High-selenium-resistant arthrobacter darwendi N-15 and application thereof

Abstract

The invention discloses arthrobacter gandavensis N-15 with high selenium resistance and application of the arthrobacter gandavensis N-15, the arthrobacter gandavensis N-15 with high selenium resistance is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M20242146, and the preservation date is October 9, 2024. The arthrobacter gandavensis N-15 has the advantages that the arthrobacter gandavensis N-15 can be used for preparing the selenium-resistant arthrobacter gandavensis N-15; the selenium resistance of the arthrobacter dabarangensis N-15 reaches 128 mmol / L, and the arthrobacter dabarangensis N-15 has stable adaptability to high selenium concentration and is convenient for biological preparation of nano-selenium. The arthrobacter dawlini N-15 can be used for reducing 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, the nano-selenium has relatively good antioxidant capacity. The arthrobacter dawlini N-15 and the nano-selenium produced by the arthrobacter dawlini N-15 can be widely applied to the fields of medicine, food and agriculture.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a highly selenium-resistant Arthrobacterium gandavanensis N-15 and an application thereof. Background Art

[0002] Selenium is an essential trace element for the human body. It is known for its powerful antioxidant properties. It can effectively remove free radicals in the body and protect cells from oxidative damage. It participates in the activation of multiple enzymes in the human body, helps the immune system to enhance its defense, and is essential for cardiovascular health, thyroid function and reproductive function. Appropriate selenium intake helps prevent cancer and delay aging. Through a reasonable diet, such as eating seafood, meat and whole grains and other selenium-rich foods, the human body can maintain an appropriate level of selenium and promote overall health.

[0003] Nano-selenium is a new form of selenium prepared by biotechnology, which has extremely high biological activity and safety. Through microbial transformation, inorganic selenium is converted into nano-scale organic selenium, which not only greatly improves the bioavailability of selenium, but also significantly reduces its toxicity. Nano-selenium has become a new star in the field of nutritional supplements and functional foods because of its unique nano-size effect, which can more effectively play the role of antioxidant, immunomodulatory and anti-cancer health benefits. Its green and environmentally friendly production process is also in line with modern people's pursuit of health and sustainable development.

[0004] Through the transformation of microorganisms, inorganic selenium can be effectively converted into organic selenium, especially nano-selenium, which is more easily absorbed and utilized by the human body. This not only significantly improves the bioavailability of selenium, but also greatly reduces the potential toxicity of selenium, making it safer and more reliable. In addition, the process of microbial conversion of selenium is relatively mild, does not require extreme conditions such as high temperature and high pressure, and does not produce harmful substances. Therefore, the screening, identification and development and utilization of natural microbial strains with high selenium resistance and selenium conversion have important research value and broad application prospects. Summary of the invention

[0005] The invention provides a highly selenium-resistant Arthrobacterium gandamansis N-15 and an application thereof. The Arthrobacterium gandamansis N-15 can still grow normally on a culture medium with a high concentration of selenite, and the Arthrobacterium gandamansis N-15 can reduce selenite into nano-selenium.

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

[0007] The second aspect of the present invention provides the use of the highly selenium-resistant Arthrobacter gandavensis N-15 for producing nano-selenium or products containing nano-selenium.

[0008] Products containing nano-selenium can be feed, fermented food, health products, agricultural preparations (such as plant nutrient solution, etc.), etc.

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

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

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

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

[0013] Optionally, the preparation method of the seed solution for inoculating Arthrobacter gandavensis N-15 as the selected one is as follows: streak the strain N-15 on LB solid medium for activation, pick a single clone of the strain after activation and transfer it to LB liquid medium, and culture it under constant temperature shaking at 180 rpm and 37°C. When the OD 600 When it is about 0.8, it is used as seed liquid.

[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 present invention has the following beneficial effects:

[0017] (1) The selenium resistance of the Arthrobacterium gandavensis N-15 of the present invention reaches 128 mmol / L, has stable adaptability to high selenium concentrations, and is convenient for the biological preparation of nano-selenium.

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

[0019] (3) The Gandaman Arthritis Bacillus N-15 of the present invention has low toxicity and multiple biological activities, including antibacterial, anticancer, anti-leishmanial, hemolytic, protein kinase inhibition and antioxidant activities, and is widely used in the fields of medicine, food, agriculture, etc.

[0020] (4) The nano-selenium produced by the Arthrobacterium gandavensis N-15 of the present invention has functional properties such as antibacterial, anti-tumor, and immune regulation. In addition, nano-selenium not only has special effects on the human body, but also can regulate plant growth, activate plant defense mechanisms, and reduce the negative impact of insect pests on plants. Nano-selenium has broad development prospects in the fields of medicine, food, agriculture, etc. due to its high bioavailability and safety. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 is the phylogenetic tree of strain N-15;

[0023] Figure 3 This is the growth curve of Arthrobacter gandavanensis N-15 in medium containing different concentrations of sodium selenite;

[0024] Figure 4 The growth of Arthrobacter gandamansis N-15 in medium containing different concentrations of sodium selenite;

[0025] Figure 5 is the reduction rate of Arthrobacterium gandavanensis N-15 to different concentrations of sodium selenite added;

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

[0027] Figure 7 The response surface diagram and contour diagram of the effects of inoculum size and temperature on the sodium selenite reduction rate of Arthrobacter gandamansis N-15;

[0028] Figure 8 The response surface diagram and contour diagram of the effects of shaker speed and temperature on the sodium selenite reduction rate of Arthrobacter gandavensis N-15;

[0029] Fig. 9 The response surface diagram and contour diagram of the effect of inoculation amount and shaking speed on the sodium selenite reduction rate of Arthrobacter gandavensis N-15;

[0030] Fig.10 is the particle size and potential distribution diagram of nano-selenium, A is the particle size of nano-selenium, B is the potential distribution of nano-selenium;

[0031] Fig.11 is the scavenging rate of DPPH free radicals by Arthrobacter gandamansis N-15;

[0032] Fig.12 is the scavenging rate of ABTS free radicals by Arthrobacter gandavanensis N-15;

[0033] Fig.13 The morphology and elemental composition of nano-selenium, A is the scanning electron microscope image, B is the EDS spectrum image;

[0034] Fig.14 This is the FTIR spectrum of nano-selenium. DETAILED DESCRIPTION

[0035] The present invention is further described below by way of examples, but the present invention is not limited thereto. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0036] The present invention provides a method for producing nano-selenium by using the highly selenium-resistant Arthrobacter gandavensis N-15. The Arthrobacter gandavensis N-15 is inoculated into a culture medium containing sodium selenite and cultured to synthesize nano-selenium.

[0037] Furthermore, 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 to 32 mmol / L.

[0039] In some examples, the inoculation amount of Arthrobacter gandavensis N-15 is 1% to 5%.

[0040] In some examples, the preparation method of the seed solution for inoculating Arthrobacter gandavensis N-15 as the selected strain is as follows: streak the strain N-15 on LB solid medium for activation, pick a single clone of the strain after activation and transfer it to LB liquid medium, and culture it at a constant temperature of 180 rpm and 37°C. 600 When it is about 0.8, it is used as seed liquid.

[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 the present invention, the reagents and instrument equipment information used are as follows:

[0044] M5 Taq HiFi PCR Mix (2×): Polymer Corporation;

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

[0046] Hydrochloric acid (super pure): Wuhan Xinshenshi Chemical Technology Co., Ltd.;

[0047] ZEALWAY-GI54DS high pressure sterilizer: American Zhiwei (Xiamen) Instrument Co., Ltd.;

[0048] Neo 15R refrigerated high-speed centrifuge: Eppendorf, Germany;

[0049] AG223331 PCR amplifier: Eppendorf, Germany;

[0050] UV1802G UV-visible spectrophotometer: Shanghai Spectrum Instrument Co., Ltd.;

[0051] JN-mini Pro low-temperature ultrahigh-pressure continuous flow cell disruptor: Guangzhou Juneng Nano Biotechnology Co., Ltd.; BENANO 180 ZETA PRO zeta potential and nanoparticle size analyzer: Dandong Better Instrument Co., Ltd.;

[0052] -80℃ ultra-low temperature freezer: Thermo Fisher Scientific Inc.

[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, the materials and reagents used in the following examples can be obtained from commercial sources. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Example 1 Isolation, purification and preservation of 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), shake at 30°C and 200 rpm for 30 min, and according to the 10-fold gradient dilution method, take 50 µL of the diluted bacterial solution and evenly spread it on LB solid culture medium containing 5 mmol / L sodium selenite, and culture it at 37°C to observe the colony morphology.

[0057] Use a sterile inoculation loop to pick up red colonies of different forms for isolation and purification. This type of strain can reduce inorganic selenite to red nano-selenium; after streaking purification on LB solid culture medium (containing 5 mmol / L sodium selenite), it is frozen and stored in a -80°C refrigerator.

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

[0059] The strain N-15 was streaked onto LB solid medium for activation. After the first generation of activation, a single clone was picked out and streaked onto an LB solid plate and an LB solid plate containing 1 mmol / L sodium selenite to continue growing.

[0060] like Figure 1 As shown, the bacterial colonies are yellow, with a bulge in the middle, a smooth and opaque surface, and a colony diameter ranging from 0.2 to 0.3 mm. They are positive after Gram staining. On a solid culture medium containing 1 mmol / L of selenite, the Gandavanian Arthritis Bacterium N-15 strain reduces selenite into red elemental selenium, showing as red colonies surrounded by a bright yellow outer circle.

[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 enzyme including dNTPs (2.5 mmol / L) 10.0 µL, 10 × Buffer 15.0 µL, Taq enzyme (5.0 U / µL) 1 µL, H 2 O 8.5 µL.

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

[0065] The PCR product was detected by 1% agarose gel electrophoresis and a specific band of about 1500 bp appeared.

[0066] After the PCR products were sent for sequencing, the sequencing results were subjected to Blast homology analysis on NCBI, and the sequence information of strains with homology greater than 98% and named model bacteria were selected to construct a phylogenetic tree using MEGA7.0 software.

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

[0068] Example 4 Growth curve determination of Arthrobacter gandamansis N-15

[0069] The Arthrobacter gandamansis N-15 was streaked and activated in LB solid medium. After activation, a single clone was picked out and transferred to LB liquid medium. The culture was shaken at 180 rpm and 37°C until the OD 600 About 0.8, made into seed liquid.

[0070] Before inoculation, take 1 mL of the seed solution and dilute it with sterile PBS buffer to an optical density (OD 600 ) was about 0.6, and then the bacterial solution was inoculated into 100 mL LB liquid culture medium at a volume fraction of 1% and cultured at 37°C and 180 rpm. The optical density (OD) of the bacterial solution at 600 nm was measured every 3 h. 600 ), 3 parallels per group, with OD 600 The value is the vertical axis and the culture time is the horizontal axis (h), and the growth curve is drawn.

[0071] In this experiment, the growth curve of strain N-15 was measured and plotted within 60 h. Figure 3 As shown in the figure, the growth of Arthrobacterium gandavanense N-15 was slow from 0 to 6 h, and the growth was rapid from 6 to 12 h. 600 The value increased from 0.1 to 1.5, and then at 15 h, the growth rate of the strain slowly entered the growth stable phase, OD 600 The value reaches around 2.0.

[0072] Example 5 Determination of the ability of Arthrobacter gandavensis N-15 to tolerate sodium selenite

[0073] The isolated Arthrobacterium gandavanensis N-15 was inoculated into LB liquid medium and cultured at 37°C and 180 rpm until the logarithmic phase. Then 200 µL of the above bacterial solution was added into a bacterial culture bottle containing 20 mL (1% inoculation volume) of LB liquid medium containing different concentrations of sodium selenite (32, 64, 128 mmol / L), and cultured at 37°C and 180 rpm for 36 h, and the color change of the liquid was 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 showed a distinct red color; 36 hours after inoculation, the fermentation broth with a sodium selenite concentration of 128 mmol / L turned dark red, indicating that within 36 hours, the selenium resistance of the Gandaman Arthritis Bacterium N-15 strain reached 128 mmol / L.

[0075] Example 6 Determination of the selenite reducing ability of Arthrobacter gandavensis N-15

[0076] The determination of selenite reduction rate of Bacillus gandavensis 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 selenium-rich bacteria in soil [J]. Soils, 2018, 50(06): 1203-1207. DOI: 10.13758 / j.cnki.tr.2018.06.023.). Pick a single colony on the LB solid medium and inoculate it into 50 mL LB liquid medium, place it in a shaking incubator at 37°C and 180 rpm and culture it overnight until the logarithmic growth phase. Take a 1% live bacterial suspension (absorbance value at 600 nm wavelength is 0.6-0.8) and inoculate it into LB liquid medium with initial concentrations of 2, 4, 8, 16, and 32 mmol / L of selenite, respectively. Set 3 replicates for each concentration, shake and culture at 37°C and 180 rpm for 60 h. After the culture solution is centrifuged at 10,000 rpm for 10 min, the concentration of Se(IV) in the precipitate is determined by 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 can be seen that at low selenium concentrations of less than 4 mmol / L and the same culture time, the reduction rate of selenite by Bacillus gandavensis N-15 was improved with the increase of selenite concentration, but at selenite concentrations of less than 4 mmol / L, the same culture time had no significant effect on the reduction rate of Bacillus gandavensis N-15. Compared with the reduction rate at 2 mmol / L selenium concentration, the reduction rates of 16 mmol / L and 32 mmol / L were significantly reduced, but the difference between the latter two was not significant, indicating that Bacillus gandavensis 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 Bacillus sp. N-15: Bacillus sp. N-15 was inoculated with 2 mmol / L sodium selenite TSB medium at a 2% inoculation rate 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 determined.

[0081] Effect of shaking speed on the production of nano-selenium by Bacillus gandavensis N-15: Bacillus gandavensis N-15 was inoculated with 2% inoculum into 2 mmol / L sodium selenite TSB medium and cultured at 140, 160, 180, 200 and 220 rpm at 37°C for 48 h, and the sodium selenite reduction rate of each group was determined.

[0082] Effect of sodium selenite addition on the production of nano-selenium by Bacillus sp. N-15: Bacillus sp. N-15 was inoculated with 2% inoculum into sodium selenite TSB medium with sodium selenite contents of 1, 2, 3, 4, and 5 mmol / L, respectively. After shaking culture at 37°C and 180 rpm for 48 h, the sodium selenite reduction rate of each group was determined.

[0083] Effect of inoculation size on the production of nano-selenium by Bacillus sp. N-15: The strain was inoculated in 2 mmol / L sodium selenite TSB medium at an inoculation size of 1%, 2%, 3%, 4%, and 5%. The sodium selenite reduction rate of each group was measured after cultured at 37°C and 180 rpm for 48 h.

[0084] Depend on Figure 6It can be seen that the reduction rate of sodium selenite by Bacillus gandavensis N-15 reached the maximum when the concentration of sodium selenite was 2 mmol / L, and the reduction rate gradually decreased with the increase of the concentration of sodium selenite; the optimal temperature for the reduction of sodium selenite by Bacillus gandavensis N-15 was 37℃; when the shaker speed was 180 rpm, the reduction rate of sodium selenite by Bacillus gandavensis N-15 was significantly higher than that of other groups; when the inoculation amount was between 1% and 5%, the reduction rate of sodium selenite by Bacillus gandavensis N-15 had an obvious trend of first increasing and then decreasing.

[0085] Example 8 Response surface experiment

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

[0087] The PB experiment was carried out, and each experiment was repeated three times. The results were statistically analyzed with the average value, and the results are shown in Table 3. The significance of the four factors was obtained. The main effect factors (P value < 0.05) were screened from all the single factors and used as the influencing factors of the response surface experimental 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 significant difference; ** indicates extremely significant difference. 2 =0.97, Adj R 2 =0.94, Pred R 2 =0.84;

[0095] On the basis of the PB experiment, the culture temperature, shaker speed and inoculation size were used as factors, and the reduction rate of sodium selenite was used as the evaluation index. The Box-Behnke design of the Design-Expert 13 software was used to design a response surface experiment with three factors and three levels. The factor levels of the response surface experiment are shown in Table 4.

[0096] Table 4 Factor levels of response surface experiment

[0097]

[0098] Table 5 Box-Benhnken experimental design and its results

[0099]

[0100] The results in the table were analyzed by regression analysis using Design Expert 13 software, and a quadratic regression equation was established between the sodium selenite reduction rate (Y) and the temperature (A), inoculation amount (B), and shaking table 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 variance analysis are shown in Table 6. The experimental model P<0.001 is extremely significant, the lack of fit term P=0.58>0.05 is not significant, the P values ​​of factors A and B are <0.01, and the P value of factor C is <0.05, indicating that the temperature, inoculation amount, and shaking speed have a significant effect on the reduction rate of sodium selenite.

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

[0104] The response surface diagram and contour diagram of the interaction between different factors on the reduction rate of sodium selenite are shown in Figure 7-9 As shown, Figure 7 The effect of inoculation amount 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 is shown in Figure 2. Fig. 9 This is the effect of inoculation size and shaking speed on the reduction rate of sodium selenite.

[0105] Table 6 Analysis of variance of regression model

[0106]

[0107] Example 9 Preparation of Nano-Selenium

[0108] The seed liquid was inoculated at a 1% inoculation rate into 100 mL of LB medium containing 2 mmol / L sodium selenite, and cultured at 37°C and 180 rpm for 48 h. The bacterial liquid was centrifuged at 12000 rpm for 10 min, and the precipitate was collected. After washing twice, a small amount of water was added to resuspend it. The resuspended liquid was treated with a cell disruptor (1200 bar), and the disrupted liquid was centrifuged at 8000 rpm for 10 min. 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 nano-selenium synthesized by strain N-15 were determined using a nanoparticle size potential analyzer.

[0111] Nano-selenium was prepared according to Example 9, and 2 mmol / L sodium selenite was added to the LB medium. After culturing for 24 h, the final SeNPs particle size was between 100-300 nm. The SeNPs particle size obtained after culturing for 48 h was between 200-400 nm. The Zeta potential results are shown in Fig.10 As shown, SeNPs particles are negatively charged, and the absolute value of the potential measured after 24 h of culture is between 52.2±1.17 mV on average. Zeta potential can reflect the strength of mutual repulsion or attraction between particles. The higher its absolute value, the more stable the system. The absolute value of the potential of SeNPs synthesized by Bacillus gandavensis N-15 of the present invention is higher than 40 mV, indicating that SeNPs have good stability.

[0112] Example 11 Antioxidant Experiment of Nano-Selenium

[0113] 1. DPPH free radical scavenging assay:

[0114] Take 2.0 mL of nano-selenium sample solution with different concentrations (0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL) and mix 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-visible 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-(A 1 -A 2 ) / A 0 ]×100%

[0116] In the formula, A0 is the blank absorbance (DPPH solution without sample); A 1 Represents the absorbance of the sample mixture; A 2 It represents the absorbance of the sample solution without DPPH.

[0117] like Fig.11 As shown in the figure, the nano-selenium produced by reducing sodium selenite by Arthrobacterium gandavensis N-15 has the ability to scavenge DPPH free radicals. When the concentration of nano-selenium is 1 µg / mL, the scavenging rate of DPPH free radicals reaches (17.41±0.8)%.

[0118] 2. ABTS free radical scavenging assay:

[0119] ABTS solution (7.0 mmol / L) and potassium persulfate (K 2 S 2 O 8 , 2.45 mmol / L) were mixed to prepare ABTS radical cation (ABTS + ), and the mixture was reacted at 25°C in the dark for 12 hours. Then, ABTS + The absorbance of the solution at 734 nm was diluted to 0.70±0.02. + The solution (3.0 mL) was mixed with 1.0 mL of nano-selenium sample solution of different concentrations (0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL) and reacted for 6 min in the dark. The absorbance was measured at 734 nm and used as a positive control. The ABTS free radical scavenging activity (%) was calculated using the following formula: ABTS free radical scavenging activity (%) = [1−(A 1 −A 2 ) / A 0 )]×100%

[0120] In the formula, A 0 is the blank absorbance (ABTS + Ascorbic acid solution does not contain sample); A 1 Represents the absorbance of the sample mixture; A 2 Indicates that it does not contain ABTS + The absorbance of the sample solution.

[0121] like Fig.12As shown in the figure, the nano-selenium produced by reducing sodium selenite by Arthrobacterium gandavanensis N-15 has a certain ability to scavenge ABTS free radicals. With the increase of nano-selenium concentration, the ABTS free radical scavenging rate gradually increased. In the concentration range of 0.05~1 µg / mL, the ABTS free radical scavenging rate is product concentration-dependent. When the concentration of nano-selenium is 1 µg / mL, the scavenging rate of ABTS free radicals reaches (60.01±2.13)%, which has a strong free radical scavenging ability.

[0122] Example 12 Scanning electron microscope and EDS spectrometer measurement

[0123] A small amount of freeze-dried nano-selenium sample was fixed on a conductive glue, and after gold spraying, it was observed at different magnifications in a scanning electron microscope, and the EDS sample was used for elemental analysis.

[0124] Nano-selenium synthesized by microorganisms converts inorganic selenium into nano-selenium in cells and then gradually secretes it outside the cells. The morphology of nano-selenium can be observed by scanning electron microscopy. The results of scanning electron microscopy are as follows: Fig.13 -A shows that the SeNPs obtained by reducing Bacillus gandavanensis N-15 are spherical, with a large amount of nano-selenium appearing in a spherical distribution, and the particle size is between 200-400 nm, which is basically consistent with the results of the particle size analyzer measurement. Fig.13 -B and Table 7 show that, in addition to Se, there are also a small amount of other elements in the main components, indicating that the spherical particles are nano-selenium, and the source of P and S may be the culture medium. The bacteria have a strong ability to enrich selenium. In addition to a small amount existing inside the cell, most of it will be secreted outside the cell, thus making the bacteria and the culture fluid appear red, and the longer the culture time, the darker the color.

[0125] Table 7 Spectral element analysis results

[0126]

[0127] Example 13 Fourier spectrometer measurement

[0128] Potassium bromide and nano-selenium freeze-dried samples were ground in a mass ratio of 100:1, mixed evenly, and compressed on a tablet press at 4000-400 cm -1 The freeze-dried nano-selenium sample was scanned at a wave number of .

[0129] like Fig.14 As shown, nano-selenium is at 3300.6 cm -1 There is a strong broad peak at 2926.8 cm, which is caused by the contraction vibration of the NH group of the amide group. Amide bonds in organisms connect amino acids to form protein molecules. A large number of amide groups indicate that protein molecules may be bound to the surface of nano-selenium. -1The absorption peak at 1653.5 cm is caused by the stretching vibration of the alkyl CH bond. -1 The characteristic peak at 1541.7 cm is attributed to the contraction vibration of carbonyl C=O and carboxyl COO-. -1 The absorption peak at 1397.8cm -1 、1235.5cm -1 、1068.4cm -1 The absorption peak at may be the stretching vibration of CO or CH in the polysaccharide molecule. This indicates that organic matter such as protein and polysaccharide may be bound to the surface of SeNPs, which is basically consistent with other reports on the synthesis of nano-selenium by microorganisms.

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

Claims

1. A highly selenium-resistant Arthrobacter gandavensis N-15, characterized in that: The highly selenium-resistant Arthrobacter gandavensis N-15 has been deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: M20242146 and the deposit date being October 9, 2024.

2. The use of the highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 1, characterized in that: Used for producing nano-selenium or products containing nano-selenium.

3. A method for producing nano-selenium using the highly selenium-resistant Arthrobacter gandavensis N-15 of claim 1, characterized in that: The Arthrobacter gandavensis N-15 is inoculated into a culture medium containing sodium selenite for culture to synthesize nano-selenium.

4. The method for producing nano-selenium by using highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 3, characterized in that: The cultured mixture is centrifuged, the precipitate is collected, crushed, centrifuged again, and the precipitate is collected to obtain the nano-selenium.

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

6. The method for producing nano-selenium by using highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 3, characterized in that: The inoculation amount of the Arthrobacter gandavensis N-15 is 1% to 5%.

7. The method for producing nano-selenium by using highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 3, characterized in that: The culture temperature is 28-40°C.

8. The method for producing nano-selenium by using highly selenium-resistant Arthrobacter gandavensis N-15 according to claim 3, characterized in that: The culture rotation speed is 140-220 rpm.

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