A lactobacillus jensenii exopolysaccharide selenium nanoparticle, a preparation method and application thereof

Abstract

The present application relates to the technical field of nanobiomedicine, and discloses a lactobacillus jensenii exopolysaccharide selenium nanoparticle, a preparation method and application thereof, the nanoparticle comprises lactobacillus jensenii exopolysaccharide and selenium nanoparticles dispersed therein, and the selenium is amorphous zero-valent selenium. The preparation method comprises the following steps: mixing a lactobacillus jensenii exopolysaccharide solution and a sodium selenite solution, adding an ascorbic acid solution as a reducing agent, and performing a reduction reaction under light-proof heating conditions to separate and obtain the lactobacillus jensenii exopolysaccharide selenium nanoparticle. The exopolysaccharide effectively inhibits the aggregation and crystallization of the nanometer selenium as a matrix, not only significantly improves the structural stability and slow-release performance of the nanoparticle, but also synergistically enhances the phagocytic ability of macrophages, and can effectively intervene in the polarization direction of macrophages by regulating the secretion of pro-inflammatory and anti-inflammatory factors, and has important application value in the preparation of immunomodulatory and anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedicine technology, and in particular to a selenium nanoparticle of extracellular polysaccharide from Lactobacillus chiayi, its preparation method, and its application. Background Technology

[0002] Probiotics play a crucial role in maintaining bodily health by regulating the host's immune system, suppressing inflammatory responses, and improving metabolic disorders. Previous studies have shown that live *Lactaseibacillus chiayiensis* can significantly alleviate pathogen-induced infection symptoms, effectively reduce intestinal inflammation, and exert a preventative and protective effect. Further research has revealed that, compared to the bacteria themselves, their extracellular metabolites—extracellular polysaccharides—as typical prebiotics, exhibit stronger biological activity and application value due to their high safety, outstanding anti-inflammatory and immunomodulatory properties.

[0003] Selenium, an essential trace element for organisms, participates in the regulation of immune homeostasis through multiple mechanisms, such as regulating macrophage polarization and reducing the release of inflammatory mediators. However, the direct application of inorganic selenium is limited by problems such as a narrow safe dose window, high toxicity risk, and low bioavailability; while organic selenium has relatively low toxicity, it still faces challenges such as excessive selenium loading and insufficient nanoparticle stability in traditional preparation processes.

[0004] In recent years, polysaccharide-selenium nanoparticles synthesized using polysaccharides as a matrix have shown unique advantages: the hydroxyl groups of polysaccharide molecules can inhibit the aggregation of selenium particles through hydrogen bonding, thereby improving the stability of the nanoparticles, and they also exhibit better bioavailability and lower toxicity compared to pure organic selenium. However, existing polysaccharide-selenium nanoparticles mostly aim for high selenium loading, making it difficult to balance "low selenium toxicity" and "high bioactivity". Therefore, there is an urgent need in this field to develop a precise and intelligent preparation strategy to achieve synergistic optimization of low selenium dosage, high structural stability, and high immunomodulatory activity. Summary of the Invention

[0005] This invention provides a method for preparing and applying selenium nanoparticles made from extracellular polysaccharides of Lactobacillus chilii. By encapsulating and stabilizing zero-valent selenium nanoparticles with extracellular polysaccharides from Lactobacillus chilii, the physicochemical stability and bioavailability of the selenium nanoparticles are significantly improved, while achieving efficient immune intervention and regulation of macrophage polarization.

[0006] This invention provides selenium nanoparticles of Lactobacillus chiayi exopolysaccharide, comprising Lactobacillus chiayi exopolysaccharide and selenium nanoparticles dispersed in the Lactobacillus chiayi exopolysaccharide; wherein the selenium in the selenium nanoparticles is amorphous zero-valent selenium.

[0007] Furthermore, in the X-ray photoelectron spectroscopy, the electron binding energy peak of the Se 3d orbital of the selenium nanoparticles contains 55.1 eV ± 0.2 eV; in the X-ray diffraction pattern, there is a broad diffraction peak at a diffraction angle 2θ of 20.96° ± 0.2°.

[0008] Furthermore, in the Fourier transform infrared spectrum of the *Lactobacillus chiiense* extracellular polysaccharide selenium nanoparticles, the OH stretching vibration peak is located at 3144.99 cm⁻¹. -1 ±4cm -1 At this point, the C=O stretching vibration peak is located at 1631.48 cm⁻¹. -1 ±4cm -1 Place.

[0009] This invention also provides a method for preparing the above-mentioned Lactobacillus chiayi extracellular polysaccharide selenium nanoparticles, comprising the following steps:

[0010] (1) Prepare a solution of extracellular polysaccharide of Lactobacillus chilii and mix it with sodium selenite solution to obtain a mixture;

[0011] (2) Add ascorbic acid solution as a reducing agent to the mixture and carry out a heating reduction reaction under light-protected conditions;

[0012] (3) After the reaction was completed, centrifugation was performed, the precipitate was collected and freeze-dried to obtain selenium nanoparticles of extracellular polysaccharide from Lactobacillus chiayi.

[0013] Furthermore, in the reaction system composed of the *Lactobacillus chilii* extracellular polysaccharide solution, sodium selenite solution, and ascorbic acid solution, the final concentration of the *Lactobacillus chilii* extracellular polysaccharide is 10-20 mg / mL, and the final concentration of the sodium selenite is 2.5-20 mmol / L.

[0014] Further, in step (2), the volume ratio of the sodium selenite solution to the ascorbic acid solution is 1:1 to 1:5, the heating reaction temperature is 30~50℃, and the time is 20~60min.

[0015] Further, in step (1), the preparation of the *Lactobacillus chiayiensis* extracellular polysaccharide solution specifically includes:

[0016] The fermentation broth of Lactobacillus chiiensis from Chiayi was taken, the bacterial cells were removed and concentrated, and then ethanol was added for alcohol precipitation. The precipitate was collected to obtain crude polysaccharide.

[0017] The crude polysaccharide was subjected to deproteinization treatment;

[0018] The deproteinized polysaccharide was purified by sequentially using a cellulose chromatography column and a gel chromatography column to obtain purified extracellular polysaccharide of Lactobacillus chiayi.

[0019] The purified extracellular polysaccharide of Lactobacillus chiayi was dissolved in a solvent to obtain the Lactobacillus chiayi extracellular polysaccharide solution.

[0020] The present invention also provides the application of the above-mentioned Lactobacillus chiayi extracellular polysaccharide selenium nanoparticles in the preparation of drugs or formulations that regulate macrophage polarization.

[0021] Furthermore, the regulation of macrophage polarization includes: inhibiting macrophage secretion of pro-inflammatory factors, and / or regulating macrophage secretion of anti-inflammatory factors;

[0022] The pro-inflammatory factor is selected from interleukin-6 and tumor necrosis factor-α, and the anti-inflammatory factor is selected from interleukin-10 and transforming growth factor-β.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention utilizes extracellular polysaccharide from *Lactobacillus chiiense* as a matrix to prepare a polysaccharide-selenium nanoparticle complex via an optimized carrier template reduction method. This effectively avoids the aggregation, sedimentation, and inactivation crystallization phenomena that easily occur in nano-selenium under conventional conditions, ensuring that selenium remains stably in a highly bioactive amorphous zero-valent state. This complex not only significantly enhances the structural stability and sustained-release performance of nanoparticles by leveraging the steric hindrance and higher-order structural recombination of polysaccharide macromolecules, but also synergistically enhances the phagocytic efficiency of selenium nanoparticles by macrophages. Simultaneously, these polysaccharide-selenium nanoparticles can effectively intervene in the polarization direction of macrophages by inhibiting the secretion of pro-inflammatory factors and regulating the release of anti-inflammatory factors. Under the premise of low dosage and low toxicity, it achieves a multi-target synergistic enhancement of high nanoparticle stability and strong immunomodulatory activity, showing broad application prospects in the preparation of drugs or formulations for treating intestinal inflammation and regulating the immune microenvironment. Attached Figure Description

[0025] Figure 1 This is a Pareto plot of the single-factor experiment results and the Plackett-Burman (PB) experiment in this invention.

[0026] Figure 2 The images show the 3D diagram and contour plot of the Box-Behnken response surface experimental design (BBD) in this invention.

[0027] Figure 3 These are stability assessment, morphology, and structural characterization diagrams of LC EPS-SeNPs placed for 14 days in this invention.

[0028] Figure 4 This figure shows the effect of LC EPS-SeNPs on the bioactivity of RAW 264.7 macrophages in this invention.

[0029] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0030] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0031] Example 1: Characterization of the microstructure and physicochemical properties of selenium nanoparticles containing extracellular polysaccharides from Lactobacillus casei of Chiayi.

[0032] This embodiment provides a selenium nanoparticle product of Lactobacillus chiayi exopolysaccharide, the product comprising Lactobacillus chiayi exopolysaccharide (LCEPS) and selenium nanoparticles (SeNPs) dispersed in the polysaccharide, wherein the selenium in the selenium nanoparticles is amorphous zero-valent selenium.

[0033] The Lacticaseibacillus chiayiensis GX6 involved in this invention was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on November 7, 2025, with accession number GDMCC No:67236.

[0034] To clarify the physicochemical characteristics and microstructure of the *Lactobacillus chiiense* extracellular polysaccharide selenium nanoparticles, the following characterization methods were used:

[0035] 1. Fourier Transmission Infrared Spectroscopy (FT-IR) Analysis

[0036] The infrared spectra of the samples were determined using a Fourier transform infrared spectrometer (Vertex 70). The test conditions were: wavenumber range 4000–400 cm⁻¹. -1 The KBr tableting method was used (the sample to KBr mass ratio was approximately 1:100, and after being ground evenly, it was pressed into a transparent sheet), with a resolution of 4 cm⁻¹. -1 The number of scans was 32.

[0037] Test results are as follows Figure 3 As shown in Figure J, infrared spectroscopy revealed that the LC EPS was at 3384.45 cm⁻¹. -1 The broad and strong OH stretching vibration peak that appeared at [location] redshifted to 3144.99 cm⁻¹ after the formation of LC EPS-SeNPs. -1 Furthermore, the peak shape becomes significantly sharper. This is because the formation and dispersion of SeNPs disrupts or reorganizes the original strong hydrogen-bonded association structure of the polysaccharide. Some hydroxyl groups are released from the strong hydrogen bonds, and the chemical environment becomes more homogeneous, leading to a slight decrease in the OH bond force constant (red shift) and a weakening of vibrational coupling (peak sharpening). The C=O stretching vibration peak in LC EPS is located at 1643.05 cm⁻¹. -1In LC EPS-SeNPs, this peak red-shifts to 1631.48 cm⁻¹. -1 This may be an indirect result of changes in the hydroxyl hydrogen bond network, leading to adjustments in the electron distribution or intramolecular constraints of the carbonyl group. Due to instrument system errors, the infrared absorption peak may vary by ±4 cm⁻¹. -1 The instrument's allowable error range. Furthermore, the LCEPS is located at 761.74 cm. -1 and 852.38cm -1 The fingerprint peaks are attributed to the out-of-plane bending vibrations of the CH ring in the pyranose ring and the characteristic vibrations of the galactose and mannose residues. These two vibrations completely disappear in the LC EPS-SeNPs spectrum, indicating that the LC EPS polysaccharide chains have undergone significant conformational rearrangement and higher-order structural reorganization to encapsulate and stabilize the SeNPs.

[0038] 2. Ultraviolet-Visible Spectroscopic Characterization

[0039] The LC EPS-SeNPs stock solution (2 mg / mL) prepared according to the optimized process was diluted with deionized water to a suitable concentration. Its absorbance was then measured using a UV-Vis spectrophotometer (UV-2550), scanning the wavelength range of 200–800 nm, and the characteristic absorption peaks were recorded. The results are as follows: Figure 3 As shown in Figure F, LC EPS-SeNPs exhibit a characteristic absorption peak of selenium in the 200-300 nm range, proving that Se was successfully introduced into LC EPS-SeNPs.

[0040] 3. Morphology and structural characterization of nanoparticles

[0041] Its three-dimensional morphology was observed using a field emission scanning electron microscope (FE-SEM, SU8010) (accelerating voltage 5kV, gold sputtering treatment). Figure 3 As shown in C and D, the SEM images show that LC EPS-SeNPs are spherical nanoparticles with good overall dispersion and smooth surfaces.

[0042] The elemental distribution and composition of nanoparticles were analyzed using energy-dispersive X-ray spectroscopy-elemental mapping (EDX-Mapping, mounted on FE-SEM). For example... Figure 3 As shown in Figure E, C, O, and Se elements were detected, with a Se mass fraction of 0.51%.

[0043] Phase analysis of LC EPS-SeNPs powder was performed using X-ray diffraction (XRD, D8 Advance), with a focus on the crystal form of selenium. Test conditions: Cu Ka radiation (λ = 0.15406 nm), scan range 5°–90°, scan rate 10° / min, tube voltage 40 kV, tube current 40 mA. Test results are as follows: Figure 3 China I and Figure 3As shown by the middle Lager diffraction peaks, LC EPS-SeNPs only exhibited diffraction peaks representing the crystalline region of Se at a diffraction angle of 2θ of 20.96°. However, these peaks were flat and broad, and typical Bragg diffraction peaks were not observed, indicating that the selenium in LC EPS-SeNPs is in an amorphous state, ruling out the possibility of its transformation into the non-biologically active or crystalline gray element selenium. Due to instrument system errors, a reasonable error range of ±0.2° is generally allowed for the above diffraction angles.

[0044] The valence state of selenium in LC EPS-SeNPs powder was analyzed using X-ray photoelectron spectroscopy (XPS, Escalab 250 Xi). Test conditions: monochromatic Al Ka ​​X-ray source (energy 1486.6 eV), power 150 W, vacuum level in the analysis chamber < 5 × 10⁻⁶. -9 mbar; charge correction was performed using the C1s peak at 284.8 eV as an internal standard, with a binding energy error of ±0.2 eV. Results are as follows... Figure 3 As shown in Figures G and H, the XPS full spectrum exhibits three peaks: C1s, O1s, and Se 3d. The electron binding energy peak position of the Se 3d orbital, fitted by peak sub-scores, is 55.12 eV. Referring to the NIST X-ray Photoelectron Spectroscopy Database - Standard Reference Database 20 (Version 4.1), the 55.1 eV electron binding energy peak position for selenium is the electron binding energy peak position for zero-valent selenium, and the 59.1 eV electron binding energy peak position for sodium selenite is not present, indicating a complete reaction. This confirms that LC EPS can bind the Se ions of Na₂SeO₃. 4+ Completely restored to Se 0 Elemental, and excluding Se. 4+ This could be a possibility for Lewis acids to form chemical coordination bonds with oxygen-containing functional groups.

[0045] Example 2: Preparation method and process optimization of selenium extracellular polysaccharide nanoparticles from Lactobacillus chiayi

[0046] This embodiment provides a method for preparing selenium nanoparticles of extracellular polysaccharide from *Lactobacillus chiiense* and its parameter optimization process. The specific steps are as follows:

[0047] 1. Preparation of Lactobacillus casei extracellular polysaccharide (LC EPS)

[0048] Add 20mL of Chiayi Lactobacillus GX6 bacterial culture (10 9The culture medium (CFU / mL) was inoculated into 2L of MRS medium and cultured for 24 hours. The supernatant (culture medium) was collected by centrifugation. The culture medium was concentrated to 500mL, and ethanol was added to the concentrate to achieve a concentration of 75% (v / v). Crude polysaccharide was then obtained and deproteinized using enzymatic and TAE methods. Subsequently, the polysaccharide was purified using a DEAE-52 cellulose column (2.6cm × 30cm) and a Sephadex G-100 column (2.6cm × 90cm). Finally, after concentration and lyophilization, purified *Lactobacillus chiayi* extracellular polysaccharide (LC EPS) was obtained.

[0049] 2. Preliminary preparation of Lactobacillus chilii extracellular polysaccharide selenium nanoparticles (LC EPS-SeNPs)

[0050] 10 mL of the prepared LC EPS solution was vortexed with an equal volume of fresh sodium selenite solution (0.01 mol / L) and magnetically stirred for 5 min. Simultaneously, an equal volume of ascorbic acid solution (0.04 mol / L) was added as a reducing agent. The mixture was heated and stirred in the dark for 20 min. After the reaction was complete, the mixture was centrifuged at 8000 r / min for 30 min, and this process was repeated three times. The precipitate was redissolved in distilled water and centrifuged three more times. After freeze-drying, LC EPS-SeNPs were obtained.

[0051] 3. Single-factor experiments and analysis

[0052] Using the reduction method described above, the effects of five factors—LC EPS addition amount, sodium selenite concentration, sodium selenite to ascorbic acid solution volume ratio, reaction temperature, and reaction time—on LC EPS-SeNPs particle size, zeta potential, and PDI were determined and analyzed. The experimental factors and levels are shown in Table 1 below.

[0053] The results of the single-factor experiment are as follows Figure 1 As shown in A to E:

[0054] Effect of reaction time ( Figure 1 A): Under constant conditions, the particle size and Zeta potential reach their minimum values ​​at a reaction time of 40 min. As the reaction time increases, the particle size begins to increase because prolonged heating weakens the system's stability (consumption of reducing agent, increased thermal motion, solvent evaporation), leading to particle agglomeration. Ultimately, the thermodynamically driven trend towards size homogenization further promotes the increase in particle size.

[0055] Effect of sodium selenite concentration ( Figure 1 B): When the sodium selenite concentration is 10 mmol / L, the particle size reaches its minimum value, and the particle size begins to increase as the concentration continues to increase.

[0056] The effect of volume ratio Se2O3:VC Figure 1 C): The minimum particle size is reached when the volume ratio is 1:2. High concentrations of vitamin C accelerate the reduction reaction, leading to explosive nucleation in a short time. However, excessive vitamin C or factors such as oxidation byproducts, decreased pH, and changes in ionic strength can disrupt particle stability and increase particle size.

[0057] Effect of reaction temperature Figure 1 D): The particle size reaches its minimum at a reaction temperature of 20℃. Low temperature increases the stability of the reaction system by slowing down the reduction rate, reduces agglomeration, and at the same time reduces the thermodynamic driving force to inhibit coarsening, ultimately keeping SeNPs in a small size and narrow distribution state.

[0058] Effect of LC EPS concentration ( Figure 1 E): When the concentration of LC EPS is 15 mg / mL, the particle size of LC EPS-SeNPs reaches its minimum.

[0059] 4. Plackett-Burman (PB) experimental design for screening

[0060] Using the particle size (Y) of LC EPS-SeNPs as an indicator, a Plackett-Burman experiment was designed using Design Expert 8.0.6 software to determine significant factors. The factors and levels of the PB experiment design are shown in Table 2 below:

[0061] The experimental design and response values ​​of the PB test are shown in Table 4 below:

[0062] Analysis of variance (ANOVA) was performed on the PB experiment data, and Lenth's method was used to identify significant effects. The results of the ANOVA are shown in Table 5, and the Pareto plot of the effects is shown in [Table 5]. Figure 1 F: Statistical analysis shows that the standard deviation (Std.Dev.) = 6.37, the multiple correlation coefficient R² = 0.9901, the adjusted coefficient of determination (AdjR²) = 0.9636, the predicted coefficient of determination (PredR²) = 0.8014, the mean (Mean) = 224.12, the coefficient of variation (CV%) = 2.84, and the precision (Adeq Precision) = 13.257. The model p-value of 0.0064 < 0.05 indicates that the regression model is significant and has a good fit. The CV value of 2.84% indicates good reliability and accuracy, and the precision of 13.257 (greater than 4.0 is considered reasonable).

[0063] Depend on Figure 1As shown in Table 5, factors B (LC EPS concentration), D (reaction time), and E (reaction temperature) have a negative effect on particle size, meaning that particle size decreases with increasing concentration; while factor C has a positive effect. Comparing the p-values, the significance of B, E, and D is higher than that of C (Na2SeO3 concentration). Therefore, subsequent response surface methodology (BBD) experiments will focus on three factors—LC EPS concentration (A), reaction time (B), and reaction temperature (C)—for further analysis.

[0064] 5. Box-Behnken Design (BBD) Response Surface Optimization

[0065] A three-factor, three-level BBD experimental design was conducted for the three significant variables selected. The variable and level coding table is shown in Table 3 below: BBD designed a total of 17 test sites. The test matrix, actual response values, and model prediction values ​​are shown in Table 6 below:

[0066] Using Design-Expert software, a multivariate regression fitting analysis was performed, and the quadratic polynomial regression equation between the nanoparticle size (Y) and independent variables A, B, and C was obtained as follows:

[0067] Y=191.48+0.55A+51.13B+2.59C-2.46AB-19.40AC+14.42BC+35.28A 2 +59.97B 2 +23.94C 2

[0068] The analysis of variance (ANOVA) was performed on the quadratic regression model, and the results are shown in Table 7 below:

[0069] The model's coefficient of determination R 2 =0.9855, Corrected coefficient of determination AdjR²=0.9668, Predicted coefficient of determination PredR²=0.8972, Coefficient of variation CV%=4.08. Based on the analysis results, the model P<0.05 and the lack-of-fit term was not significant (P=0.5453>0.05), indicating high reliability of the regression model. Experimental variance analysis showed that the order of influence of each factor on particle size was: reaction time (B) > reaction temperature (C) > LC EPS concentration (A).

[0070] Figure 2 The 3D response surface plot and contour plot show the pairwise interactions of each factor:

[0071] The interaction between A and B ( Figure 2 (AB): When the reaction temperature (C) is at zero, the contour plot of the interaction between LC EPS concentration (A) and reaction time (B) is elliptical, indicating a significant interaction. As both increase, the particle size of LC EPS-SeNPs first increases and then decreases, with a steep response surface slope. The optimal particle size corresponds to an LC EPS concentration between 12 and 18 mg / mL, and a reaction time between 20 and 40 min.

[0072] Interaction between A and C ( Figure 2 CD): When the reaction time (B) is at zero, the contour plot of the interaction between LC EPS concentration (A) and reaction temperature (C) is circular, indicating a significant interaction. As both increase, the particle size first increases and then decreases, with the optimal values ​​between LC EPS concentrations of 12 and 18 mg / mL and reaction temperatures of 35 and 45 °C.

[0073] The interaction between C and B ( Figure 2 EF): When the LC EPS concentration (A) is at zero, the contour plot of the interaction between reaction temperature (C) and reaction time (B) is elliptical, indicating a significant interaction. As both increase, the particle size first increases and then decreases, with a steep slope. The optimal values ​​are between 35 and 45°C at the reaction temperature and between 20 and 40 minutes at the reaction time.

[0074] 6. Optimal process parameter adjustment and verification

[0075] The theoretically optimal conditions were derived using Design-Expert software. Considering practical feasibility, the process parameters were adjusted to: LC EPS concentration of 15 mg / mL, reaction time of 31 min, and reaction temperature of 41℃. Table 8 below shows the results of the process adjustment and experimental measurements.

[0076] An improved adaptive neurofuzzy inference system (ANFIS) model was further introduced to characterize the evolution law. The results show that, compared with the RSM model, the artificial intelligence-based ANFIS model can more accurately capture the nonlinear mapping relationship between LC concentration, reaction time, reaction temperature and response value, better obtain the optimal conditions of the analytical equation, and effectively avoid the waste of raw materials and labor.

[0077] The stability of LC EPS-SeNPs prepared under optimal conditions was immediately determined after 0, 7, and 21 days at 25°C. The results are as follows: Figure 3As shown in Figures A and B, the LC EPS-SeNPs prepared based on the ANFIS model optimization exhibited superior stability: after 14 days of storage at room temperature, the particle size increased only from 174 nm to 220 nm, and the particle size change and aggregation degree were significantly smaller than those of the pure SeNPs group that had already undergone severe agglomeration.

[0078] Example 3: Application of Lactobacillus chiayi extracellular polysaccharide selenium nanoparticles in regulating macrophage polarization

[0079] This embodiment provides the application of *Lactobacillus chiiense* extracellular polysaccharide selenium nanoparticles in the preparation of drugs or formulations that regulate macrophage polarization. Specific cell assay procedures and activity regulation effect analysis are as follows:

[0080] 1. Cell scratch assay (to evaluate cell migration ability)

[0081] RAW264.7 macrophages were seeded in 12-well plates and cultured for 12 hours. The original culture medium was then aspirated, and the cells were washed twice with sterile PBS. Using a 200µL sterile pipette tip, two horizontal and vertical lines were drawn along a ruler in each well, intersecting at the center of the well. The cells were washed three times with PBS to remove any scraped cells. Drug-containing culture medium was added, and the plates were incubated at 37°C with 5% CO2. Samples were taken at 0, 12, 24, and 48 hours, and the average distance between the scratched cells was measured and statistically analyzed.

[0082] The results are as follows Figure 4 As shown in C and D, the experiment was divided into a control group (CC), a simple LC EPS group, and a LC EPS-SeNPs drug group. At 12h, 24h, and 48h time points, the cell migration rate (Migration rate%) of the LC EPS-SeNPs group was significantly higher than that of the control group and the pure polysaccharide group, demonstrating a remarkable beneficial effect in promoting macrophage migration and repair.

[0083] 2. Effects of drugs on the secretion of cell polarizing factors

[0084] The experiment was conducted in 24-well cell plates, divided into the following groups:

[0085] Negative control group: DMEM medium was added;

[0086] M1 macrophage positive control group: 1 µg / mL LPS and 10 ng / mL IFN-γ were added;

[0087] M2 macrophage positive control group: 20 ng / mL IL-4 added;

[0088] Drug group: Equal volumes of LC EPS-SeNPs solutions of different concentrations were added, and three concentration groups were set up: high, medium and low, with final concentrations of 2500µg / mL, 1250µg / mL and 625µg / mL, respectively.

[0089] After incubating the cells in each group for 24 hours, the supernatant was collected, and the contents of pro-inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as well as anti-inflammatory and repair factors interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected using an ELISA kit.

[0090] Analysis results as follows Figure 4 As shown in E:

[0091] Regulation of pro-inflammatory factors: The secretion of pro-inflammatory factors IL-6 and TNF-α decreased in a dose-dependent manner with increasing LC EPS-SeNPs concentration, and the differences between each concentration group (625, 1250, 2500 µg / mL) and the LPS+IFN-γ stimulation group were extremely significant (P < 0.01), suggesting that the nanoparticles do not promote macrophage polarization into M1 type pro-inflammatory macrophages.

[0092] Regulation of anti-inflammatory and repair factors: The secretion of anti-inflammatory and repair factors IL-10 and TGF-β also decreased in a dose-dependent manner with increasing LC EPS-SeNPs concentration, indicating that they have a clear intervention and regulatory effect on the secretion spectrum of M2 cytokines.

[0093] Conclusion: In summary, LC EPS-SeNPs intervened in the polarization direction of macrophages by synergistically regulating the secretion of M1 pro-inflammatory factors (IL-6, TNF-α) and M2 anti-inflammatory factors (IL-10, TGF-β), and this regulatory effect has a significant concentration-dependent characteristic.

[0094] 3. Cell uptake assay (fluorescently traced phagocytic capacity)

[0095] To prepare fluorescently labeled nanoparticles, 1 mg of FITC was first dissolved in 0.5 mL of DMSO. Then, before adding sodium selenite, the FITC solution was slowly added dropwise to 10 mL of LC EPS solution in 50 µL increments, with continuous stirring for 2 hours. After preparation, to remove unbound FITC, the fluorescently labeled LC EPS-SeNPs were centrifuged at 10000 rcf for 15 minutes, the supernatant was discarded, and the resuspension step was repeated twice to obtain LC EPS-SeNPs-FITC. Unmodified SeNPs-FITC was prepared using the same method as a control.

[0096] Simple SeNPs-FITC and modified LC EPS-SeNPs-FITC were added to 12-well cell plates containing RAW264.7 macrophages and incubated for 18 h. Cell nuclei were stained with DAPI, washed twice with PBS, and then an anti-fluorescence quencher was added. The phagocytosis of fluorescent substances was observed using a fluorescence microscope, and the fluorescence intensity of intracellular fluorescence in RAW264.7 cells was detected using a microplate reader (Ex=562.0 nm, Em=610.0 nm).

[0097] The results are as follows Figure 4 As shown in Figures A and B, the FITC channel (green) traces the subcellular distribution of SeNPs, while the DAPI channel (blue) labels the cell nucleus to locate cell positions. The results showed that the pure SeNPs-FITC group exhibited green fluorescence at the baseline phagocytic level; while the LC EPS-SeNPs-FITC group showed higher fluorescence brightness and denser distribution, with a significantly higher fluorescence intensity than the former (P < 0.05). Further incubation for 24 hours revealed that the pure SeNPs-FITC group had no obvious particle morphology (suggesting that pure nanoparticles are highly susceptible to aggregation, sedimentation, and inactivation), but the LC EPS-SeNPs-FITC group maintained good nano-dispersion. This phenomenon directly confirms that LC EPS modification can maintain the structural stability of nanoparticles, synergistically enhance the sustained-release performance of SeNPs, thereby efficiently enhancing the phagocytic capacity of macrophages for SeNPs and synergistically improving its sustained-release effect and bioavailability.

[0098] The aforementioned cellular-level active intervention effects are highly consistent with its preventive and protective effects in vivo, which significantly alleviates infection symptoms induced by Salmonella typhimurium in C57BL / 6 mice, improves survival rate, promotes the expression of intestinal antimicrobial peptides, and reduces intestinal inflammation. This provides a solid foundation for the preparation of novel macrophage immunomodulatory drugs or preparations.

[0099] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, apparatus, article, or method that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, apparatus, article, or method. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, apparatus, article, or method that includes that element.

[0100] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A type of selenium nanoparticle containing extracellular polysaccharide from Lactobacillus chilii, characterized in that, It includes extracellular polysaccharide of Lactobacillus chiayi and selenium nanoparticles dispersed in the extracellular polysaccharide of Lactobacillus chiayi; wherein the selenium in the selenium nanoparticles is amorphous zero-valent selenium.

2. The selenium nanoparticles of *Lactobacillus chiiense* extracellular polysaccharide according to claim 1, characterized in that, In X-ray photoelectron spectroscopy, the peak position of the electron binding energy of the Se 3d orbital of the selenium nanoparticles contains 55.1 eV ± 0.2 eV; In the X-ray diffraction pattern, there is a broad diffraction peak at a diffraction angle of 2θ of 20.96°±0.2°.

3. The selenium nanoparticles of *Lactobacillus chiiense* extracellular polysaccharide according to claim 1, characterized in that, In the Fourier transform infrared spectrum of the selenium nanoparticles containing extracellular polysaccharide from *Lactobacillus casei* of Chiayi, the OH stretching vibration peak is located at 3144.99 cm⁻¹. -1 ±4cm -1 At this point, the C=O stretching vibration peak is located at 1631.48 cm⁻¹. -1 ±4cm -1 Place.

4. A method for preparing selenium nanoparticles of extracellular polysaccharide from *Lactobacillus chiayiensis* according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Prepare a solution of extracellular polysaccharide of Lactobacillus chilii and mix it with sodium selenite solution to obtain a mixture; (2) Add ascorbic acid solution as a reducing agent to the mixture and carry out a heating reduction reaction under light-protected conditions; (3) After the reaction was completed, centrifugation was performed, the precipitate was collected and freeze-dried to obtain selenium nanoparticles of extracellular polysaccharide from Lactobacillus chiayi.

5. The method for preparing selenium nanoparticles of Lactobacillus chiayi exopolysaccharide according to claim 4, characterized in that, In the reaction system composed of the *Lactobacillus chilii* extracellular polysaccharide solution, sodium selenite solution, and ascorbic acid solution, the final concentration of the *Lactobacillus chilii* extracellular polysaccharide is 10-20 mg / mL, and the final concentration of sodium selenite is 2.5-20 mmol / L.

6. The method for preparing selenium nanoparticles of Lactobacillus chiayi exopolysaccharide according to claim 4, characterized in that, In step (2), the volume ratio of the sodium selenite solution to the ascorbic acid solution is 1:1 to 1:5, the heating reaction temperature is 30~50℃, and the time is 20~60min.

7. The method for preparing selenium nanoparticles of Lactobacillus chiayi exopolysaccharide according to claim 4, characterized in that, In step (1), the preparation of the *Lactobacillus chiayiensis* extracellular polysaccharide solution specifically includes: The fermentation broth of Lactobacillus chiiensis from Chiayi was taken, the bacterial cells were removed and concentrated, and then ethanol was added for alcohol precipitation. The precipitate was collected to obtain crude polysaccharide. The crude polysaccharide was subjected to deproteinization treatment; The deproteinized polysaccharide was purified by sequentially using a cellulose chromatography column and a gel chromatography column to obtain purified extracellular polysaccharide of Lactobacillus chiayi. The purified extracellular polysaccharide of Lactobacillus chiayi was dissolved in a solvent to obtain the Lactobacillus chiayi extracellular polysaccharide solution.

8. The use of the Lactobacillus chiayi exopolysaccharide selenium nanoparticles according to any one of claims 1-3 in the preparation of a drug or formulation for regulating macrophage polarization.

9. The application of the *Lactobacillus chiiense* extracellular polysaccharide selenium nanoparticles according to claim 8 in the preparation of drugs or formulations for regulating macrophage polarization, characterized in that... The regulation of macrophage polarization includes: inhibiting macrophage secretion of pro-inflammatory factors, and / or regulating macrophage secretion of anti-inflammatory factors; The pro-inflammatory factor is selected from interleukin-6 and tumor necrosis factor-α, and the anti-inflammatory factor is selected from interleukin-10 and transforming growth factor-β.

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