Selenomonas rifym and use thereof

Abstract

The application provides a pantoea agglomerans and application, and belongs to the technical field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 36122. The strain has high selenium tolerance and conversion capacity, and can convert high-toxicity inorganic selenium (such as Se (IV)) into low-toxicity organic selenium (such as selenomethionine and selenocystine) and zero-valent nano selenium (Se 0 ) in a culture medium containing sodium selenite. The pantoea agglomerans obtained in the application has the characteristics of selenium-loving microorganisms, and has potential application values in selenium bioconversion, microbial synthesis of nano selenium and crop growth promotion and selenium enrichment. The pantoea agglomerans can promote plant growth, and the liquid fermentation process developed by using the strain has a highest organic selenium conversion rate of 92.5% and a nano selenium synthesis efficiency of 88.3%. The pantoea agglomerans is helpful for the development of organic selenium agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a selenium-tolerant pantothenic bacterium and its application in promoting selenium enrichment in plant growth. Background Technology

[0002] Pantothecinia clumps ( Pantoea agglomerans Pantotheca acuminata is a Gram-negative rod-shaped bacterium with a cell size of 0.5-1.0 × 1-3 micrometers. It has peritrichous flagella and produces yellow pigment. Most strains are facultative anaerobic chemoheterotrophic bacteria. Since Pantotheca acuminata is a Gram-negative rod-shaped bacterium, its cell wall is relatively thin. It has been reported that Pantotheca acuminata can synthesize nano-selenium. However, due to its low selenium concentration tolerance, according to the inventors' previous experiments, the highest selenium concentration that Pantotheca acuminata can grow at is 2 mM. Its selenium conversion rate is also significantly lower than that of other microorganisms. It can only convert a small amount of selenium into organic selenium or nano-selenium at lower concentrations.

[0003] Selenium (Se), an essential micronutrient for humans and animals, plays a crucial physiological role in antioxidation, immune regulation, thyroid hormone metabolism, and cancer prevention. Selenium deficiency in the human body can directly lead to various diseases such as Keshan disease and Kashin-Beck disease, while maintaining adequate selenium intake is key to maintaining health. Selenium in nature exists primarily in two forms: inorganic selenium (such as selenate Se(VI) and selenite Se(IV)) and organic selenium (such as selenoamino acids, selenoproteins, and selenium polysaccharides). Inorganic selenium, especially selenite, not only has low bioavailability but also high toxicity, and its narrow safety window severely limits its direct application in the nutritional field. In contrast, organic selenium and zero-valent nano-selenium (Se)... 0 It exhibits higher bioavailability and significantly reduced cytotoxicity, and is therefore widely regarded as an ideal and safe form of dietary selenium supplementation.

[0004] Currently, the main technical approaches to obtaining organic selenium and nano-selenium include chemical synthesis, plant transformation, and microbial transformation. Chemical synthesis typically uses strong reducing agents (such as hydrazine and sodium borohydride) to reduce and prepare nano-selenium, or generates selenoamino acids through organic synthesis reactions. While this method allows for mass production, it inevitably has several drawbacks: the chemical reduction process may pose environmental and safety risks; the synthesized nano-selenium particles are prone to aggregation and uneven size distribution, affecting their bioactivity; and the organic synthesis route is cumbersome, costly, and may leave harmful organic solvent residues, making it difficult to meet the stringent safety requirements of the food and pharmaceutical industries. Plant transformation (i.e., plant selenium enrichment technology) involves applying selenium fertilizer and utilizing high-selenium-polymer crops such as edible fungi or grains to convert inorganic selenium into organic selenium. While this method is more natural, it suffers from inherent drawbacks such as a long conversion cycle, susceptibility to geographical environment and seasonal climate, large fluctuations in selenium content within plants, and a low proportion of organic selenium, making it difficult to achieve standardized and large-scale production.

[0005] Microbial transformation utilizes the metabolic functions of microorganisms such as yeast, lactic acid bacteria, and fungi to reduce inorganic selenium to nano-selenium or assimilate it to organic selenium. Due to its advantages such as mild reaction conditions, environmental friendliness, good biocompatibility of the products, and ease of process control, it has become a current research hotspot. For example, existing patents disclose the use of *Lactobacillus acidophilus* for the synthesis of nano-selenium or *Candida utilis* for the production of selenium-enriched yeast. However, existing microbial technologies still face a series of severe challenges: the primary bottleneck is that many strains have limited tolerance to selenium concentrations, and their growth is significantly inhibited under high selenium pressure, leading to a sharp decline in conversion efficiency. For example, the growth of some lactic acid bacteria is severely inhibited when the sodium selenite concentration exceeds 2 mM. Secondly, existing strains tend to have single functions, often only proficient in synthesizing nano-selenium or accumulating organic selenium, making it difficult to achieve the targeted and efficient synthesis of two products in the same fermentation system through simple condition control. More importantly, microbial strains with both high selenium conversion capabilities and multiple plant growth-promoting functions (such as secreting growth hormones, dissolving phosphorus and potassium, and fixing nitrogen) are extremely scarce, which greatly limits their integrated application prospects in functional agriculture. In addition, most of the nano-selenium synthesized by microorganisms is an intracellular product, and its extraction and crushing processes are complex. Moreover, nanoparticles are easily oxidized and aggregated after extraction, and maintaining their long-term dispersion stability remains a major challenge for achieving large-scale application.

[0006] Against this backdrop, the present invention successfully isolated a strain of Pantoea agglomerans AKFX-001 with extremely high selenium tolerance and transformation efficiency from selenium-rich soil in Naore Village, Ziyang County. This strain can not only efficiently and directionally synthesize organic selenium or nano-selenium according to culture conditions, but also possesses a variety of significant plant growth-promoting characteristics, breaking through multiple bottlenecks in existing technologies and providing a brand-new solution for the microbial resource utilization of selenium. Summary of the Invention

[0007] The purpose of this invention is to provide a *Pantotheca acuminata* strain AKFX-001 and its applications. This strain can grow rapidly in sodium selenite medium with a concentration of 2-100 mM and can convert some of the sodium selenite into beneficial microorganisms. It can be used to prepare microbial fertilizers. This strain was deposited at the China General Microbiological Culture Collection Center on September 30, 2025. Pantoea agglomerans The accession number is: CGMCC No.36122, Latin name: Pantoea agglomerans, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Tel: 010-64807596.

[0008] The bacterial strain preserved in this invention was collected from surface soil samples collected from a selenium-rich tea garden in Naore Village, Ziyang County, Ankang City, Shaanxi Province. Specifically, 1 g of soil sample was added to 100 mL of LB liquid medium (containing 0.5 mM sodium selenite) and cultured at 30°C and 180 rpm for 48 h with shaking. The culture solution was serially diluted and spread onto LB solid plates (containing 1 mM sodium selenite), and incubated upside down at 30°C for 48 h. Single colonies were picked and purified repeatedly.

[0009] The purified strains were inoculated onto LB plates containing different concentrations of sodium selenite (0, 5, 10, 20, 50, 100 mM) and incubated at 30°C for 48 h.

[0010] The selected pantothecin clumps could still grow on plates containing 100 mM sodium selenite, indicating that they have extremely strong selenium tolerance. The pantothecin clumps were then enhanced to obtain the pantothecin clumps AKFX-001.

[0011] The aforementioned pantothecin AKFX-001, fermented in a selenium-containing medium, can convert inorganic selenium (sodium selenite) into organic selenium (such as selenomethionine) and red elemental selenium nanoparticles. By optimizing fermentation conditions (such as selenium concentration, temperature, pH, and time), the organic selenium conversion rate can reach up to 92.5%, and the nanoparticle selenium synthesis efficiency reaches 88.3%. The prepared nanoparticles are uniformly distributed with an average particle size of 200-500 nm.

[0012] In addition, this strain possesses a variety of plant growth-promoting characteristics, such as secreting indoleacetic acid (IAA), producing siderophores, and dissolving inorganic phosphorus.

[0013] The strains and processes provided by this invention have advantages such as high selenium conversion efficiency, low cost, environmental friendliness, and wide application range, and have broad application prospects in functional agriculture, food additives, biomedicine and other fields. Beneficial effects

[0014] First, this invention provides a *Pantotheca agglutinosa* strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 36122. This strain exhibits high selenium tolerance and transformation capabilities, enabling it to convert highly toxic inorganic selenium (such as Se(IV)) into less toxic organic selenium (such as selenomethionine and selenocysteine) and zero-valent nano-selenium (Se) in a culture medium containing sodium selenite. 0 ).

[0015] Secondly, the clump-forming pantothenic bacteria obtained in this invention exhibit selenophilic microbial characteristics, and have potential applications in selenium biotransformation, microbial synthesis of nano-selenium, and selenium enrichment for crop growth promotion. The liquid fermentation process developed using this strain achieves an organic selenium conversion rate of up to 92.5% and a nano-selenium synthesis efficiency of 88.3%.

[0016] Finally, the Pantothecin bacteria preserved in this invention have strong environmental adaptability, can grow rapidly in an inorganic selenium environment, and can convert inorganic selenium into organic selenium with high absorption rate, which is of great help to the development of organic selenium agricultural products. Attached Figure Description

[0017] Figure 1 This shows the colony morphology of strain AKFX-001 on LB plates (after 48 h of incubation).

[0018] Figure 2 Different dilution gradients of soil suspension (10) -4 10 -5 10 -6 The difference in colony morphology and quantity is shown in the figure.

[0019] Figure 3 The OD values ​​of bacterial cultures at different culture times in mediums containing 2.5 mg / mL and 5 mg / mL sodium selenite are... 600 Value distribution chart.

[0020] Figure 4 The OD values ​​of bacterial cultures at different culture times in mediums containing 2.5 mg / mL and 5 mg / mL sodium selenite are... 600 Value distribution chart.

[0021] Figure 5 The colony morphology of potential selenium-enriched microorganisms obtained for further screening is shown in the following diagrams: (A) strains 2-4: multi-type myxobacteria; (B) strains 5-3: pantothecin; (C) strain 7-1: mycosis fungiformis; (D) strain 12-4: agaricus bacteria.

[0022] Figure 6The images show a comparison of the apparent colors of bacterial cultures after 48 h of cultivation at different sodium selenite concentrations. (A) Sodium selenite concentration: 100 μg / mL; (B) Sodium selenite concentration: 150 μg / mL; (C) Sodium selenite concentration: 200 μg / mL; (D) Sodium selenite concentration: 250 μg / mL; Sodium selenite concentration: 300 μg / mL.

[0023] Figure 7 The effect of sodium selenite concentration on the growth of the strain (OD600).

[0024] Figure 8 The red phenotype of the bacterial culture is shown as a stage change during adaptive evolution. (A) 100 μg / mL; (B) 150 μg / mL; (C) 200 μg / mL; (D) 250 μg / mL; (E) 300 μg / mL.

[0025] Figure 9 The red colony phenotype represents the stage-wise changes during adaptive evolution. (A) 100 μg / mL; (B) 150 μg / mL; (C) 200 μg / mL; (D) 250 μg / mL; (E) 300 μg / mL.

[0026] Figure 10 The growth curves are shown for different concentrations of sodium selenite stress.

[0027] Figure 11 This is a comparison diagram showing the growth of *Panthera clumps* preserved in this invention and other *Panthera clumps* under different concentrations of sodium selenite stress. Detailed Implementation

[0028] The present invention will be described in detail below with reference to examples. All methods and techniques, unless otherwise specified, are conventional. Example

[0029] This embodiment provides a cluster of pantothecin ( Pantoea agglomerans The Latin name of this strain is: Pantoea agglomerans, This strain was deposited on September 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36122. The address is Room H129, CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101, China. The telephone number is 010-64807596.

[0030] The strain preserved in this embodiment was isolated from a surface soil sample collected from a selenium-rich tea garden in Naore Village, Ziyang County, Ankang City, Shaanxi Province. After purification, enhancement, and identification, a cluster of pantothenic bacteria was obtained.

[0031] The method for bacterial identification was as follows: Gram staining (negative, short rod-shaped), physiological and biochemical tests were performed on strain AKFX-001, and genomic DNA was extracted. The 16S rRNA gene was amplified using universal primers 27F / 1492R and sequenced. The sequence was then compared with the NCBI database using BLAST. Pantoea agglomerans The XYZ strain showed the highest homology, reaching 99.8%. This strain was identified as *Pantotheca acuminata* (…). Pantoea agglomerans It was named AKFX-001.

[0032] Partial 16S rRNA gene sequence (1403 bp) of strain AKFX-001:

[0033] Example

[0034] Because some soils in the selenium-rich areas of Ankang City, Shaanxi Province have high selenium content, the inventors' research group hypothesizes that, during long-term adaptive evolution, strains with strong inorganic selenium tolerance and transformation capabilities have emerged in the soil. This embodiment provides a method for screening and enhancing the inorganic selenium transformation dominant strain AKFX-001, as follows.

[0035] First, soil samples with high selenium content were collected from selenium-rich areas in Ankang City, Shaanxi Province. During sampling, fields, roadsides, and locations with special terrain were avoided. The depth and amount of soil samples were kept consistent at different collection points. A total of 22 soil samples with a depth of 20 cm were collected.

[0036] The selenium-rich soil samples were brought back to the laboratory, large plant debris and stones were removed, the soil was ground through a 0.85 mm sieve, stored in a sealed bag, and kept in a 4°C refrigerator for later use.

[0037] The screening method for strain AKFX-001 includes the following steps.

[0038] (1) Weigh 22 selenium-rich soil samples, each accurately weighed 1 g, and place them in sterile containers. Add 9 mL of TSA liquid culture medium to each container. After sealing, incubate in a shaker at 37℃ and 200 rpm for 8 h. Then, dilute the resulting suspensions to 10 mL. −5 Concentration gradients were established, and 100 μL of the diluted soil suspension was then pipetted onto TSA solid plates containing 1 mg / mL and 2.5 mg / mL sodium selenite, respectively.

[0039] (2) Select individual colonies of varying sizes and morphologies from plates containing 1 mg / mL sodium selenite and inoculate them into TSA liquid medium containing 2.5 mg / mL sodium selenite. Incubate under suitable conditions for 24 h. During the incubation period, measure the OD of the bacterial culture using an enzyme-linked immunosorbent assay (ELISA) reader every 12 h. 600 Values ​​were filtered to select OD values ​​at 12 h and 24 h. 600 Thirty-nine tubes were obtained from bacterial cultures with values ​​all within the top 50. Subsequently, 10 μL of each of these 39 tubes was inoculated into TSA liquid medium containing 5 mg / mL sodium selenite and cultured for another 36 h. During the culture, the OD values ​​of the bacterial cultures were measured using a microplate reader at 24 h and 36 h. 600 The values ​​were ultimately selected based on OD values ​​at 24 h and 36 h. 600 The data for bacterial suspensions with values ​​all in the top 10 are shown in Figure 3.

[0040] (3) Select individual colonies of varying sizes and morphologies from plates containing 2.5 mg / mL sodium selenite and inoculate them into TSA liquid medium containing 2.5 mg / mL sodium selenite. Incubate under suitable conditions for 24 h. During the incubation period, measure the OD of the bacterial culture using an enzyme-linked immunosorbent assay (ELISA) reader every 12 h. 600 Values ​​were filtered to select 12-hour and 24-hour OD values. 600 Forty tubes were obtained from bacterial cultures with values ​​all within the top 50. Subsequently, 10 μL of each of these 40 tubes was inoculated into TSA liquid medium containing 5 mg / mL sodium selenite and cultured for 36 h. During the culture, the OD values ​​of the bacterial cultures were measured at 24 h and 36 h. 600 Values, selecting 24-hour and 36-hour OD values. 600 The data for bacterial cultures with values ​​all in the top 10 are shown in Figure 4.

[0041] (4) The bacterial cultures of the 20 strains selected in 2) and 3) were mixed with 60% sterile glycerol solution at a volume ratio of 1:1 (v / v), and then dispensed into sterile cryovials, 1.5 mL per tube. Ten tubes of each strain were dispensed and stored in an ultra-low temperature freezer at -80℃.

[0042] (5) Sterilize the inoculation loop by flame, and after cooling, dip it into 20 μL of potentially selenium-enriched microbial culture solution and streak it on TSA solid medium without Na2SeO3. Then, invert the medium and place it in a 37°C incubator for 24 h to obtain activated single colonies.

[0043] (6) Select activated single colonies and inoculate them into 4 mL of TSA liquid medium containing different concentrations of sodium selenite (100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL and 300 μg / mL, respectively). Set up three parallel control experiments for each concentration. Incubate the inoculated medium in a shaker at 37℃ for 48 h, and measure the OD of the bacterial solution every 24 h during the incubation period. 600 In addition, prepare TSA liquid medium with the same concentrations as above (100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL and 300 μg / mL), without colony inoculation, and culture for the same duration under the same conditions as a blank control group, and select the strain with the best growth.

[0044] The experimental results are as follows.

[0045] As shown in Figure 2, the number of colonies on the plate gradually decreased with increasing sodium selenite concentration and soil suspension dilution gradient, and the microbial tolerance to sodium selenite also decreased accordingly. When the dilution gradient was 10... −5At this time, the number and morphology of single colonies are most ideal, making it convenient to observe and study the growth status of individual colonies.

[0046] Single colonies exhibited optimal growth at sodium selenite concentrations of 1 mg / mL and 2.5 mg / mL. Based on a comprehensive comparative analysis of colony growth, the optimal dilution gradient was determined to be 10-1. −5 The suitable sodium selenite concentrations in the culture medium are 1 mg / mL and 2.5 mg / mL.

[0047] After different single colonies were inoculated into liquid culture medium containing different concentrations of sodium selenite (TSA), the four strains with the best growth and the highest number of colonies were selected. They were (A) strain 2-4: multi-type myxobacterium; (B) strain 5-3: pantothecin; (C) strain 7-1: mycosis fungoides; (D) strain 12-4: agaricus.

[0048] As shown in Figure 6, when the sodium selenite concentration was 100 μg / mL, compared to the higher concentration treatment group, the bacterial solution did not show the characteristic red change, and the bacterial precipitate remained white. This phenomenon indicates that at a sodium selenite concentration of 100 μg / mL, the bacterial strain's sodium selenite metabolic threshold was not reached, and the selenate reductase system was not fully activated. At this point, the bacteria may metabolize low-concentration selenate through non-reductive pathways such as methylation, resulting in insufficient production of red selenium nanoparticles (SeNPs). Based on the color performance of the bacterial solution, subsequent adaptive evolutionary experiments (ALE) will use 100 μg / mL sodium selenite as the initial selection pressure.

[0049] As shown in Figure 7, strain 5-3 exhibited significantly better growth and redness values ​​than strains 2-4, 7-1, and 12-4 in TSA medium with continuously increasing sodium selenite concentrations. Figure 6 shows that when the sodium selenite concentration exceeded 100 μg / mL, the bacterial culture turned significantly red, indicating that strain 5-3 possesses excellent tolerance and transformation ability to inorganic selenium, and can efficiently synthesize red elemental selenium and selenium nanoparticles (SeNPs) via the sodium selenite reduction pathway.

[0050] Finally, a strain 5-3, known as Pantotheca cumulus, with high tolerance to inorganic selenium was successfully bred. An adaptive laboratory evolution method with gradient concentrations was used to further enhance its tolerance to sodium selenite (Na2SeO3).

[0051] The inventors further enhanced the selected strains of Pantothenia gravis with high inorganic selenium tolerance, using the following specific methods:

[0052] (1) The 5-3 bacterial suspension was taken out of the -80℃ freezer and, using sterilized inoculation tools, was streaked in three zones on TSA solid medium without sodium selenite. Then, the medium was placed in a 37℃ incubator for 24 h to obtain single colonies of strain 5-3. Next, the obtained single colonies were picked and inoculated into 3 mL of TSA liquid medium and cultured in a shaker at 37℃ for 24 h to successfully prepare a bacterial suspension of strain 5-3.

[0053] (2) Using a pipette, take 5-3 of the bacterial suspension and, through appropriate dilution or concentration, determine its OD value. 600 The value was adjusted to 0.05. Next, 100 μL of OD was accurately pipetted. 600 A bacterial suspension with a OD value of 0.05 was inoculated into a container containing 4 mL of TSA liquid medium containing 100 μg / mL sodium selenite, with three parallel control groups. The containers were incubated in a shaker at 37°C for 24 h, and the OD value of the bacterial suspension was measured every 12 h during the incubation period. 600 OD value and redness (a). After cultivation, fermentation broths with a small ratio of OD value to redness (a) value and an OD value of 0.7 or higher were selected and diluted to 10⁻⁶. −6 and 10 −7 A concentration gradient was established, and then appropriate amounts of each dilution were spread onto TSA solid plates containing 100 μg / mL sodium selenite. The plates were then inverted and incubated at 37°C for 24 h.

[0054] (3) Repeat the above-mentioned adaptive evolution experiment steps, gradually increasing the concentration of sodium selenite in the culture medium to 150 μg / mL, 200 μg / mL, 250 μg / mL and 300 μg / mL. After multiple rounds of screening and cultivation, the inorganic selenium tolerance of strain 5-3 was significantly enhanced. Specifically, a stepwise sodium selenite concentration acclimatization strategy was adopted, with a concentration increment of 50 μg / mL, starting from an initial concentration of 100 μg / mL and gradually increasing to a final concentration of 300 μg / mL. With the help of this gradual selection pressure, the inorganic selenium tolerance of strain 5-3 was specifically enhanced.

[0055] As shown in Figures 8 and 9, with the stepwise increase in sodium selenite selection pressure (incrementing by 50 μg / mL) during adaptive evolution, the redness value of the bacterial culture continuously increased, and the colony color gradually deepened. This significant trend indicates that with the increase of Na2SeO3 concentration, the efficiency of strain 5-3 in synthesizing elemental selenium or selenium nanoparticles (SeNPs) was effectively improved. Therefore, adaptive evolution successfully enhanced the tolerance of strain 5-3 to inorganic selenium.

[0056] The successfully enhanced strain 5-3 was isolated, purified, identified, and preserved to obtain the pantothecin clumps described in Example 1. AKFX-001. Example

[0057] This embodiment provides the pantothecin clumps preserved according to the present invention. AKFX-001 Growth curves at sodium selenite concentrations of 0 mM, 2.0 mM, 20 mM, and 100 mM are shown in the figure. Figure 10 As shown: Clumps of Pantothenia glutinosa AKFX-001 Images of *Pantotheca cumulus* 20-3, screened by our research group, cultured under sodium selenite concentrations of 0 mM, 2.0 mM, 20 mM, and 100 mM are shown below. Figure 11 As shown.

[0058] The results show that the pantothenic agglomerates preserved in this invention are effective. AKFX-001 It can grow rapidly at concentrations of 2-100 mM and convert inorganic selenium into nano-selenium, while Pantothenia glutinosa 20-3 hardly grows at concentrations of 2-100 mM. This demonstrates that the Pantothenia glutinosa preserved in this invention... AKFX-001 It has a high selenium tolerance. Example

[0059] This embodiment provides a method for determining the plant growth-promoting characteristics using strain AKFX-001, as detailed below.

[0060] ①Indoleacetic acid (IAA) production capacity: After incubation at 30°C for 48 h in a medium containing tryptophan, the IAA production capacity of strain AKFX-001 was determined by the Salkowski colorimetric method to be 45.2 ± 2.1 mg / L.

[0061] ② Phosphorus solubility: After culturing on PVK solid plates for 7 days, a clear phosphorus solubility zone appeared around the colonies, and the phosphorus solubility index was 2.5.

[0062] ③ Siderogenic capacity: After culturing on CAS test plates for 48 h, the appearance of an orange-yellow halo around the colony indicates that it has the ability to produce siderogenic substances. Example

[0063] This embodiment provides a method for synthesizing organoselenium using strain AKFX-001, as detailed below.

[0064] Fermentation medium: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, MgSO4 0.2 g / L, K2HPO4 1 g / L, sodium selenite 2 mM (0.3459 g / L), pH 7.0.

[0065] Fermentation method: The activated AKFX-001 strain was inoculated into the fermentation medium at an inoculum of 2% and cultured at 30℃ and 180rpm for 60 h with shaking.

[0066] 1. Determination of total selenium (refer to national standard fluorescence spectrophotometry)

[0067] Accurately weigh 0.5g~3g (accurate to 0.001g) of lyophilized bacterial strain, or accurately pipette 1.00mL~5.00mL of liquid sample into an Erlenmeyer flask. Add 10mL of nitric acid-perchloric acid mixture (9+1) and a few glass beads, cover with a watch glass, and digest overnight. The next day, heat on a hot plate, adding nitric acid as needed. When the solution becomes clear and colorless with the appearance of white fumes, continue heating until the remaining volume is about 2mL. Do not evaporate to dryness. After cooling, add 5mL of hydrochloric acid solution (6mol / L) and continue heating until the solution becomes clear and colorless with the appearance of white fumes. Continue heating until the remaining volume is about 2mL and cool. Simultaneously prepare a reagent blank. Add hydrochloric acid solution (1+9) to 5mL of the digested sample solution and blank solution, then add 20mL of EDTA mixture. Adjust the pH to a pale reddish-orange (pH 1.5~2.0) with ammonia solution (1+1) and hydrochloric acid solution (1+9). The following steps were performed in a darkroom: Add 3 mL of DAN reagent (1 g / L), mix well, heat in a boiling water bath for 5 min, remove and cool, add 3 mL of cyclohexane, shake for 4 min, transfer the entire solution to a separatory funnel, discard the aqueous layer after separation, and carefully pour the cyclohexane layer (upper layer) from the top of the separatory funnel into a capped test tube, ensuring no water droplets are mixed into the cyclohexane. Transfer the cyclohexane extract to a quartz cuvette, set the excitation wavelength to 376 nm and the emission wavelength to 520 nm on a fluorescence spectrophotometer, and measure its fluorescence intensity. Calculate the total selenium content in the sample based on the simultaneously measured selenium standard curve.

[0068] The lyophilized bacterial strain (0.1–0.2 g) was dissolved in 10 mL of hydrochloric acid (3 mol / L), sonicated for 15 minutes, and then heated at 100°C for 15 minutes. After cooling, the supernatant was obtained by centrifugation at 8000 × g for 15 minutes. The residue was further extracted twice using the above extraction process, and the resulting supernatants were combined. Subsequent measurement procedures were the same as for total selenium determination. The inorganic selenium content was determined using the same analytical methods as for total selenium determination. The organic selenium content was obtained by subtracting the inorganic selenium content from the total selenium content.

[0069] Selenium content (µg / ml) = Selenium bioconversion rate =

[0070] C: Selenium concentration (µg / mL) of the sample solution obtained from the standard curve.

[0071] C0: Selenium concentration in the blank solution (µg / mL) obtained from the standard curve.

[0072] V: Total volume of sample extract (mL)

[0073] m: Sample mass (ug)

[0074] Results: The organic selenium content in the fermentation broth accounted for 92.5% of the total selenium content, indicating that strain AKFX-001 can efficiently convert inorganic sodium selenite into organic selenium selenomethionine. Example

[0075] This embodiment provides a method for synthesizing amorphous selenium nanoparticles using strain AKFX-001, as detailed below.

[0076] Fermentation medium: TSA medium with added sodium selenite 3 mM (i.e. 0.414 g / L), pH 7.0.

[0077] Fermentation method: The activated strain was inoculated at a rate of 5% and cultured at 30℃ and 150 rpm for 96 h with shaking. The culture medium was observed to gradually change from colorless to orange-red, indicating the formation of zero-valent selenium nanoparticles.

[0078] The method for determining nano-selenium is as follows.

[0079] Centrifuge the culture medium at 13,500 × g for 20 minutes at 4°C. Wash the precipitate with PBS buffer, discard the supernatant, and collect the bacterial cell pellet. Resuspend the washed bacterial pellet in sterile water. Add 100 μL of lysozyme solution and incubate at 37°C for 3 hours. Then, sonicate on ice (40 minutes, 2 seconds on, 2 seconds off) to completely lyse the cells and release SeNPs. Resuspend the lysed mixture in 1.5 M Tris-HCl buffer (pH 8.3, containing 1% SDS) and wash several times, then centrifuge at 12,500 × g for 10 minutes at 4°C. Resuspend the pellet in sterile water, add 30% 1-octanol solution, mix thoroughly, and centrifuge at 2,000 × g for 5 minutes at 4°C. The precipitate was washed sequentially with deionized water, 70% ethanol, 100% ethanol, and chloroform. Finally, it was centrifuged at 13,500 × g for 10 minutes at 4°C to obtain a pure SeNPs precipitate. The SeNPs were directly characterized and confirmed using a combination of morphological observation and elemental analysis.

[0080] Extraction and Characterization: Bacterial cells were collected by centrifugation, washed with deionized water, and then subjected to ultrasonic disruption to release intracellular selenium nanoparticles. The cells were centrifuged again to collect the red precipitate (zero-valent selenium nanoparticles). Transmission electron microscopy (TEM) revealed that the selenium nanoparticles were spherical, uniformly distributed, and had an average particle size of 300 nm. X-ray diffraction (XRD) analysis confirmed that they were amorphous selenium nanoparticles.

[0081] Selenium bioconversion rate =

[0082] According to the test, its synthesis efficiency (conversion rate) of zero-valent nano-selenium is 88.3%.

Claims

1. A type of pantothecin AKFX-001, characterized in that: The Latin name of the clustered pantothecin AKFX-001 is: Pantoea agglomerans It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36122.

2. The use of the AKFX-001 clump-forming bacteria as described in claim 1, characterized in that: The AKFX-001 clump-forming pantothecin is used to convert inorganic selenite into organic selenium selenite in a 2-100 mM sodium selenite medium.

3. The use of the AKFX-001 clump-forming bacteria as described in claim 1, characterized in that: The aforementioned Pantotheca agglomerata AKFX-001 is used to convert inorganic selenite sodium selenite into amorphous nano-selenium in a 2-100 mM sodium selenite medium.

4. The use of the AKFX-001 clump-forming bacteria as described in claim 1, characterized in that: The AKFX-001 pantothecin bacteria is used to produce indoleacetic acid, phosphate solubilizers, and iron carriers.

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