Bacillus subtilis for co-production of selenium polysaccharide-nano-selenium and application thereof

Abstract

The invention discloses bacillus subtilis for co-production of selenium polysaccharide-nano-selenium and application of the bacillus subtilis, and belongs to the technical field of microbial synthesis and bioengineering. The preservation number of the bacillus subtilis is GDMCC (China General Microbiological Culture Collection Center) NO: 67288. Based on the strain, the invention establishes an efficient and green co-production process for synchronously preparing polysaccharide-stabilized nano-selenium (EPS-SeNPs) and selenium polysaccharide (Se-EPS) by a one-pot method, and the process can synchronously and efficiently produce the selenium polysaccharide (703.87 g / kg) with high polysaccharide content and the polysaccharide-stabilized nano-selenium with high selenium load and strong antioxidation from a single fermentation system. The process is simplified, meanwhile, selenium resources are efficiently utilized, product functions are synergistically interacted, and an efficient, low-cost and environment-friendly solution is provided for developing novel functional selenium-based materials.

Description

Technical Field

[0001] This invention relates to the field of microbial synthesis and bioengineering technology, and in particular to a strain of Bacillus subtilis that co-produces selenium polysaccharide-nano-selenium and its applications. Background Technology

[0002] Selenium, an essential trace element for the human body, possesses various biological activities, including antioxidant, antitumor, heavy metal toxicity antagonism, and immune system regulation. Currently, selenium exists in nature primarily in inorganic, organic, and elemental forms. Among these, nano-selenium, due to its excellent biocompatibility and activity, shows great promise in the fields of biomedicine and nutritional fortification. However, nano-selenium is prone to aggregation and oxidation in aqueous systems due to its high surface energy, leading to decreased stability and loss of biological activity, severely limiting its practical applications. To improve the stability of nano-selenium, researchers typically use polysaccharides as natural stabilizers, forming "polysaccharide-nano-selenium" complexes through physical coating or chemical bonding to enhance its dispersibility and biological activity. Selenization of polysaccharides can combine the biological functions of polysaccharides with the bioactive advantages of nano-selenium; however, current technologies often require separate steps for the synthesis of selenium polysaccharides and the preparation of nano-selenium, resulting in cumbersome processes that can easily introduce harmful reagents, making it difficult to achieve green and efficient production.

[0003] Currently, the main methods for synthesizing nano-selenium include physical methods, chemical methods, and biotransformation methods. While physical methods can prepare high-purity nano-selenium, the equipment is expensive, the reaction conditions are harsh, the yield is low, and it is prone to aggregation, making it unsuitable for large-scale applications. Chemical methods can prepare nano-selenium and control its morphology using reducing agents, but it has poor stability, is easily converted into highly toxic gray-black elemental selenium, and the use of chemical reagents may cause environmental pollution. Biotransformation methods utilize microorganisms or plants to reduce inorganic selenium to nano-selenium, offering advantages such as environmental friendliness and high biocompatibility, and have become a research hotspot. Existing research shows that microorganisms such as Bacillus subtilis can reduce selenite to generate nano-selenium and simultaneously secrete extracellular polysaccharides. If these extracellular polysaccharides can combine with nano-selenium to form stable complexes, or simultaneously synthesize selenium polysaccharides, it is hoped that the simultaneous completion of "nano-selenium stabilization" and "polysaccharide functionalization" can be achieved in a single fermentation system. This co-production strategy not only simplifies the process and reduces production costs, but also may enhance the stability and synergistic biological activities (such as antioxidant and immunomodulatory effects) of the composite product by forming a unique structure through the in-situ interaction between extracellular polysaccharides and selenium nanoparticles. However, existing strains generally suffer from low selenium tolerance, slow reduction rates, and insufficient genetic stability, which limits the yield and efficiency of selenium nanoparticles and selenium polysaccharides, making it difficult to meet the needs of industrial production.

[0004] Therefore, developing a strain with high selenium tolerance, genetic stability, and the ability to efficiently synthesize selenium polysaccharides and nano-selenium simultaneously, and establishing a simple and green co-production process, has become a key challenge in promoting the industrial application of selenium-based functional raw materials. Summary of the Invention

[0005] The purpose of this invention is to provide a Bacillus subtilis strain that co-produces selenium polysaccharide-nano-selenium and its applications, thereby solving the problems existing in the prior art. This invention provides a highly selenium-tolerant and genetically stable Bacillus subtilis strain B590, and establishes a one-pot co-production process based on it. This process simultaneously and efficiently produces selenium polysaccharide with high polysaccharide content (703.87 g / kg) and polysaccharide-stabilized nano-selenium with high selenium loading and strong antioxidant properties (selenium content reaches 2.02 × 10⁻⁶ g / kg). 5 (mg / kg) provides a new, efficient, and green solution for the development of functional selenium materials.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a strain of Bacillus subtilis B590 that co-produces selenium polysaccharide-nano selenium. The Bacillus subtilis B590 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2025, with accession number GDMCC NO: 67288.

[0008] The present invention also provides an application of the aforementioned Bacillus subtilis B590 in the simultaneous preparation of selenium polysaccharide-nano selenium.

[0009] The present invention also provides a method for the simultaneous preparation of selenium polysaccharide-nano selenium, comprising the step of culturing the aforementioned Bacillus subtilis B590 in a fermentation medium containing selenite.

[0010] Optionally, the Bacillus subtilis B590 is fermented in the fermentation medium until OD reaches [a certain value]. 600 After reaching 1.0, the selenite is added for selenization culture; the fermentation broth after selenization culture is subjected to solid-liquid separation to obtain precipitate and supernatant; the precipitate is washed to obtain polysaccharide-stabilized selenium nanoparticles EPS-SeNPs; alcohol is added to the supernatant for precipitation, and the precipitate is collected to obtain selenium polysaccharide Se-EPS.

[0011] Optionally, the fermentation conditions are 37°C and 200 rpm.

[0012] Optionally, the selenization culture time is 24 hours.

[0013] Optionally, the concentration of the selenite in the fermentation medium is 5 mM.

[0014] Optionally, the fermentation medium comprises 400 g / L sucrose, 10 g / L yeast extract, and 10 g / L sodium chloride.

[0015] The present invention also provides a method for preparing polysaccharide-stabilized selenium nanoparticles and selenium polysaccharides.

[0016] This invention also provides the application of the polysaccharide-stabilized selenium nanoparticles and selenium polysaccharides in the preparation of functional selenium-based materials.

[0017] The present invention discloses the following technical effects:

[0018] This invention provides a strain of Bacillus subtilis B590 with extremely high selenium tolerance and excellent genetic stability. This strain exhibits a tolerance to sodium selenite (Na2SeO3) up to 520 mM, and its nano-selenium yield remains highly stable (35.84%~36.38%) after nine consecutive generations of subculturing, indicating its stable characteristics and ability to reliably meet the needs of large-scale continuous industrial production.

[0019] Based on the aforementioned strains, this invention establishes a highly efficient and green one-pot co-production process for the simultaneous preparation of polysaccharide-stabilized selenium nanoparticles (EPS-SeNPs) and selenium polysaccharides (Se-EPS). This process overcomes the technical bottleneck of traditional stepwise synthesis, featuring a simple procedure that directly yields two high-value-added products from a single fermentation system. The prepared Se-EPS contains a polysaccharide content as high as 703.87 g / kg. Simultaneously, the core co-produced product, EPS-SeNPs, exhibits an extremely high selenium loading ((2.02±0.01)×10⁻⁶). 5 It exhibits excellent antioxidant activity (mg / kg), achieving efficient utilization of selenium resources and synergistic enhancement of product functions while simplifying the process, providing an efficient, low-cost, and environmentally friendly solution for developing new functional selenium-based materials. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The growth of Bacillus subtilis MEB 44 on LB plates with (A) and (B) 5 mM sodium selenite, and the growth of Bacillus subtilis MEB 44 on LB plates with (C) and (D) 120 mM sodium selenite.

[0022] Figure 2 The effect of mutagenesis time on the lethality of Bacillus subtilis MEB 44;

[0023] Figure 3 The growth status of the mutant strain on LB plates containing 120 mM Na2SeO3;

[0024] Figure 4 The conversion efficiency of some strains to Na2SeO3 after ARTP screening is shown; where A represents the OD before and after selenization culture. 600 The change; B is the verification of the SeNPs yield;

[0025] Figure 5 Survival rate of mutant strain B5 under different concentrations of Na2SeO3;

[0026] Figure 6 Figure showing the results of genetic stability verification of Bacillus subtilis B590;

[0027] Figure 7 The bacterial growth and selenium metabolism of Bacillus subtilis B590 in LB medium containing 5 mM Na2SeO3;

[0028] Figure 8 The effect of sucrose addition on EPS-SeNPs (A), Se-EPS (B) and protein content (C);

[0029] Figure 9 The effect of Na2SeO3 addition on EPS-SeNPs (A), Se-EPS (B) and protein content (C);

[0030] Figure 10 The images show the morphology of EPS-SeNPs and Se-EPS; where (A) and (B) are TEM images and diffraction patterns of EPS-SeNPs, respectively; (C) is a SEM image of EPS; and (D) is a SEM image of Se-EPS.

[0031] Figure 11 Fourier transform infrared spectra of EPS-SeNPs;

[0032] Figure 12 Fourier transform infrared spectra of EPS and Se-EPS;

[0033] Figure 13 DPPH radical scavenging rates of EPS-SeNPs (A) and Se-EPS (B);

[0034] Figure 14 ABTS radical scavenging rates of EPS-SeNPs (A) and Se-EPS (B);

[0035] Figure 15 The hydroxyl radical scavenging rates of EPS-SeNPs (A) and Se-EPS (B) are given. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Example 1: Screening and domestication of Bacillus subtilis producing selenium polysaccharide-nano selenium

[0042] 1. Starting strain and culture medium

[0043] This embodiment uses Bacillus subtilis MEB 44, which was screened and identified in the laboratory, as the starting strain. This strain was isolated from Liubao tea in Guangxi. LB medium was used for cultivation, with the following composition: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride; when preparing the solid medium, an additional 15 g / L of agar was added.

[0044] 2. Strain activation and seed culture preparation

[0045] One loopful of Bacillus subtilis MEB 44 was taken from a glycerol tube and streaked onto an LB agar plate. The plate was then incubated upside down at 37°C for 24 hours. A single colony with good morphology was picked and inoculated into an Erlenmeyer flask containing 30 mL of LB liquid medium. The flask was then incubated at 37°C and 200 rpm with shaking for 12 hours to obtain the primary seed culture. Subsequently, the primary seed culture was transferred at an inoculation rate of 3% (v / v) to an Erlenmeyer flask containing 30 mL of fresh LB medium and incubated under the same conditions for 12 hours to obtain the secondary seed culture for later use.

[0046] 3. Evaluation of selenium tolerance of the starting strain

[0047] The minimum inhibitory concentration (MIC) of sodium selenite against strain MEB 44 was determined using the broth dilution method. The specific steps were as follows: In a 96-well plate, the sodium selenite solution was serially diluted with LB medium to achieve a gradient distribution of final sodium selenite concentrations in each well. The bacterial suspension (OD) was then... 600 =0.60±0.02, corresponding to a viable bacterial concentration determined by the dilution plating method) at 5×10 5 The final concentration of sodium selenite (CFU / mL) was added to each well. A control containing no selenium and a blank control containing only selenium were set up. The 96-well plate was incubated at 37°C for 24 hours and then observed. The minimum sodium selenite concentration that completely inhibited bacterial growth was defined as the MIC.

[0048] Experimental results are as follows Figure 1 As shown, compared with the control group, the colonies became smaller under Na2SeO3 stress, and the colonies turned orange-red due to the production of nano-selenium. MIC assay results indicated that strain MEB 44 had high tolerance to selenite, and SeNPs could be observed even at a sodium selenite concentration of 120 mM. To further improve the tolerance of Bacillus subtilis MEB 44 derived from Liubao tea to sodium selenite, ARTP mutagenesis was used to obtain a strain with high sodium selenite tolerance.

[0049] 4. ARTP mutagenesis and initial screening

[0050] To enhance the selenium tolerance of the strain, MEB 44 was mutagenized by ambient pressure room temperature plasma (ARTP). Activated Bacillus subtilis MEB 44 was inoculated at a rate of 3% (v / v) into 30 mL of LB medium and cultured at 37°C and 200 rpm for 12 h. One mL of the culture was then centrifuged at 8000 rpm for 10 min at 4°C, washed three times with sterile physiological saline, and resuspended in 5% glycerol (v / v) to obtain a bacterial concentration of 10⁻⁶. 7CFU / mL bacterial suspension. 10 μL of the bacterial suspension was evenly spread onto a sterile metal slide and placed in the reaction chamber of the ARTP mutagenesis instrument. The mutagenesis parameters were set as follows: working gas 99.999% pure helium, gas flow rate 10 SLM, radio frequency power 120 W, and treatment distance 2 mm. Mutagenesis times were set to 0 s, 5 s, 10 s, 20 s, 30 s, 40 s, 50 s, and 60 s. After treatment, the slide was immediately immersed in 990 μL of LB medium to elute the bacteria, and incubated at 37°C in the dark for 4 h. The incubated bacterial suspension was serially diluted, spread onto LB plates, and incubated at 37°C for 24 h. Colony counts were recorded to plot the lethality curve and determine the mutagenesis time.

[0051] .

[0052] The lethality of Bacillus subtilis MEB 44 under different mutagenesis times is as follows: Figure 2 As shown, when the mutagenesis time is 0-20 s, the bacterial mortality rate increases with the increase of mutagenesis time; when the mutagenesis time is 20 s, the lethality of MEB 44 reaches 99.6%. This invention selects 20 s as the optimal mutagenesis time for strain MEB 44.

[0053] The mutagenized bacterial suspension was plated onto LB selection plates containing 120 mM sodium selenite and incubated upside down at 37°C for 36 hours. 102 smooth, well-defined, red, circular colonies were obtained. Figure 3 As shown, this indicates that it can reduce selenite to nano-selenium. These red single colonies were picked, purified, cultured, and preserved.

[0054] 5. Secondary screening and acquisition of highly selenium-tolerant mutants

[0055] The mutant strains obtained from the initial screening were re-screened to evaluate their sodium selenite conversion rate. Single colonies of the starting strain MEB 44 and each mutant strain obtained from the initial screening were incubated in LB medium at 37°C and 200 rpm for 14 h. After centrifugation, the cells were dispersed into 96-well plates containing 5 mM sodium selenite in LB medium. Uninoculated medium was used as a blank control, and the plates were incubated at 37°C for 12 h. The OD values ​​before and after incubation were compared. 600 Change in value (△OD) 600 ( ), strains capable of rapidly transforming Na2SeO3 were screened and repeatedly validated, some results are shown below. Figure 4 As shown in the figure. The strain B5, which achieved the highest conversion rate, was selected, showing a conversion rate of 36.21% for 5 mM Na₂SeO₃ within 24 h. The MIC of B5 was determined again using the microbroth dilution method to be 250 mM.

[0056] To obtain a strain with stronger selenium tolerance, laboratory adaptive evolution (ALE) was performed on B5. The activated strain was streaked on LB agar plates and cultured overnight. Single colonies were picked and re-inoculated into four 40 mL test tubes containing 5 mL of LB medium. After culturing for 6 h, Na2SeO3 was added to three of the tubes to a final concentration of 250 mM, while the other tube served as a control.

[0057] Once the culture medium turns red, indicating the formation of nano-selenium, transfer it to fresh LB medium, subculture twice, and then re-inoculate it into four 40 mL test tubes. Repeat the above steps, iterating the sodium selenite concentration in a gradient of 30 mM until the strain cannot grow and the culture medium no longer turns red.

[0058] ALE results are as follows Figure 5 As shown, with the gradual increase of Na₂SeO₃ concentration, the survival rate of strain B5 gradually decreased from 67.40% to 51.65%. This indicates that the toxicity of Na₂SeO₃ to the strain increases with increasing Na₂SeO₃ concentration. However, even under the high concentration stress of 520 mM Na₂SeO₃, half of the cells still survived, demonstrating its strong Na₂SeO₃ tolerance. The domestication process ended when the Na₂SeO₃ concentration reached 520 mM. This iterative domestication process shows that strain B5 can still survive and reduce selenium at a high concentration of 520 mM sodium selenite.

[0059] 6. Strain identification and preservation

[0060] Genomic DNA was extracted from strain B5 and used as a template for PCR amplification using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') for the bacterial 16S rRNA gene. The PCR reaction mixture (50 μL) consisted of 25 μL of premixed mix, 22 μL of ddH2O, 1.0 μL each of forward and reverse primers, and 1.0 μL of DNA template. The reaction program was as follows: 94℃ pre-denaturation for 2 minutes; 94℃ denaturation for 30 seconds, 54℃ annealing for 30 seconds, and 72℃ extension for 1 minute, for a total of 30 cycles; and a final extension at 72℃ for 5 minutes. After the PCR amplification reaction, the sample was sent to Sangon Biotech (Shanghai) Co., Ltd. for amplicon sequencing. Phylogenetic analysis of the sequencing results using MEGA 12 software confirmed that the strain belonged to Bacillus subtilis and was named Bacillus subtilis B590. Bacillus subtilis B590 was deposited on November 13, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC NO: 67288.

[0061] 7. Verification of genetic stability

[0062] Because mutagenic strains have self-repair mechanisms and the risk of reversion mutations, only strains with good genetic stability can meet the needs of large-scale industrial production. Therefore, the genetic stability of the mutagenic strain B590 was verified by continuous subculturing and using SeNPS yield as the evaluation index.

[0063] Bacillus subtilis B590 was continuously passaged in LB medium containing 5 mM Na2SeO3, transferred every 24 h, for a total of 9 passages. Samples were taken every other generation to determine the yield of selenium nanoparticles (SeNPs) in the fermentation broth. Results are as follows: Figure 6 As shown, after nine consecutive generations, the transformation rate of SeNPs remained stable between 35.84% and 36.38%, indicating that strain B590 has good genetic stability and is suitable for industrial application.

[0064] Example 2: Growth and Selenium Reduction Kinetics Analysis of Bacillus subtilis B590

[0065] 1. Monitoring of growth and reduction processes

[0066] The activated Bacillus subtilis B590 seed culture was inoculated into LB medium containing 5 mM sodium selenite at a rate of 3% (v / v), with a control of LB medium without selenium. The medium was cultured at 37°C with shaking at 200 rpm. Samples were taken every 2 hours from the start of inoculation.

[0067] (1) Bacterial growth (OD) 600 Take an appropriate amount of culture medium and measure its absorbance (OD) at 600 nm. 600 ), and plot the growth curve.

[0068] (2) Determination of nano-selenium (SeNPs) content

[0069] Selenium content standard curve plotting:

[0070] Principle: Hydroxylamine, acting as a reducing agent, can reduce selenite to amorphous red selenium, and its oxidation end product does not interfere with the assay system. The reaction principle is as follows:

[0071] ,

[0072] A certain amount of 5 mg / mL sodium selenite solution was added to a 2 mL centrifuge tube to achieve selenium concentrations of 0, 3, 6, 9, and 12 μmol. Then, 40 μmol of hydroxylamine hydrochloride was added. According to the above formula, this concentration of hydroxylamine is sufficient to ensure the presence of SeO3 in the system. 2- Completely reduced to Se 0 Subsequently, the centrifuge tubes were dried in a 50°C oven. 1 mL of 1M Na2S solution was added to each centrifuge tube and gently mixed. After standing at room temperature for 2 hours, the tubes were centrifuged, and the absorbance of the supernatant at 500 nm was measured. A standard curve was then plotted.

[0073] Determination of SeNP content:

[0074] Take 1 mL of culture medium, centrifuge at 12000 rpm for 5 min, and collect the precipitate (containing bacterial cells and SeNPs). Wash the precipitate three times with 1M NaCl solution to remove impurities, disperse it ultrasonically, add 1 mL of 1M Na2S solution, and shake at room temperature for 2 h to dissolve the SeNPs and form a selenosulfate complex. After centrifugation at 8000 rpm for 15 min, take the supernatant and measure the absorbance at 500 nm. Calculate the SeNP content in the sample according to the pre-plotted selenium content standard curve (using Na2SeO3 as the standard, reduced by hydroxylamine hydrochloride and then treated in the same way).

[0075] (3) Residual selenite (SeO3) 2- Content determination

[0076] The colorimetric assay of 3,3'-diaminobenzidine (DAB) was used to analyze the SeO3 in the culture medium. 2- The content of Se(IV) was determined. Under acidic conditions, Se(IV) reacts with DAB to produce a yellow selenium-containing complex. Based on the principle of "like dissolves like," the selenium-containing complex can be extracted with organic solvents such as toluene under neutral conditions, and the extract exhibits maximum absorbance at 420 nm. Within a certain concentration range, the content of Se(IV) is directly proportional to the absorbance value.

[0077] Construction of the selenium standard curve: Add 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0 mL of 1 mg / L selenium standard solution to six volumetric flasks, respectively. Make up to 50 mL with distilled water, adjust the pH to 2–3, then add 1 mL of 5% EDTA-2Na to mask interfering ions. Mix thoroughly, then add 4 mL of freshly prepared 0.5% DAB solution, shake well, and react in the dark for 1 h. Adjust the pH to neutral with NaOH solution, transfer to a separatory funnel, add 6 mL of toluene, and shake for 1 min to ensure complete extraction of Se-DAB by the toluene layer. Let stand for 10 min, and measure the absorbance of the toluene layer at 420 nm. Using toluene as a blank, plot the standard curve.

[0078] Determination of SeO3 in the sample 2- content:

[0079] Bacillus subtilis B590 culture medium was centrifuged at 12000 rpm and 4 ℃ for 10 min, filtered through a 0.22 μm filter membrane, diluted as needed, and 1 mL was transferred to a volumetric flask. Subsequent procedures were performed as per the standard curve analysis. The SeO3 concentration in the sample was calculated based on the standard curve. 2- content.

[0080] 2. Results Analysis

[0081] like Figure 7 As shown, Bacillus subtilis B590 exhibited a lag phase of approximately 0-2 hours in LB medium containing 5 mM Na₂SeO₃, followed by a logarithmic growth phase, and then entered a stationary phase after approximately 12 hours. Within 8 hours of adding 5 mM Na₂SeO₃ to the medium, bacterial growth was lower than the control group, indicating that this Na₂SeO₃ concentration inhibited the proliferation of strain B590 to some extent. 2- After entering the body, it can react with sulfhydryl groups in proteins, generating free radicals that can cause severe cell damage, thus affecting bacterial growth. Residual selenium analysis revealed that under 5mM Na₂SeO₃ conditions, the formation of SeNPs was observed in strain B590 after 2 hours of selenization. After 8 hours of selenium-added culture, the transformation of SeNPs essentially ceased, indicating that the bacteria were in a stationary phase. This suggests that Na₂SeO₃ reduction occurs concurrently with the growth of the strain. Furthermore, from... Figure 7 It can be seen that the concentration of SeNPs increases as the concentration of Na2SeO3 decreases, but the reduced Na2SeO3 is not completely converted into SeNPs, indicating that the reduced Na2SeO3 may exist in other forms, such as selenoproteins, selenopolysaccharides, etc.

[0082] Example 3: One-pot process for co-producing polysaccharide-stabilized selenium nanoparticles (EPS-SeNPs) and selenium polysaccharides (Se-EPS).

[0083] 1. Optimization of co-production culture medium and culture conditions

[0084] Using a fermentation medium (with a basic composition of sucrose concentration gradient, yeast extract 10 g / L, and sodium chloride 10 g / L), the effects of key parameters on co-production were studied.

[0085] Sucrose concentration optimization: Bacillus subtilis B590 seed culture was inoculated at a 3% inoculum into fermentation media with sucrose contents of 200, 250, 300, 350, 400, and 450 g / L, respectively, and cultured at 37℃ and 200 rpm until OD reached. 600 When the concentration reached 1.0, Na2SeO3 was added to a final concentration of 5 mM, and the mixture was cultured for another 24 h and 48 h. The yields of EPS-SeNPs and Se-EPS were then measured.

[0086] Sodium selenite concentration optimization: At the optimal sucrose concentration, the seed culture was inoculated into the fermentation medium at a 3% inoculum and cultured until OD... 600 When the concentration was 1.0, Na2SeO3 was added to make the final concentration 0, 2.5, 5, 7.5 and 10 mM respectively, and the culture was continued for 24 h and 48 h. The yields of EPS-SeNPs and Se-EPS were then measured.

[0087] The effects of sucrose addition and Na2SeO3 concentration on EPS-SeNPs and Se-EPS yields and protein content are as follows: Figure 8 and Figure 9 As shown.

[0088] 2. Determine the optimal co-production conditions and product separation

[0089] Based on comprehensive yield indicators, the optimal co-production conditions were determined to be: using a fermentation medium containing 400 g / L sucrose, 10 g / L yeast extract, and 10 g / L sodium chloride. The B590 seed culture was inoculated at a 3% inoculum and cultured at 37℃ and 200 rpm until OD reached [value missing]. 600 =1.0, then add Na2SeO3 to a final concentration of 5 mM, and continue selenization culture for 24 h.

[0090] After the culture was completed, the fermentation broth was centrifuged at 8000 rpm for 20 min to obtain two parts: precipitate and supernatant.

[0091] Extraction of EPS-SeNPs: The precipitate mainly consists of bacterial cells and their bound selenium nanoparticle complexes (EPS-SeNPs). Washing the precipitate several times with deionized water or buffer solution yields crude EPS-SeNPs, which can be further purified by ultrasonic dispersion and centrifugation.

[0092] Extraction of Se-EPS: The supernatant contained selenium polysaccharides (Se-EPS) secreted extracellularly. Three volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand overnight at 4°C to precipitate the polysaccharides. The next day, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes. The precipitate was washed sequentially with anhydrous ethanol and acetone, and then vacuum dried to obtain crude Se-EPS.

[0093] Under these optimal conditions, the yield of EPS-SeNPs is 49.25%, and the yield of Se-EPS can reach 252.61 g / L, of which the protein content is 20.94 g / L.

[0094] 3. Chemical composition analysis of the product

[0095] The prepared EPS-SeNPs and Se-EPS were subjected to component analysis.

[0096] Polysaccharide content determination: The phenol-sulfuric acid method was used.

[0097] Protein content determination: BCA protein quantification kit was used.

[0098] Selenium content determination: The selenium content was determined by inductively coupled plasma mass spectrometry (ICP-MS) according to the first method of the national standard GB5009.268.

[0099] The analysis results are shown in Table 1.

[0100] Table 1 Chemical composition of EPS-SeNPs and Se-EPS

[0101]

[0102] 4. Characterization Results

[0103] 4.1 Morphological Analysis

[0104] The prepared EPS-SeNPs and Se-EPS were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the results are as follows: Figure 10 As shown in (A), EPS-SeNPs are approximately spherical with a particle size distribution ranging from 80 nm to 450 nm. They exhibit good dispersibility and no obvious agglomeration. Figure 10The absence of obvious diffraction rings in the diffraction pattern of (B) indicates that the nanoparticle has an amorphous structure.

[0105] Figure 10 (C) and (D) show that the EPS surface of the control group has a rough and porous morphology, while the Se-EPS surface obtained after selenization treatment is relatively smooth.

[0106] 4.2 Infrared Spectroscopy Analysis

[0107] EPS-SeNPs, EPS, and Se-EPS were tested at 4000 ~ 400 cm⁻¹. -1 Fourier transform infrared spectroscopy was performed within the range to analyze its characteristic functional groups.

[0108] The infrared spectrum of EPS-SeNPs is as follows: Figure 11 As shown. 3260cm -1 The absorption peak at 3050 cm⁻¹ is attributed to the OH / NH stretching vibration; -1 With 2910cm -1 The absorption peak at 1720 cm⁻¹ originates from the CH vibration; -1 The peak at 1650 cm⁻¹ represents the C=O stretching vibration. -1 1530cm -1 and 1230cm -1 The locations correspond to amide I, II, and III bands, respectively; 1390cm -1 The absorption peak at 1080 cm⁻¹ can be attributed to the CO stretching vibration or the CH₂ / CH₃ bending vibration; -1 The absorption peak indicates the presence of C=O. The above spectral characterization results show that the surface of EPS-SeNPs contains organic components such as polysaccharides, proteins, and lipids.

[0109] EPS and Se-EPS have similar infrared spectra characteristics, such as Figure 12 As shown. Both are at 3300cm. -1 (OH stretching vibration), 2940cm -1 (CH stretching vibration), 1650cm -1 (Amide I band), 1540cm -1 (Amide II band), 1240cm -1 (Amide III band), 1450cm -1 (CH bending / CO vibration), 1070cm -1 (glycosidic bond COH / COC vibration) and 835cm -1A characteristic absorption peak appears near the (β-glycosidic bond vibration). In the Se-EPS spectrum, some absorption peaks are red-shifted relative to EPS, indicating that the introduction of selenium may form Se-O bonds with polysaccharide molecules without disrupting the basic skeletal structure of the polysaccharide.

[0110] Example 4 Evaluation of the in vitro antioxidant activity of the co-product

[0111] The scavenging abilities of EPS-SeNPs, unselenized extracellular polysaccharides (EPS, extracted from selenium-free cultures), and Se-EPS against DPPH radicals, ABTS cation radicals, and hydroxyl radicals were determined, with vitamin C (Vc) used as a positive control. All samples were prepared into solutions of different concentrations using deionized water for testing.

[0112] 1. Determination of DPPH free radical scavenging ability

[0113] Take 1.0 mL of sample solutions of different concentrations, add 2.0 mL of freshly prepared 0.04 mg / mL DPPH-ethanol solution, mix well, and react at room temperature in the dark for 40 min. Centrifuge at 8000 rpm for 10 min, and measure the absorbance of the supernatant at 517 nm (A1). Simultaneously measure the absorbance of the control group (A0) with an equal volume of anhydrous ethanol replacing the sample solution, and the absorbance of the sample blank group (A2) with an equal volume of anhydrous ethanol replacing the DPPH solution. The clearance rate is calculated using the following formula:

[0114] .

[0115] The results are as follows Figure 13 As shown, the scavenging effect of EPS-SeNPs on DPPH radicals was concentration-dependent within the range of 0.2 mg / mL to 1.0 mg / mL, with the highest scavenging rate of 65.64% at a concentration of 1.0 mg / mL. Se-EPS showed a similar effect, with a scavenging rate of 53.58% at 4.0 mg / mL, which was 1.47 times that of EPS, indicating that the introduction of Se enhanced the scavenging ability of EPS against DPPH radicals.

[0116] 2. Determination of ABTS free radical scavenging ability

[0117] Preparation of ABTS working solution (OD) 734=0.70±0.02). Mix 0.4 mL of sample solution with 3.6 mL of ABTS working solution and react at room temperature in the dark for 5 min. After centrifugation at 8000 rpm for 10 min, measure the absorbance of the supernatant at 734 nm (A1). Use an equal volume of deionized water instead of the sample as the control group (A0), and an equal volume of anhydrous ethanol instead of the ABTS working solution as the blank group (A2). The clearance rate is calculated using the same method as the DPPH method.

[0118] The results are as follows Figure 14 As shown, the ABTS scavenging rate of EPS-SeNPs increased with increasing sample concentration. When the concentration increased from 0.2 mg / mL to 1.0 mg / mL, the ABTS radical scavenging rate increased from 38.75% to 54.42%. The ABTS radical scavenging rates of EPS and Se-EPS showed a trend of first increasing and then gradually stabilizing with increasing sample concentration, and the scavenging effect of Se-EPS was better than that of EPS. Ultimately, the ABTS radical scavenging rates of 4.0 mg / mL EPS and Se-EPS were 65.06% and 70.98%, respectively.

[0119] 3. Determination of hydroxyl radical scavenging ability

[0120] Add 1.0 mL of sample solution, 1.0 mL of 9 mmol / L FeSO4 solution, and 1.0 mL of 9 mmol / L salicylic acid-ethanol solution sequentially to a test tube. After mixing, add 1.0 mL of 9 mmol / L H2O2 solution to initiate the reaction. After thorough mixing, incubate at 37°C in the dark for 40 min, and measure the absorbance at 510 nm (A1). Use an equal volume of deionized water instead of the sample as the damage control group (A0), and use an equal volume of deionized water instead of H2O2 solution as the undamaged group (A2). The clearance rate is calculated as before.

[0121] The results are as follows Figure 15 As shown, the scavenging effect of EPS-SeNPs at different concentrations on hydroxyl radicals showed a similar trend to that of DPPH and ABTS, with a scavenging rate of 71.56% at a concentration of 1.0 mg / mL. In contrast, the scavenging effect of Se-EPS on hydroxyl radicals was less affected by concentration changes, with scavenging rates ranging from 58.00% to 68.38% within a concentration range of 0.25 mg / mL to 4.0 mg / mL.

[0122] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus subtilis B590 that co-produces selenium polysaccharide-nano-selenium, characterized in that, The Bacillus subtilis B590 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2025, with accession number GDMCC NO: 67288.

2. The application of Bacillus subtilis B590 as described in claim 1 in the simultaneous preparation of selenium polysaccharide-nano selenium.

3. A method for simultaneous preparation of selenium polysaccharide-nano selenium, characterized in that, The method includes the step of culturing Bacillus subtilis B590 as described in claim 1 in a fermentation medium containing selenite.

4. The method according to claim 3, characterized in that, The Bacillus subtilis B590 was fermented and cultured in the fermentation medium until OD100. 600 After reaching a value of 1.0, the selenite is added for selenization culture. The fermentation broth after selenization culture was subjected to solid-liquid separation to obtain precipitate and supernatant; the precipitate was washed to obtain polysaccharide-stabilized selenium nanoparticles EPS-SeNPs. Alcohol was added to the supernatant to precipitate the product, and the precipitate was collected to obtain selenium polysaccharide Se-EPS.

5. The method according to claim 4, characterized in that, The fermentation conditions were 37°C and 200 rpm.

6. The method according to claim 4, characterized in that, The selenization culture time was 24 hours.

7. The method according to claim 3, characterized in that, The concentration of the selenite in the fermentation medium is 5 mM.

8. The method according to claim 3, characterized in that, The fermentation medium consists of 400 g / L sucrose, 10 g / L yeast extract, and 10 g / L sodium chloride.

9. A polysaccharide-stabilized selenium nanoparticle and selenium polysaccharide prepared by the method according to any one of claims 3-8.

10. The application of the polysaccharide-stabilized selenium nanoparticles and selenium polysaccharide as described in claim 9 in the preparation of functional selenium-based materials.

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