevity, all possible combinations of the technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of technical features, each technical feature in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope disclosed in the present specification.

[0026] The present application provides a high-efficiency selenium-tolerant and nanoselenium biosynthesis strain of Pantoea agglomerans, which is classified and named as Ochrobactrum sp.RS1, which is screened from selenium-rich soil in Youyi Village, Chengguan Town, Zhenping County, Xi'an City, Shaanxi Province, China, and preserved in China Center for Type Culture Collection on December 15, 2025, with the preservation number CCTCC NO: M20252876.

[0027] The present application will be further described in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the description of the present application, and these equivalent forms also fall within the scope defined by the claims attached hereto.

[0028] The following examples use apparatus and equipment that are conventional in the art. The methods used in the following examples, where not specified, are generally according to conventional conditions, or according to the conditions recommended by the manufacturer. The LB liquid medium used in the following examples is formulated as follows: peptone 10.0 g, yeast extract 5.0 g, sodium chloride 10.0 g, distilled water 1 L, pH 7. The LB solid medium: peptone 10.0 g, yeast extract 5.0 g, sodium chloride 10.0 g, agar 15 g, distilled water 1 L, pH 7. All media are used after sterilization in an autoclave at 121°C for 20 min. Other various raw materials used, unless otherwise specified, are conventional commercially available products, which are of conventional specifications in the art.

[0029] (I) Isolation and identification of Ochrobactrum sp. RS1 1. Isolation and screening of strains 1.1 Isolation of strains: 10 g of rhizosphere soil sample of Coptis chinensis collected from the base of 10,000 mu of Coptis chinensis in the forest of Youyi village, Chengguan town, Zhenping county, Ankang city, Shaanxi province, China was weighed and placed in a conical flask containing 90 mL of sterilized deionized water, and shaken at 28°C and 180 rpm for 30 min, and then left to stand for 10 min. 2 mL of supernatant was added to Luria-Bertani liquid medium containing 20 mM selenium for enrichment culture. The mixed bacterial solution after enrichment was separated by dilution plating method, and 100 μL of liquid of 10 -4 , 10 -5 , 10 -6 , 10 -7 dilution was plated on Luria-Bertani (LB) solid medium without selenium and placed in a 28°C incubator for 4 d to obtain single colonies.

[0030] 1.2 Screening of strains: the growth conditions and types of growing strains were observed, and single colonies with different colony morphological characteristics and good growth vigor were picked and repeatedly separated and purified by plate streaking method until multiple repeated single colonies appeared on the culture medium. The purified different bacterial species were identified by 16S rDNA sequencing, and then inoculated in LB solid medium containing different concentrations (20, 50, 70, 90, 100, 150, 200, 300 mM) of sodium selenite, and placed in a 28°C incubator for 48 h. The color change was observed to determine the selenium tolerance strength of each strain, so as to obtain the strain with the highest selenium tolerance strength. For example, the strain with the highest selenium tolerance strength was obtained by the following method: 10 μL of 16S rDNA sequencing positive control strain was inoculated in 10 mL of LB liquid medium containing 20 mM sodium selenite, and placed in a 28°C incubator for 48 h. The culture was centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded. The precipitate was resuspended in 1 mL of sterilized deionized water, and 1 mL of sterilized deionized water was added to the precipitate to dilute it to 10 Figure 1As shown, colonies appear red on LB solid agar plates containing different concentrations of sodium selenite. To improve the selenium tolerance of the original strain, a series of LB liquid agar plates containing sodium selenite at concentration gradients of 200, 250, 300, 350, and 400 mM were prepared. The bacteria were subcultured three times in each concentration gradient to ensure bacterial adaptability. Finally, a bacterium capable of tolerating a 300 mM sodium selenite concentration was obtained. This bacterium exhibited milky-white colonies of varying sizes, mostly round in shape, with neat edges, a smooth and moist surface, a slightly convex center, opaque colonies, a viscous texture, and was easily picked up. A preliminary strain suspected to be *Bacillus anthocyanin* was obtained and named RS1.

[0031] 2. Strain identification 2.1 Morphological observation The single colony of strain RS1 obtained in step 1 was streaked onto LB solid agar plates. The plates were then inverted in a constant temperature incubator and incubated at 28°C for 3 days for morphological observation.

[0032] As shown in Figure 2, RS1 colonies are round or nearly round, viscous, smooth, slightly raised, milky white, and moist.

[0033] 2.2 Molecular biological identification Table 1 Nucleotide Sequence List

[0034] The obtained strain RS1 was identified by 16S rRNA gene sequencing, and the identification results are shown in SEQ ID NO.1 in Table 1. To explore the evolutionary history and interspecies relationships of RS1, we constructed a phylogenetic tree of RS1 by analyzing the similarity and differences in gene sequences (Figure 2). RS1 and... Ochrobactrum RPTAtOch1 is the most genetically related species, with a 16S rRNA sequence similarity of 99.93%, and the self-support between the two species is 98%, indicating a very close genetic relationship. Secondly, RS1 and... Brucellathiophenivorans HY2-1 and Ochrobactrum quorumnocens A44 is relatively closely related, with a self-exhibition support rate of 97%.

[0035] Based on comprehensive morphological and molecular biological identification results, and integrating phylogenetic and evolutionary analysis, strain RS1 was identified as *Bacillus pallida*, and named *Bacillus pallida* RS1, with the Latin name [missing information]. Ochrobactrum sp. RS1 was deposited on December 15, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number: CCTCC NO: M20252876.

[0036] 2.3 Drawing the growth curve The strain RS1 screened was activated, inoculated into 100 mL sterilized LB liquid medium at an inoculation amount of 1%, and placed in a constant temperature shaker at 28°C and 180 rpm. Samples were taken at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, and 48 h, and the OD of the bacterial liquid was measured by a UV spectrophotometer 600 , and the growth curve of the strain RS1 under different sodium selenite concentrations (0, 20, 50, 100, 150, and 200 mM) within 0-48 h was drawn Figure 3 .

[0037] As can be seen from Figure 3 , in the absence of sodium selenite, the OD 600 of the strain RS1 remained relatively stable within the first 6 h, and the growth rate was extremely slow. During this period, the strain adapted to the environmental conditions, and the growth was in the lag phase; from 6 h to 12 h, the OD 600 of the strain increased significantly, and after 6 h, the strain entered the logarithmic growth phase, at which time the strain was in the rapid proliferation stage. With the increase of time, the bacterial growth speed increased, and reached the maximum at 36 h. From 36 h to 42 h, the OD 600 of the strain changed little, and the strain grew stably, being in the plateau phase. After 42 h, the OD 600 of the strain began to decrease, and the strain entered the decline phase, and the bacterial density gradually decreased. Since the higher the concentration of sodium selenite, the greater the toxic effect of the bacteria, it was observed that under the concentration of 20-100 mM sodium selenite, the logarithmic growth phase of RS1 was 12-18 h, under the concentration of 150 mM sodium selenite, the logarithmic growth phase was extended to 18-24 h, and under the concentration of 200 mM, the logarithmic growth phase of RS1 was 24-30 h.

[0038] (II) Characterization of the synthesis of nano-selenium (RS1-SeNPs) by the strain RS1 1. Scanning electron microscope observation LB liquid medium with 20, 50, 100 mM sodium selenite concentration was prepared respectively, and 1% volume of Ochrobactrum RS1 bacterial liquid was added, and cultured at 28°C, 180 r / min oscillation, and when the culture solution was red, 5 mL bacterial liquid was taken, centrifuged at 5000 r / min for 5 min, and the supernatant was removed, and the precipitate was washed with cold PBS buffer (pH 7.4) for 3 times to remove residual culture medium and other impurities. Then, 1 mL of 2.5% glutaraldehyde solution was added to the bacterial-containing precipitate for fixation treatment, and then washed with PBS buffer for 3 times. Then, 30%, 50%, 70%, 80%, 95% ethanol was used for gradient dehydration, and then 100% ethanol was used for dehydration twice, each for 20 min, and finally 100% acetone was used for replacement twice, each for 20 min. Before electron microscope observation, the sample needs to be treated by a series of processes such as centrifugation, dehydration, drying, gold spraying, etc., and finally observed by scanning electron microscope.

[0039] The scanning electron microscope results are shown in Figure 5 Ochrobactrum RS1 is rod-shaped, and granular objects can be observed on the surface of the bacterial body, and it is preliminarily determined that the substance is RS1-SeNPs particles formed by reduction of Ochrobactrum RS1, which shows that under the concentration of 20, 50, 100 mM sodium selenite, Ochrobactrum RS1 can convert sodium selenite into nanoselenium and release it to the outside of the cell.

[0040] 2. Transmission electron microscope observation, particle size analysis and infrared spectrum analysis The morphology and structure of the nanoselenium synthesized by Ochrobactrum RS1 and its functional group composition were characterized and analyzed by transmission electron microscope and Fourier infrared transform spectrum. The separation and purification of nanoselenium used sucrose density gradient centrifugation method: the above culture solution was centrifuged at 8000 r / min for 10 min, the precipitate was washed with ultrapure water for 3 times to remove the residual culture medium, and the precipitate was resuspended with sterile 1M PBS buffer. Sterile 50% and 70% sucrose solution was prepared. The precipitate resuspension was transferred to the upper layer of the sucrose density gradient solution, and the bacteria were separated by centrifugation at 15000×g for 20 min, and the red precipitate was collected after 2 times of gradient centrifugation, and washed with ultrapure water for 3 times to remove residual sucrose. The obtained precipitate was dried by supercritical drying, and then observed by transmission electron microscope, measured by particle size, and analyzed by Fourier infrared transform spectrum. When analyzing the structure of the sample by Fourier transform infrared spectrometer, the nanoselenium powder was mixed with potassium bromide, and the absorption spectrum of the nanoselenium was determined after pressing the tablet.

[0041] The transmission electron microscope results are shown in Figure 5As shown, when the sodium selenite concentration was 20 mM, the average particle size of the synthesized RS1-SeNPs was 282.5 nm, with 30.67% of the RS1-SeNPs having a particle size between 300 and 400 nm. When the sodium selenite concentration was 50 mM, the average particle size of the synthesized RS1-SeNPs was 203.5 nm, with 46.65% of the RS1-SeNPs having a particle size concentrated between 100 and 200 nm. At a sodium selenite concentration of 100 mM, the average particle size of the synthesized RS1-SeNPs was 190.0 nm, with the particle size concentrated between 100 and 200 nm, accounting for 55.41% of the total. In summary, the sodium selenite concentration affects the particle size of RS1-SeNPs synthesized by *Ailuropoda spp.* RS1. Within a certain range, the higher the sodium selenite concentration, the smaller the particle size of the synthesized RS1-SeNPs, and the more concentrated the particle size range.

[0042] Fourier transform infrared spectroscopy results are as follows Figure 6 As shown, the Fourier transform infrared spectra are at 3389, 2938, 1633, 1539, 1435, 1238, and 1068 cm⁻¹. -1 It exhibits a typical absorption peak at 3389 cm⁻¹. -1 The strong absorption peak at 2938 cm⁻¹ is caused by the stretching vibrations of the NH group of the amide group and the intermolecular and intramolecular -OH groups. -1 The characteristic peak at 1633 cm⁻¹ is caused by the symmetric and asymmetric vibrational stretching of aliphatic groups (such as -CH₂ and -CH₃ present in protein side chains and lipids). -1 The strong absorption peak at 1539 cm⁻¹ is caused by the stretching vibration of the C=O group (amide I band) of the amide group in different randomly coiled proteins, and is a characteristic spectral band of proteins. -1 The absorption peak at 1435 cm⁻¹ is attributed to the amide II band, while the peak at 1435 cm⁻¹ is attributed to the amide II band. -1 The absorption peak at 1238 cm⁻¹ is due to the bending deformation of aliphatic CH₄. -1 The absorption peak at 1068 cm⁻¹ corresponds to the bending vibration of -OH, and is mostly caused by polysaccharides. -1 The absorption peak at the α-position may be caused by the CN vibration in the amine. Therefore, organic matter such as proteins, lipids, and carbohydrates are attached to the surface of RS1-SeNPs, and it is speculated that these organic matter plays an important role in stabilizing the morphology, structure, and function of RS1-SeNPs.

[0043] (III) Optimization of conditions for the transformation of sodium selenite by Bacillus anguillarum RS1 1. Single-factor experiment The effects of single-factor experiments on the growth of *Aureobacterium tumefaciens* RS1 and the conversion rate of sodium selenite were investigated from four aspects: temperature, initial pH, culture time, and sodium selenite concentration.

[0044] 1.1 Effects of different culture temperatures on the growth of *Bacillus angustifolius* RS1 and the conversion rate of sodium selenite Prepare LB liquid medium with a pH of 7.0. After autoclaving, add the aseptically filtered sodium selenite stock solution to the LB medium Erlenmeyer flasks under a sterile operating table to a final concentration of 20 mM. Inoculate activated *Bacillus angulata* RS1 bacterial suspension at a 1% inoculum and incubate at 25, 30, 35, 40, and 45°C with shaking at 180 r / min. Each treatment has three replicates. The control is *Bacillus angulata* reactivated under the same conditions without the addition of sodium selenite. Ochrobactrum RS1 bacterial culture. The control group and the treatment group with added sodium selenite were co-cultured for 72 h, and the OD of the bacterial culture was measured. 600 Conversion rate of sodium selenite.

[0045] The bacterial culture was inoculated at a ratio of 1% into LB liquid medium at pH 7.0, with a sodium selenite concentration of 20 mM. The culture was incubated at 180 r / min for 72 h at different temperatures, and its OD was measured. 600 The bacterial density curve was plotted. Simultaneously, the supernatant was centrifuged and digested, and the selenium content was determined using an atomic absorption spectrophotometer with a selenium standard solution as the standard. The conversion rate curve of sodium selenite was then plotted.

[0046] Test results as follows Figure 7 As shown in Figure A, it can be seen that, under the same conditions, 35℃ is the optimal temperature for bacterial growth, while 40℃ is unsuitable for the growth of *Ailuropoda oryzae* RS1. With increasing temperature, the conversion rate of sodium selenite first increases and then decreases, reaching its highest value at 30℃. Therefore, in subsequent experimental designs, the temperature range was set between 25℃ and 35℃.

[0047] 1.2 Effects of different initial pH values ​​on the growth of *Aureobacterium tumefaciens* RS1 and the conversion rate of sodium selenite. To investigate the effect of pH on the conversion efficiency of sodium selenite by bacteria, LB liquid medium was prepared, and the pH was adjusted to 4, 5, 6, 7, 8, and 9, respectively. Sterilized sodium selenite stock solution was added to the sterilized LB liquid medium in an aseptic operating table to a final concentration of 20 mM, and pre-activated *Bacillus anguillarum* RS1 bacterial suspension (1% inoculum) was inoculated. The culture conditions were 28℃, 180 r / min, and shaking incubation for 72 h. The OD of the bacterial suspension was then measured. 600and the conversion rate of sodium selenite by bacteria. Each treatment was set with 3 replicates. The control group was the bacterial solution without sodium selenite under the same conditions.

[0048] From Figure 7 As can be seen from Table B, the pH value of 6-9 is more suitable for the growth of A. hydrophila RS1, and the conversion rate of sodium selenite is also higher. However, since the conversion rate of sodium selenite is significantly reduced at pH 9, the pH value range of 6-8 is selected for the subsequent experiment.

[0049] 1.3 Effect of different culture times on the growth of A. hydrophila RS1 and the conversion rate of sodium selenite First, LB liquid medium was prepared and its pH value was adjusted to 7.0. After sterilization, the filtered and sterilized sodium selenite stock solution was added to the LB liquid medium in a sterile operation table to make its final concentration reach 20 mM. Then, the pre-activated A. hydrophila RS1 bacterial solution was inoculated into the medium at an inoculation amount of 1%. All cultures were carried out at 28°C, and were shaken at 180 r / min for 12, 24, 36, 48, 60, and 72 h, respectively. The OD 600 and the conversion rate of sodium selenite by bacteria, each treatment was set with 3 replicates. The control group was the bacterial solution without sodium selenite under the same conditions.

[0050] From Figure 7 As can be seen from Table C, with the extension of time, A. hydrophila RS1 enters the logarithmic growth phase at 12-48 h, and gradually stabilizes at about 60 h, reaching the growth plateau. The period of 0-24 h is the key period for the conversion of sodium selenite by bacteria, and after 24 h, the bacteria continue to convert, and the conversion efficiency is the highest at 60 h, about 45%.

[0051] 1.4 Effect of different sodium selenite concentrations on the growth of A. hydrophila RS1 and the conversion rate of sodium selenite LB liquid medium with a pH value of 7.0 was prepared. The prepared medium was filled into three-necked flasks, 100 mL per flask. After sterilization, the filtered and sterilized sodium selenite stock solution was added in a sterile operation table to make its final concentration reach 1, 2, 3, 4, 5, 10, 20, 50, and 100 mM, respectively, and the pre-activated A. hydrophila RS1 bacterial solution was inoculated at an inoculation amount of 1%. The culture temperature was 28°C, and the cultures were shaken for 72 h on a shaking table, each treatment was set with 3 replicates. The control group was the bacterial solution without sodium selenite under the same conditions. The OD 600 and the conversion rate of sodium selenite were measured.

[0052] Sodium selenite has strong biological toxicity, and its inhibitory effect on microbial growth becomes more and more significant with the increase of concentration. For example, Figure 7As shown in FIG. 5, the bacterial density decreased sharply when the concentration of sodium selenite increased from 20 mM to 100 mM. Within a certain concentration range, the amount of selenium nanoparticles synthesized by the microorganism increased with the increase of the concentration of inorganic sodium selenite, and the color of the bacterial solution gradually deepened and became red. In this process, the microorganism could convert inorganic selenium into selenium nanoparticles. The results showed that the conversion rate of sodium selenite by the bacterium RS1 was the highest when the concentration of sodium selenite was 5 mM, and the conversion rate decreased significantly when the concentration of sodium selenite was higher than 10 mM, indicating that too high concentration of inorganic selenium would inhibit the growth of the microorganism and weaken the ability of the bacterium RS1 to synthesize RS1-SeNPs. In summary, 5 mM of sodium selenite was selected in the subsequent response surface optimization experiment, and the concentration of sodium selenite had less inhibition on the growth of the bacterium RS1 and had a higher conversion rate.

[0053] 2. Optimization of the conditions for synthesizing RS1-SeNPs by the bacterium RS1 by the response surface method 2.1 Selection of factor levels Based on the analysis of the results of the four single-factor experiments, the three main factors affecting the conversion rate of sodium selenite by the bacterium RS1, i.e., pH (A), temperature (B), and culture time (C), were optimized by the Box-Behnken experimental design, and the three-factor and three-level response surface analysis method was used to design the experiments. The selected relevant factors and levels are shown in Table 2.

[0054] Table 2. Factor and level table for the response surface experiment

[0055] The average value of the conversion rate of sodium selenite was used as the response value, and the experimental scheme was designed. For the three key factors of pH, temperature, and culture time, a total of 17 experiments were designed. The specific experimental design and results are shown in Table 3.

[0056] Table 3. Experimental scheme and results of the response surface experiment

[0057] 2.2 Establishment of regression equation Multiple linear regression and quadratic term fitting were performed on the response value and each factor. The binary regression equation about the conversion rate Y of sodium selenite by the bacterium RS1 was obtained as follows: Y = 72.25 - 0.67A + 2.38B + 3.78C + 0.85AB + 0.36AC + 0.99BC - 6.33A 2 - 7.01B 2 - 9.62C 2 The regression coefficients of the constructed model Y, the significance of the regression equation and the fitting degree were tested (Table 4). The influence of each variable in the regression equation on the numerical value of the response surface was analyzed, and the smaller the P value, the greater the significance of the corresponding variable. According to the P value, the order of influence on the conversion rate of sodium selenite was: culture time > temperature > pH value. For the two factors of temperature and culture time, the P value was less than 0.05, indicating that the influence of these two factors on the conversion rate of sodium selenite was significant. The P value of pH was 0.3954, indicating that within the range of pH 6 to 8, the influence of pH on the conversion rate of sodium selenite was not significant. In addition, the P values of the square terms of pH, temperature and culture time were all less than 0.01, indicating that the quadratic effects of these factors on their conversion ability were extremely significant. However, the pH value, the interaction of pH value and temperature or culture time, and the interaction of temperature and culture time had no effect on the conversion rate.

[0058] Table 4 Analysis of variance of sodium selenite conversion rate model

[0059] According to the results of the analysis of variance of the sodium selenite conversion regression model, the F value of the model was 25.90, and the corresponding P value was 0.0001. This indicated that the model used had extremely significant statistical significance, could effectively reflect the experimental data, and had high credibility and application value. The lack of item P value of the model and experimental data fitting degree was 0.9309, indicating that the lack of item was not significant, further verifying the rationality of the model. The determination coefficient R 2 of the model was 0.9708, and the adjusted determination coefficient R 2 adj was 0.9334, both indicating that the fitting effect of the model on the experimental data was good. The response value was mainly determined by the three independent variables: pH value, temperature and culture time, and the regression equation could accurately reflect the internal relationship between the three factors and the response value. The above results showed that the model was reliable and could well fit the optimization process of sodium selenite conversion.

[0060] 2.3 Optimization analysis and verification test by response surface method The pH value was 6.773, the temperature was 29.265℃, the culture time was 66.456h, and the conversion rate of sodium selenite reached 70.732%. Considering the actual operation feasibility factors, the conditions for reducing sodium selenite to RS1-SeNPs by Ochrobactrum sp. RS1 were set as follows: pH value of 7.0, temperature of 30℃, and culture time of 66h. The measured conversion rate was 70.20%±0.39%. The experimental results showed that the constructed simulation model could effectively predict the relationship between each factor and the conversion rate of sodium selenite. Based on the fitting function, response surface design and analysis were performed by Design-Expert version 12 software, and a response surface graph was generated. The response surface graph showed the influence of the interaction between each single factor on the conversion rate of sodium selenite. Figure 8 As can be seen from Figure 8 , the influence trend of the pH-temperature interaction on the conversion rate is a parabolic surface with a large longitudinal span and an elliptical contour line, indicating that the interaction of the two factors has a significant effect on the conversion rate of sodium selenite. With the increase of pH value and temperature, the conversion rate of sodium selenite first increases and then decreases. The suitable conditions are as follows: pH value of 6.5-7.5 and temperature of 27-33℃, which can significantly improve the conversion rate of sodium selenite. As can be seen from Figure 8 B, with the increase of pH value and culture time, the conversion rate first increases and then decreases, and the trend of change with culture time is more significant, indicating that the culture time has a more significant effect on the conversion rate than the pH value. When the pH value is 6.5-7.5 and the culture time is 56-70h, it is the best conversion condition under the joint action of the two factors. Figure 8 In C, the optimal culture conditions for the conversion of sodium selenite under the influence of temperature and culture time are concentrated in the combination of temperature of 28-34℃ and culture time of 56-70h.

[0061] Overall, the interaction between pH value, temperature, and culture time is significant, indicating that when analyzing the conversion efficiency or response change, the influence of single factor should not be considered alone, but the synergistic effect of each factor and its interaction should be considered comprehensively. In actual operation, the interaction of each factor and its combination should be evaluated comprehensively to ensure that each factor can play a synergistic role, so as to achieve the best experimental results.

[0062] (Four) Effect of RS1-SeNPs on the growth of Salvia miltiorrhiza To investigate the effect of Ochrobactrum sp. RS1 isolated from selenium-rich soil and its biosynthesized RS1-SeNPs on the growth of Salvia miltiorrhiza, we treated Salvia miltiorrhiza seedlings with Ochrobactrum sp. RS1, purified RS1-SeNPs, and Ochrobactrum sp. RS1 and RS1-SeNPs.

[0063] The experiment on the effects of different treatments on the physiological indicators of Salvia miltiorrhiza seedlings was conducted. Three treatment groups were set up, with the water treatment group as the blank control, as follows: CK: water treatment group; RS1: Resuspend activated Pantoea agglomerans RS1 with sterile water, control OD 600 at 0.8-1.0; SeNPs: Resuspend the obtained RS1-SeNPs with sterile water, control the final concentration at about 10 mg / L; RS1+SeNPs: Cultivate according to the above method for synthesizing biological selenium nanoparticles using Pantoea agglomerans RS1, then centrifuge the Pantoea agglomerans RS1 cells and RS1-SeNPs together, resuspend the precipitate with sterile water, control OD 600 at 0.8-1.0.

[0064] Spraying rules: 5 mL per plant, spraying once every two days, a total of 15 times (30 days). There are 4 groups of treatments, each group sets 8 biological replicates, a total of 32 plants. After the treatment is completed, wash the soil on the surface of the sample, dry the water with filter paper, then measure the growth indicators of Salvia miltiorrhiza plants.

[0065] Take out the whole Salvia miltiorrhiza plant from the flowerpot, carefully clean the surface soil, then wash with deionized water. Then place the washed Salvia miltiorrhiza plant on filter paper, quickly dry the surface water, take pictures, measure and record the plant height, total fresh weight, root length, root weight and root diameter of the whole Salvia miltiorrhiza plant. The measurement methods of each growth indicator are as follows.

[0066] Plant height: The plant height of Salvia miltiorrhiza refers to the vertical height of the aboveground part. Measure and record the vertical distance from the base of the Salvia miltiorrhiza stem to the top of the leaf.

[0067] Total fresh weight: The total fresh weight of Salvia miltiorrhiza refers to the sum of the fresh weight of the underground part and the fresh weight of the aboveground part. Wash the soil on the surface of the Salvia miltiorrhiza plant with deionized water, then dry the water on the surface of the plant with filter paper, immediately measure the mass of the whole plant with an electronic balance.

[0068] Root length: The root length of Salvia miltiorrhiza refers to the vertical length of the main root. Pour a small amount of water into a flat-bottomed glass dish, place the Salvia miltiorrhiza root in the water. Carefully straighten the root of Salvia miltiorrhiza with tweezers and a brush, then measure the length of the main root with a ruler.

[0069] Root weight: The root weight of Salvia miltiorrhiza refers to the fresh weight of the underground part. Use a scalpel to cut off the whole Salvia miltiorrhiza from the root and stem connection position (the base of the root), then immediately weigh the fresh weight of the underground part of each Salvia miltiorrhiza plant with an electronic balance.

[0070] Root diameter: The root diameter of Salvia miltiorrhiza refers to the diameter of the root 1 cm below the base of the root. Use a vernier caliper to measure.

[0071] Results as shown in Figure 9 and Figure 10 , RS1-SeNPs can promote the growth of root length, root weight and root thickness of Salvia miltiorrhiza, respectively significantly increased 30.3% (C), 81.8% (D), 36.5% (E), but RS1-SeNPs had no significant effect on the plant height and total fresh weight of Salvia miltiorrhiza (A and B). All the groups of Ochrobactrum RS1 and RS1+SeNPs had no significant change in plant height, total fresh weight, root weight and root thickness compared with the control group, only the group of RS1+SeNPs had significant effect on the root length of Salvia miltiorrhiza, which increased by 25.6% compared with the control group. Figure 9 Figure 9 Figure 9 Figure 9 Figure 10

[0072] The above is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.​​​​​

Claims

1. A strain of *Aureobacterium* that is highly resistant to selenate and capable of biosynthesizing nano-selenium, characterized in that, A strain of *Aureobacterium* that is highly resistant to selenate and capable of biosynthesizing nano-selenium has been classified and named... Ochrobactrum sp. RS1, deposited at the China Center for Type Culture Collection on December 15, 2025, with accession number CCTCC NO: M20252876.

2. The application of the *Alania spp.* strain, which is highly resistant to selenate and capable of biosynthesizing nano-selenium, as described in claim 1, in the synthesis of nano-selenium.

3. The application of the *Alania spp.* strain, which is highly resistant to selenate and capable of biosynthesizing nano-selenium, as described in claim 1, in promoting plant growth.

4. The application according to claim 3, characterized in that, The promotion of plant growth refers to promoting the growth of root length, root weight, and root diameter.

5. A method for synthesizing nano-selenium, characterized in that, The bacterial culture of the *Ailuropoda* strain described in claim 1, which is highly resistant to selenate and capable of biosynthesizing nano-selenium, was inoculated into LB liquid medium containing sodium selenite. When the culture medium turned red, the bacterial culture was centrifuged to remove the supernatant, washed, fixed, dehydrated in a gradient, and replaced with acetone to obtain nano-selenium.

6. A method for synthesizing nano-selenium according to claim 5, characterized in that, The incubation temperature is 25℃~35℃, and the incubation time is 36~72 h.

7. The method for synthesizing nano-selenium according to claim 5, characterized in that, The pH of LB liquid medium containing sodium selenite is 6-8.

8. A method for synthesizing nano-selenium according to claim 5, characterized in that, In LB liquid medium containing sodium selenite, the concentration of sodium selenite added is 1~10 mM.

9. The nano-selenium synthesized by the method according to any one of claims 5 to 8.

10. The nano-selenium according to claim 9, characterized in that, The average particle size of the nano-selenium is 190.0–282.5 nm.

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