A high selenium-tolerant gardnerella vaginalis n-15 and application thereof

Abstract

The application discloses a high selenium-resistant Arthrobacter gandavensis N-15 and application thereof. The high selenium-resistant Arthrobacter gandavensis N-15 has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20242146, and the preservation date is October 9, 2024. The selenium-resistant capacity of the Arthrobacter gandavensis N-15 reaches 128 mmol / L, and the Arthrobacter gandavensis N-15 has stable adaptability to high selenium concentration, so that the biological preparation of nano selenium is facilitated. The Arthrobacter gandavensis N-15 can reduce sodium selenite to generate nano selenium, and the generated nano selenium has certain ABTS free radical scavenging capacity and DPPH free radical scavenging capacity, that is, has good antioxidant capacity. The Arthrobacter gandavensis N-15 and the generated nano selenium can be widely applied to the fields of medicine, food and agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a highly selenium-resistant Gandalvianacter N-15 bacillus and its applications. Background Technology

[0002] Selenium is an essential trace element for the human body, renowned for its powerful antioxidant properties. It effectively eliminates free radicals in the body, protecting cells from oxidative damage. It participates in the activation of various enzymes, helps boost the immune system's defenses, and is crucial for cardiovascular health, thyroid function, and reproductive function. Adequate selenium intake helps prevent and fight cancer and slows aging. Through a balanced diet, including foods rich in selenium such as seafood, meat, and whole grains, the body can maintain appropriate selenium levels, promoting overall health.

[0003] Nano-selenium is a novel form of selenium prepared using biotechnology, exhibiting extremely high bioactivity and safety. Through microbial transformation, inorganic selenium is converted into nano-sized organic selenium, significantly improving selenium bioavailability and reducing its toxicity. Due to its unique nano-size effect, nano-selenium can more effectively exert its health benefits, including antioxidant, immune-regulating, and anti-cancer properties, making it a rising star in the field of nutritional supplements and functional foods. Its green and environmentally friendly production process also aligns with modern people's pursuit of health and sustainable development.

[0004] Through microbial transformation, inorganic selenium can be effectively converted into organic selenium, especially in forms more easily absorbed and utilized by the human body, such as nano-selenium. This not only significantly improves the bioavailability of selenium but also greatly reduces its potential toxicity, making it safer and more reliable. Furthermore, the microbial transformation of selenium is relatively mild, requiring no extreme conditions such as high temperature or high pressure, and does not produce harmful substances. Therefore, the screening, identification, and development of natural microbial strains with high selenium tolerance and selenium transformation capabilities have significant research value and broad application prospects. Summary of the Invention

[0005] This invention provides a highly selenium-resistant Gandavian arthritis bacillus N-15 and its application. The Gandavian arthritis bacillus N-15 can still grow normally on a culture medium with a high concentration of selenite, and the Gandavian arthritis bacillus N-15 can reduce selenite to nano-selenium.

[0006] In a first aspect, the present invention provides a highly selenium-resistant Arthrobacter gandavensis N-15, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20242146 and deposit date of October 9, 2024.

[0007] In a second aspect, the present invention provides the application of the above-mentioned highly selenium-resistant Arthrobacter gandavensis N-15 for the production of nano-selenium, or for the production of products containing nano-selenium.

[0008] Products containing nano-selenium can be used for feed, fermented foods, health products, and agricultural preparations (such as plant nutrient solutions).

[0009] A third aspect of the present invention provides a method for producing nano-selenium from the above-mentioned highly selenium-resistant Arthrobacter gandavensis N-15, wherein the Arthrobacter gandavensis N-15 is inoculated into a culture medium containing sodium selenite and cultured to synthesize nano-selenium.

[0010] Further, the cultured mixture is centrifuged, the precipitate is collected, crushed, centrifuged again, and the precipitate is collected to obtain the nano-selenium.

[0011] Furthermore, the concentration of sodium selenite in the culture medium is 2–32 mmol / L.

[0012] Furthermore, the inoculation amount of Arthrobacter gandavensis N-15 is 1% to 5%.

[0013] Optionally, the seed culture prepared using *Arthrobacter gandavensis* N-15 is prepared as follows: Strain N-15 is streaked onto LB solid medium for activation. After activation, single clones are picked and transferred to LB liquid medium. The culture is then incubated with constant temperature shaking at 180 rpm and 37°C. When the OD... 600 When the concentration is approximately 0.8, it is used as seed solution.

[0014] Furthermore, the culture temperature is 28–40°C.

[0015] Furthermore, the culture rotation speed is 140–220 rpm.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] (1) The selenium tolerance of Gandavella N-15 of the present invention reaches 128 mmol / L, and it has stable adaptability to high selenium concentration, which facilitates the bio-preparation of nano-selenium.

[0018] (2) The Gandarwinia arthritis N-15 of the present invention can reduce sodium selenite to produce nano-selenium.

[0019] (3) The Gandavella N-15 of the present invention has low toxicity and a variety of biological activities, including antibacterial, anticancer, antileishmaniasis, hemolytic, protein kinase inhibitory and antioxidant activities, and is widely used in medicine, food, agriculture and other fields.

[0020] (4) The nano-selenium produced by Gandavella N-15 of the present invention has antibacterial, antitumor, and immunomodulatory functional properties. Furthermore, nano-selenium not only has special effects on the human body, but can also regulate plant growth, activate plant defense mechanisms, and reduce the negative impact of pests on plants. Due to its high bioavailability and safety, nano-selenium has broad development prospects in the fields of medicine, food, and agriculture. Attached Figure Description

[0021] Figure 1 The colony morphology characteristics of strain N-15;

[0022] Figure 2 Phylogenetic tree of strain N-15;

[0023] Figure 3 The growth curves of Gandavella N-15 arthritis in medium containing different concentrations of sodium selenite are shown.

[0024] Figure 4 The growth of Gandavella N-15 arthritis bacteria in medium containing different concentrations of sodium selenite;

[0025] Figure 5 The reduction rate of Gandavella N-15 arthritis bacteria to different concentrations of sodium selenite;

[0026] Figure 6 The effect of different reaction conditions on the sodium selenite reduction rate of Gandavella N-15.

[0027] Figure 7 Response surface plot and contour plot showing the effect of inoculum size and temperature on the sodium selenite reduction rate of Gandavella N-15.

[0028] Figure 8 Response surface plot and contour plot showing the effect of shaker speed and temperature on the sodium selenite reduction rate of Gandavella N-15.

[0029] Figure 9 Response surface plot and contour plot showing the effect of inoculum size and shaker speed on the sodium selenite reduction rate of Gandavella N-15.

[0030] Figure 10 The diagram shows the particle size and potential distribution of nano-selenium, where A represents the particle size of nano-selenium and B represents the potential distribution of nano-selenium.

[0031] Figure 11 The scavenging rate of DPPH free radicals by Gandavella N-15.

[0032] Figure 12 The scavenging rate of ABTS free radicals by Gandavella N-15.

[0033] Figure 13 The images show the morphology and elemental composition of nano-selenium. A is a scanning electron microscope image, and B is an EDS spectrum.

[0034] Figure 14 This is the FTIR spectrum of nano-selenium. Detailed Implementation

[0035] The present invention will be further illustrated by the following embodiments, but these embodiments are not intended to limit the invention. Any changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0036] This invention provides a method for producing nano-selenium from the above-mentioned highly selenium-resistant Arthrobacter gandavensis N-15, wherein the Arthrobacter gandavensis N-15 is inoculated into a culture medium containing sodium selenite and cultured to synthesize nano-selenium.

[0037] Further, the cultured mixture is centrifuged, the precipitate is collected, crushed, centrifuged again, and the precipitate is collected to obtain the nano-selenium.

[0038] In some examples, the concentration of sodium selenite in the culture medium is 2–32 mmol / L.

[0039] In some examples, the inoculum size of the *Arthrobacter gandavensis* N-15 was 1% to 5%.

[0040] In some examples, the seed culture prepared using *Arthrobacter gandavensis* N-15 as the selected inoculum was prepared as follows: Strain N-15 was streaked onto LB solid medium for activation; after activation, single clones were picked and transferred to LB liquid medium, and cultured with constant temperature shaking at 180 rpm and 37°C. When OD... 600 When the concentration is approximately 0.8, it is used as seed solution.

[0041] In some examples, the culture temperature is 28–40°C.

[0042] In some examples, the culture rotation speed is 140–220 rpm.

[0043] In the following embodiments provided by this invention, the reagents and instruments used are as follows:

[0044] M5 Taq HiFi PCR Mix (2×): Polymertech Co., Ltd.

[0045] Sodium selenite (analytical grade), ascorbic acid, peptone, yeast extract, sodium chloride, agar powder: Sinopharm Chemical Reagent Co., Ltd.

[0046] Hydrochloric acid (superior grade): Wuhan Xinshen Chemical Technology Co., Ltd.;

[0047] ZEALWAY-GI54DS Autoclave: ZEALWAY (Xiamen) Instrument Co., Ltd., USA;

[0048] Neo 15R Refrigerated High-Speed ​​Centrifuge: Eppendorf GmbH, Germany;

[0049] AG223331 PCR Amplifier: Eppendorf GmbH, Germany;

[0050] UV1802G UV-Vis Spectrophotometer: Shanghai Spectrometer Instruments Co., Ltd.;

[0051] JN-mini Pro Low-Temperature Ultra-High Pressure Continuous Flow Cell Disruptor: Guangzhou Juneng Nanobiotechnology Co., Ltd.; BENANO 180 ZETA PRO Zeta Potential and Nanoparticle Size Analyzer: Dandong Better Instruments Co., Ltd.

[0052] -80℃ Ultra-Low Temperature Freezer: Thermo Fisher Scientific.

[0053] In some examples, LB medium (g / L): peptone 10.0, yeast extract 5.0, NaCl 10.0, pH 7.0-7.2.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, all experimental methods used in the following examples are conventional methods.

[0055] Example 1: Isolation, purification, and preservation of bacterial strains

[0056] Weigh 10 g of cow dung sample and add it to a sterile conical flask pre-filled with 10 glass beads and 90 mL of sterile PBS (phosphate buffered saline solution). Shake at 30°C and 200 rpm for 30 min. Following a 10-fold serial dilution method, take 50 µL of the diluted bacterial solution and spread it evenly on LB solid medium containing 5 mmol / L sodium selenite. Incubate at 37°C and observe the colony morphology.

[0057] Different morphological red colonies were picked using a sterile inoculation loop for isolation and purification. This type of strain can reduce inorganic selenite to red nano-selenium. After purification by streaking on LB solid medium (containing 5 mmol / L sodium selenite), the culture was stored frozen at -80°C.

[0058] Example 2: Morphological characteristics of strain N-15

[0059] Strawberry strain N-15 was activated by streaking on LB solid medium. After the first generation of activation, single clones were picked and streaked onto LB solid plates and LB solid plates containing 1 mmol / L sodium selenite for further growth.

[0060] like Figure 1 As shown, bacterial colonies are yellow, with a raised center, uniform edges, and a smooth, opaque surface. The colony diameter is between 0.2 and 0.3 mm. They are Gram-positive. On a solid culture medium containing 1 mmol / L selenite, the Gandavella N-15 strain reduces selenite to red elemental selenium, resulting in red colonies surrounded by a bright yellow outer ring.

[0061] Example 3 Molecular biological identification of strain N-15

[0062] Strain N-15 was amplified by PCR using the universal bacterial 16S rDNA primers 27F (5'-agagtttgatcctggctcag-3'), whose nucleotide sequence is shown in SEQ NO.1, and 1492R (5'-tacggctaccttgtacgactt-3'), whose nucleotide sequence is shown in SEQ NO.2.

[0063] PCR reaction system (25 µL): 1.0 µL each of primer 27F and primer 1492R (20 μmol / L); 1.0 µL template DNA; mixed enzymes including 10.0 µL dNTPs (2.5 mmol / L), 15.0 µL 10 × Buffer, 1 µL Taq enzyme (5.0 U / µL), and 8.5 µL H2O.

[0064] The PCR reaction program was as follows: 95℃ for 3 min; 94℃ for 25 s, 55℃ for 25 s, 72℃ for 30 s, 34 cycles; 72℃ for 5 min.

[0065] The PCR product showed a specific band of approximately 1500 bp after detection by 1% agarose gel electrophoresis.

[0066] After the PCR products were sent for sequencing, the sequencing results were analyzed for Blast homology on NCBI. The sequence information of strains with homology greater than 98% and the named type bacteria were selected, and a phylogenetic tree was constructed using MEGA7.0 software.

[0067] like Figure 2 As shown, strain N-15 has the highest sequence similarity to strain Arthrobacter gandavensis SR10. Based on phylogenetic analysis and molecular biological identification results, strain N-15 was identified as Arthrobacter gandavensis.

[0068] Example 4: Growth curve determination of Gandavella N-15

[0069] Gandavella N-15 was streaked onto LB solid medium for activation. After activation, single clones were picked and transferred to LB liquid medium, and cultured at 180 rpm and 37°C with constant temperature shaking until OD. 600 Approximately 0.8, prepared as seed liquid.

[0070] Before inoculation, take 1 mL of seed culture and dilute it with sterile PBS buffer to the optical density (OD). 600 The concentration of the bacterial culture was approximately 0.6. Then, at an inoculum volume fraction of 1%, the bacterial culture was inoculated into 100 mL of LB liquid medium and cultured at 37°C and 180 rpm. The optical density (OD) of the bacterial culture at 600 nm was measured every 3 hours. 600 ), 3 parallels per group, with OD 600 Plot the growth curve with the values ​​on the ordinate and the culture time (h) on the abscissa.

[0071] This experiment determined and plotted the growth curve of strain N-15 over 60 h, as follows: Figure 3 As shown, *Gandavella* N-15 grows slowly from 0 to 6 hours, and rapidly from 6 to 12 hours, with an OD... 600 The value increased from around 0.1 to around 1.5, and then, after 15 hours, the growth rate of the strain slowly entered a stationary phase. 600 The value reached around 2.0.

[0072] Example 5: Determination of the tolerance of Gandravella ulmoides N-15 to sodium selenite.

[0073] The isolated *Gandavella N-15* bacteria were inoculated into LB liquid medium and cultured at 37°C and 180 rpm until the logarithmic phase. Then, 200 µL of the bacterial culture was added to 20 mL (1% inoculum) of LB liquid medium containing different concentrations of sodium selenite (32, 64, and 128 mmol / L) in bacterial culture flasks and cultured at 37°C and 180 rpm for 36 h. The color change of the liquid was then observed.

[0074] The results are as follows Figure 4 As shown, 24 hours after inoculation, the fermentation broth with a sodium selenite concentration of 32 mmol / L turned a distinct red color; 36 hours after inoculation, the fermentation broth with a sodium selenite concentration of 128 mmol / L turned a deep red color, indicating that the selenium tolerance of Gandavella N-15 strain reached 128 mmol / L within 36 hours.

[0075] Example 6: Determination of the reducing power of Gandavella N-15 for selenite.

[0076] The determination of selenite reduction rate by Gandavella N-15 was optimized according to the method of Liao Qing et al. (Liao Qing, Liu Yongxian, Xing Ying, et al. Screening and identification of soil selenium-enriched bacteria [J]. Soil, 2018, 50(06):1203-1207.DOI:10.13758 / j.cnki.tr.2018.06.023.). Single colonies from LB solid medium were inoculated into 50 mL of LB liquid medium and cultured overnight at 37°C and 180 rpm until the logarithmic growth phase. Live bacterial suspensions (absorbance at 600 nm range of 0.6-0.8) were inoculated at initial concentrations of 2, 4, 8, 16, and 32 mmol / L selenite LB liquid medium, with three replicates for each concentration. The cultures were incubated at 37°C and 180 rpm for 60 h with shaking. After centrifugation at 10000 rpm for 10 min, the concentration of Se(IV) in the precipitate was determined using the HG-AFS method. The formula for calculating the selenite reduction rate is as follows:

[0077] Selenite reduction rate (%) = precipitated selenium content / initial total selenium × 100%.

[0078] Depend on Figure 5It was found that at low selenium concentrations (less than 4 mmol / L) and with the same culture time, the reduction rate of selenium by Gandavella N-15 increased with increasing selenium concentration. However, at selenium concentrations (less than 4 mmol / L), the same culture time had no significant effect on the reduction rate of Gandavella N-15. Compared with the reduction rate at 2 mmol / L selenium concentration, the reduction rates at 16 mmol / L and 32 mmol / L were significantly lower, but the difference between the latter two was not significant, indicating that Gandavella N-15 has a stable adaptability to high selenium concentrations.

[0079] Example 7 Single-factor experiment

[0080] Effect of temperature on the production of nano-selenium by Gandavella N-15: Gandavella N-15 was inoculated into 2 mmol / L sodium selenite TSB medium at a 2% inoculum and cultured at 28, 31, 34, 37 and 40 °C with shaking at 180 rpm for 48 h. The sodium selenite reduction rate of each group was then measured.

[0081] Effect of shaking speed on the production of nano-selenium by Gandavian arthritis N-15: Gandavian arthritis N-15 was inoculated into 2 mmol / L sodium selenite TSB medium at a 2% inoculum and cultured at 140, 160, 180, 200 and 220 rpm at 37℃ for 48 h. The sodium selenite reduction rate of each group was then measured.

[0082] Effect of sodium selenite addition on nano-selenium production by Gandavian arthritis N-15: Gandavian arthritis N-15 was inoculated at a 2% inoculum amount into sodium selenite TSB medium with sodium selenite concentrations of 1, 2, 3, 4, and 5 mmol / L, respectively. After incubation at 37℃ and 180 rpm for 48 h, the sodium selenite reduction rate of each group was measured.

[0083] Effect of inoculum size on the production of nano-selenium by Gandavella N-15: The strains were inoculated into 2 mmol / L sodium selenite TSB medium at inoculum sizes of 1%, 2%, 3%, 4%, and 5%. The sodium selenite reduction rate of each group was measured after incubation at 37℃ and 180 rpm for 48 h.

[0084] Depend on Figure 6It was found that the reduction rate of sodium selenite by Gandavella N-15 reached its maximum when the sodium selenite concentration was 2 mmol / L, and the reduction rate gradually decreased with the increase of sodium selenite concentration. The optimal temperature for the reduction of sodium selenite by Gandavella N-15 was 37℃. When the shaker speed was 180 rpm, the reduction rate of sodium selenite by Gandavella N-15 was significantly higher than that of other groups. When the inoculum amount was between 1% and 5%, the reduction rate of sodium selenite by Gandavella N-15 showed a clear trend of first increasing and then decreasing.

[0085] Example 8 Response Surface Experiment

[0086] Based on the results of the single-factor experiments, the value ranges of the four factors were obtained, as shown in Table 1. The sodium selenite addition amount was 1–3 mmol·L⁻¹. -1 The shaking speed is 160-200 rpm, the temperature is 34-40℃, and the inoculation amount is 2-4%.

[0087] PB experiments were conducted, with each experiment repeated three times. The results were statistically analyzed using the average value, and the results are shown in Table 3, revealing the significance of the four factors. Major factors (P-value < 0.05) were selected from all single-factor experiments and used as the influencing factors in the response surface methodology design.

[0088] Table 1. PB Experimental Factors and Levels

[0089]

[0090] Table 2 PB Experimental Design and Results

[0091]

[0092] Table 3. Analysis of Variance of PB Experimental Data

[0093]

[0094] * indicates a significant difference; ** indicates an extremely significant difference. R 2 =0.97, Adj R 2 =0.94, Pred R 2 =0.84;

[0095] Based on the PB experiment, with culture temperature, shaking speed and inoculum amount as factors, and sodium selenite reduction rate as evaluation index, a three-factor, three-level response surface methodology was designed using the Box-Behnke design of Design-Expert 13 software. The response surface methodology factor levels are shown in Table 4.

[0096] Table 4 Response Surface Experimental Factor Levels

[0097]

[0098] Table 5 Box-Benhnken Experimental Design and Results

[0099]

[0100] Using Design Expert 13 software, regression analysis was performed on the table results to establish a quadratic regression equation between sodium selenite reduction rate (Y) and temperature (A), inoculum size (B), and shaking speed (C):

[0101] Sodium selenite reduction rate (Y) = 0.9552 + 0.0139A + 0.0073B - 0.0244C + 0.0056AB + 0.0046AC + 0.0024BC - 0.0113A 2 - 0.0364B 2 - 0.0198C 2 .

[0102] The results of the analysis of variance are shown in Table 6. The experimental model P < 0.001, which is highly significant. The lack-of-fit term P = 0.58 > 0.05 is not significant. The P < 0.01 for factors A and B, and the P < 0.05 for factor C, indicate that temperature, inoculum size, and shaking speed have a significant effect on the reduction rate of sodium selenite.

[0103] The optimal fermentation medium predicted using Design-Expert 13 software was: temperature 38.634℃, inoculum size 3.163%, and shaker speed 182.0 rpm. Under these conditions, the maximum reduction rate was predicted to be 96%. To verify the effectiveness of the model, the optimal fermentation conditions were modified to: temperature 38℃, inoculum size 3%, and shaker speed 180 rpm. Under these conditions, the sodium selenite reduction rate of strain N-15 was 95.48%, which is basically consistent with the predicted value, indicating that the model is reliable.

[0104] Response surface plots and contour plots showing the effect of interactions between different factors on the reduction rate of sodium selenite are shown below. Figure 7-9 As shown, Figure 7 The effects of inoculum size and temperature on the reduction rate of sodium selenite. Figure 8 The effect of shaking speed and temperature on the reduction rate of sodium selenite. Figure 9 The effects of inoculum size and shaker speed on the reduction rate of sodium selenite.

[0105] Table 6. Analysis of Variance for Regression Models

[0106]

[0107] Example 9 Preparation of nano-selenium

[0108] The seed culture was inoculated at a rate of 1% into 100 mL of LB medium containing 2 mmol / L sodium selenite and cultured at 37℃ and 180 rpm for 48 h. After centrifuging the bacterial culture at 12000 rpm for 10 min, the precipitate was collected, washed twice with water, and resuspended with a small amount of water. The resuspended liquid was treated with a cell disruptor (1200 bar). The disrupted liquid was centrifuged at 8000 rpm for 10 min, and the precipitate was collected to obtain nano-selenium.

[0109] Example 10: Particle size distribution and zeta potential analysis of nano-selenium

[0110] The particle size and zeta potential of the selenium nanoparticles synthesized by strain N-15 were determined using a nanoparticle size potential analyzer.

[0111] Selenium nanoparticles were prepared according to Example 9. 2 mmol / L sodium selenite was added to LB medium, and after culturing for 24 h, the resulting SeNPs particles had a size between 100-300 nm. After culturing for 48 h, the SeNPs particles had a size between 200-400 nm; the Zeta potential results are as follows. Figure 10 As shown, the SeNPs particles are negatively charged, and the average absolute value of their potential measured after 24 h of culture is between 52.2 ± 1.17 mV. The zeta potential reflects the strength of the repulsive or attractive forces between particles; the higher the absolute value, the more stable the system. The SeNPs synthesized by *Gandavella N-15* in this invention have an absolute potential value higher than 40 mV, indicating that the SeNPs possess good stability.

[0112] Example 11 Antioxidant Experiment of Nano Selenium

[0113] 1. DPPH free radical scavenging assay:

[0114] Mix 2.0 mL of nano-selenium sample solutions of different concentrations (0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL) with 2.0 mL of DPPH-ethanol solution (0.1 mmol / L). Shake the mixture thoroughly and incubate at 25°C in the dark for 30 minutes. Measure the absorbance of the mixture at 517 nm using a UV-Vis spectrophotometer. Ascorbic acid was used as a positive antioxidant control. Calculate the DPPH free radical scavenging activity (%) using the following formula:

[0115] DPPH free radical scavenging activity (%) = [1 - (A1 - A2) / A0] × 100%

[0116] In the formula, A0 is the blank absorbance (DPPH solution without sample); A1 represents the absorbance of the sample mixture; and A2 represents the absorbance of the sample solution without DPPH.

[0117] like Figure 11 As shown, the nano-selenium produced by the reduction of sodium selenite by Gandavella N-15 exhibits scavenging ability against DPPH free radicals. When the concentration of nano-selenium is 1 µg / mL, the scavenging rate against DPPH free radicals reaches (17.41±0.8)%.

[0118] 2. ABTS free radical scavenging assay:

[0119] ABTS radical cations (ABTS) were prepared by mixing ABTS solution (7.0 mmol / L) with potassium persulfate (K2S2O8, 2.45 mmol / L). + The mixture was then reacted at 25°C in the dark for 12 hours. ABTS was then dissolved in phosphate-buffered saline (PBS, pH 7.4). + The absorbance of the solution at 734 nm was diluted to 0.70 ± 0.02. The above ABTS... + The solution (3.0 mL) was mixed with 1.0 mL of nano-selenium sample solutions of different concentrations (0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL), and reacted in the dark for 6 min. The absorbance was measured at 734 nm, and this was used as a positive control. The ABTS free radical scavenging activity (%) was calculated using the following formula: ABTS free radical scavenging activity (%) = [1 − (A1 − A2) / A0)] × 100%

[0120] In the formula, A0 is the blank absorbance (ABTS). + (Ascorbic acid solution without sample); A1 indicates the absorbance of the sample mixture; A2 indicates the absence of ABTS. + The absorbance of the sample solution.

[0121] like Figure 12 As shown, the nano-selenium produced by the reduction of sodium selenite by Gandavella N-15 exhibits a certain ABTS free radical scavenging ability. The ABTS free radical scavenging rate gradually increases with increasing nano-selenium concentration. Within the concentration range of 0.05–1 µg / mL, the ABTS free radical scavenging rate shows a product concentration dependence. When the nano-selenium concentration is 1 µg / mL, the ABTS free radical scavenging rate reaches (60.01 ± 2.13)%, demonstrating a strong free radical scavenging ability.

[0122] Example 12: Measurement using scanning electron microscopy and EDS (energy dispersive spectroscopy)

[0123] A small amount of freeze-dried selenium nanoparticles were fixed on conductive adhesive, sputtered with gold, and observed at different magnifications under a scanning electron microscope. Elemental analysis was performed using EDS samples.

[0124] Microbial-synthesized selenium nanoparticles are converted from inorganic selenium into nanoparticles within cells and then gradually secreted extracellularly. The morphology of these nanoparticles can be observed using scanning electron microscopy (SEM). The SEM results are shown below. Figure 13 As shown in Figure -A, the SeNPs obtained from the reduction of Gandavella N-15 by *Artemisininus gandavidii* are spherical, with a large number of nano-selenium particles appearing in a spherical distribution, and the particle size is between 200-400 nm. This is basically consistent with the particle size analyzer measurement results, and further... Figure 13 As can be seen from B and Table 7, the main components, besides Se, include small amounts of other elements, indicating that the spherical particles are nano-selenium, while the P and S likely originate from the culture medium. The bacteria have a strong selenium enrichment capacity; aside from a small amount existing intracellularly, most is secreted extracellularly, thus giving the bacteria and culture medium a red color, and the longer the culture time, the deeper the color.

[0125] Table 7 Elemental Analysis Results

[0126]

[0127] Example 13 Fourier Transform Spectrometry Measurement

[0128] Potassium bromide and lyophilized nano-selenium samples were ground at a mass ratio of 100:1, mixed thoroughly, and then compressed into tablets using a tablet press at 4000–400 cm⁻¹. -1 The freeze-dried selenium nanoparticles were scanned at a wavenumber of 1000.

[0129] like Figure 14 As shown, nano-selenium at 3300.6 cm⁻¹ -1 The presence of a strong, broad peak at 2926.8 cm⁻¹ is caused by the contraction vibration of the NH group in the amide group. In biological organisms, amide bonds connect amino acids to form protein molecules; the large number of amide groups suggests that protein molecules may be bound to the surface of the selenium nanoparticles. -1 The absorption peak at 1653.5 cm⁻¹ is caused by the stretching vibration of the alkyl CH bond. -1 The characteristic peak at 1541.7 cm⁻¹ is attributed to the contraction vibrations of the carbonyl group (C=O) and the carboxyl group (COO⁻). -1 The absorption peak at 1397.8 cm⁻¹ may be due to the stretching vibration of the double bond in the benzene ring; -1 1235.5cm -1 1068.4cm -1 The absorption peak at this point may be due to the stretching vibrations of CO or CH in the polysaccharide molecule. This indicates that organic matter such as proteins and polysaccharides may be bound to the surface of SeNPs, which is consistent with other reports of microbial synthesis of selenium nanoparticles.

[0130] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for producing nano-selenium using highly selenium-resistant Arthrobacter gandavensis N-15, characterized in that, The *Arthrobacter gandavensis* N-15 was inoculated into a culture medium containing sodium selenite and cultured to synthesize selenium nanoparticles. The highly selenium-tolerant *Arthrobacter gandavensis* N-15 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20242146 and deposit date of October 9, 2024.

2. The method for producing nano-selenium from highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 1, characterized in that, The cultured mixture was centrifuged, the precipitate was collected, crushed, centrifuged again, and the precipitate was collected to obtain the nano-selenium.

3. The method for producing nano-selenium from highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 1, characterized in that, In the culture medium, the concentration of sodium selenite is 2–32 mmol / L.

4. The method for producing nano-selenium from highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 1, characterized in that, The inoculation amount of Arthrobacter gandavensis N-15 is 1% to 5%.

5. The method for producing nano-selenium from highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 1, characterized in that, The culture temperature is 28–40°C.

6. The method for producing nano-selenium from highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 1, characterized in that, The culture rotation speed is 140–220 rpm.

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