Application of a resistant Bacillus subtilis strain and its extracellular polysaccharide in inducing and training immunity

Abstract

This invention discloses a strain of resistant *Bacillus halosa* and its extracellular polysaccharide (LCEPS) for use in inducing training immunity. It relates to the field of bioengineering technology. The molecular structural formula is provided, the corresponding strain is preserved, and related applications are described. The LCEPS of this invention is mainly composed of fructose linked by 2,6-Fru(f) glycosidic bonds, which can form uniformly sized nanoparticles in aqueous solution. Through a classic in vitro training immunity model, this invention found that co-culturing LCEPS with macrophages releases large amounts of TNF-α and IL-6, exhibiting good training immunity properties. Through establishing mouse respiratory bacterial and viral infection models, it was demonstrated that LCEPS can activate the training immune response to fight infection, and the effect is significantly better than β-glucan. This invention has high reference value for the development of novel training immune inducers and is suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically to the application of a resistant Bacillus subtilis strain and its extracellular polysaccharide in inducing and training immunity. Background Technology

[0002] For a long time, immune memory has been considered a unique feature of the adaptive immune system. However, research over the past decade has revealed that innate immune cells (such as monocytes, macrophages, and NK cells) can also exhibit enhanced or altered responses to subsequent heterologous stimuli after initial stimulation. This phenomenon is known as "trained immunity" or "innate immune memory." The core mechanism of trained immunity lies in inducing metabolic and epigenetic reprogramming in innate immune cells or their progenitor cells to achieve "non-specific enhancement" and "persistent reprogramming," without relying on antigen-specific receptors. This concept breaks through traditional thinking, greatly expands the understanding of immune memory, and provides new theoretical foundations and targets for novel anti-infection strategies, vaccine adjuvants, and immunomodulators.

[0003] Currently, the main training immunization inducers are BCG and β-glucan. While BCG, as a live attenuated vaccine, offers protection against various infections, it carries safety risks such as disseminated infection and serious adverse reactions, and its immunization efficacy varies among individuals. Although β-glucan has a relatively high safety profile, its extraction and purification processes are complex and costly, and its diverse structures make batch stability control difficult.

[0004] Given the shortcomings of BCG and β-glucan as current main training immune inducers, such as safety risks, structural uniformity, production standardization, and dosing flexibility, there is an urgent need to develop new training immune inducers that are safer, more efficient, have wider sources, more diverse structures, and are easy to mass-produce.

[0005] Extracellular polysaccharides (EPS) are high-molecular-weight polysaccharides secreted by bacteria into the extracellular environment or tightly attached to the cell surface during their growth and metabolism. As an important component of bacterial metabolites, EPS are widely found in bacteria, fungi, actinomycetes, cyanobacteria, and some archaea. In recent years, with the development of metabolomics and high-resolution structural analysis techniques, the relationship between the molecular composition, structural characteristics, and biological functions of EPS has gradually become a research hotspot. In particular, EPS derived from probiotics, environmental bacteria, and certain fungi are considered to be promising natural immunomodulators due to their potential low toxicity, multiple targets, and good biocompatibility.

[0006] Therefore, whether a strain of resistant Bacillus halosa and its extracellular polysaccharide can be provided for the application of induced immune training to overcome the above-mentioned technical deficiencies is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides an application of a resistant *Bacillus halophilus* strain and its extracellular polysaccharide in inducing and training immunity. This includes the resistant *Bacillus halophilus* LC-5 with training immune function, its metabolite: extracellular polysaccharide (LCEPS), a method for extracting and purifying LCEPS, and a method for activating and training immune responses against bacterial respiratory infections and viral influenza. LCEPS has the advantages of high yield and low cost, making it suitable for widespread application.

[0008] Preservation Information: Bacillus halotolerans LC-5 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC: NO.39583, and the deposit date is July 6, 2026.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] An extracellular polysaccharide, the structural formula of which is shown below: .

[0011] Preferred: average molecular weight of 3.12kDa-5.88kDa.

[0012] The present invention also provides a bacterium that secretes the above-mentioned extracellular polysaccharide, namely: Bacillus shalotolerans LC-5; accession number: CGMCC No.39583.

[0013] The present invention also provides a method for extracting and purifying the extracellular polysaccharide as described above, comprising the following steps: (1) Fermentation with the above strains to obtain bacterial broth; centrifugation of bacterial broth to obtain supernatant, addition of trichloroacetic acid, standing, centrifugation, collection of supernatant; alcohol precipitation, dialyzing to obtain crude extracellular polysaccharide; (2) Extracellular crude polysaccharide LCEPS was obtained by purification of extracellular polysaccharide using DEAE-Sepharose Fast Flow ion exchange column and Sepharose CL-6B molecular sieve.

[0014] The present invention also provides the application of the above-mentioned extracellular polysaccharides or the above-mentioned strains in the preparation of training immune inducers.

[0015] The present invention also provides the use of the above-mentioned extracellular polysaccharides or the above-mentioned strains in the preparation of drugs for the prevention and / or treatment of bacterial respiratory infections or viral influenza.

[0016] The present invention also provides a pharmaceutical composition comprising the above-described extracellular polysaccharide and a pharmaceutically acceptable carrier or excipient.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of a resistant Bacillus halophilus strain and its extracellular polysaccharide in inducing training immunity, and the technical effects achieved are as follows: This invention involves fermenting Haloxylon ammonium-tolerant Bacillus LC-5 to extract and purify extracellular polysaccharides (LCEPS), and then analyzing their structure and characterizing their physicochemical properties. The LCEPS provided by this invention is mainly composed of fructose linked by 2,6-Fru(f) glycosidic bonds, which can form uniformly sized nanoparticles in aqueous solution; This invention, through a classic in vitro immune training model, found that LCEPS, when co-cultured with macrophages, releases large amounts of TNF-α and IL-6, exhibiting good immune training properties. Furthermore, by establishing mouse respiratory bacterial and viral infection models, it was demonstrated that LCEPS can activate the trained immune response to fight infection, with significantly better effects than β-glucan. This invention has high reference value for the development of novel immune training inducers and is suitable for widespread application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 The attached figure shows the monosaccharide composition of the extracellular polysaccharide LCEPS provided by the present invention; wherein, A is the high-performance liquid chromatography peak chromatogram of the monosaccharide standard sample; and B is the high-performance liquid chromatography peak chromatogram of LCEPS.

[0020] Figure 2 The attached figure shows the molecular weight diagram of the extracellular polysaccharide LCEPS provided by the present invention.

[0021] Figure 3 The attached figure is a Fourier transform infrared spectrum of the extracellular polysaccharide LCEPS provided by the present invention.

[0022] Figure 4 The attached figure shows the experimental results of the extracellular polysaccharide LCEPS Congo red provided by the present invention.

[0023] Figure 5The attached figure is a molecular formula diagram of the extracellular polysaccharide LCEPS provided by the present invention.

[0024] Figure 6 The attached figure shows the detection diagrams of DLS particle size and Zeta potential of the extracellular polysaccharide LCEPS provided by the present invention, wherein A is the DLS particle size and B is the Zeta potential.

[0025] Figure 7 The attached figures show the LCEPS SEM scanning electron microscope results and transmission electron microscope images provided by the present invention, wherein A is a scanning electron microscope image and B is a transmission electron microscope image.

[0026] Figure 8 The attached figure shows the results of in vitro stimulation of BMDM to release TNF-α and IL-6 provided by the present invention; wherein, A is the flowchart of in vitro immune training; B is the result of ELISA detection of TNF-α secretion by BMDM stimulated by LCEPS and β-glucan; C is the result of ELISA detection of IL-6 secretion by BMDM stimulated by LCEPS and β-glucan.

[0027] Figure 9 The attached figure shows the results of LCEPS activation training of immune response against P. aeruginosa lung infection provided by the present invention; wherein, A is the experimental design flowchart; B is the statistical results of the survival rate of mice in each group infected with P. aeruginosa 48 h later; C is the statistical results of the bacterial load in the lungs, spleen and inguinal lymph nodes of mice in each group infected with P. aeruginosa 24 h later; D is the H&E staining results of the lungs of mice in each group infected with P. aeruginosa 24 h later.

[0028] Figure 10 The attached figure shows the results of LCEPS activation training of immune response against S. aureus lung infection provided by the present invention; wherein, A is the experimental design flowchart; B is the statistical results of the survival rate of mice in each group 48 h after S. aureus infection; C is the statistical results of the bacterial load in the lungs, spleen and inguinal lymph nodes of mice in each group 24 h after S. aureus infection; D is the H&E staining results of the lungs of mice in each group 24 h after S. aureus infection.

[0029] Figure 11 The attached figure shows the results of LCEPS activation training of immune response against H1N1 influenza infection provided by the present invention; where A is the experimental design flowchart; B is the weight change of mice 14 days after respiratory tract infection with influenza virus; and C is the survival status of mice 14 days after respiratory tract infection with influenza virus.

[0030] Figure 12 The attached figure shows the H&E staining results of the lungs of mice in each group 14 days after infection with H1N1 influenza, as provided by this invention. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] This invention discloses the application of a resistant *Bacillus halophilus* strain and its extracellular polysaccharide in inducing and training immunity. In the embodiments, all raw materials not mentioned are commercially available; all operating methods not mentioned are conventional. For example, SPF-grade female C57BL / 6N mice are available from Beijing Spaford Biotechnology Co., Ltd. (Beijing, China). The animal protocols comply with the animal care ethics and experimental regulations of Inner Mongolia University, and will not be elaborated further here.

[0033] Example 1 A method for extracting and purifying *Salicylic Acidis* and its extracellular polysaccharides is as follows: In the early stages of this invention, a strain of halophilic Bacillus was obtained through screening and named LC-5, which was then deposited under patent procedures. All subsequent experiments were based on this strain.

[0034] 1. Fermentation Bacillus halophilus LC-5 was inoculated into BHI solid medium and activated in an incubator at 37°C for 24 h. Then, 1% (v / v) inoculum was added to 50 mL of BHI liquid medium and fermented in an incubator at 37°C for 48 h. The fermented culture was centrifuged at 10000 rpm at 4°C for 15 min, and the bottom precipitate was discarded. Trichloroacetic acid was slowly added to the supernatant to adjust the concentration to 40 mg / mL. After standing at 4°C for 12 h, the culture was centrifuged at 10000 rpm at 4°C for 15 min, the bottom precipitate was discarded, and the supernatant was collected. Anhydrous ethanol was added to the supernatant to 80% of its total volume, and the culture was precipitated at 4°C for 24 h. After centrifugation at 10,000 rpm for 15 min, collect all the precipitate at the bottom. Dissolve the precipitate in distilled water at 60℃, centrifuge at 10,000 rpm for 15 min, and discard the water-insoluble precipitate at the bottom. Transfer the supernatant into a dialysis bag with a molecular weight cutoff of 3 kDa, ensuring the bag is filled to no more than two-thirds full with dialysate. Replace the dialysate every 8 hours and perform dialysis continuously for 3 days. Collect all the solution in the dialysis bag and freeze-dry under vacuum to obtain crude extracellular polysaccharide.

[0035] 2. DEAE-Sepharose Fast Flow ion exchange column purification of extracellular polysaccharides The well-preserved DEAE-Sepharose Fast Flow packing material was packed into a Column XK 26 / 40 column (height: 40 cm, inner diameter: 26 mm). Packing was stopped when the packing material was 5 cm from the top of the column. The column was equilibrated sequentially with ultrapure water and Tris-HCl solution (55 mM, pH 7.8) at a flow rate of 2 mL / min, eluting for 3 column volumes. 100 mg of crude extracellular polysaccharide was weighed and dissolved in 10 mL of Tris-HCl, filtered through a 0.22 μm filter membrane, loaded onto the column, and allowed to stand for 30 min before elution. Elution was performed sequentially with Tris-HCl eluent and 1 M NaCl eluent at a flow rate of 2 mL / min, collecting 6 mL from each tube to obtain the purified crude extracellular polysaccharide from the DEAE column. The polysaccharide content in the eluent was determined by the phenol-sulfuric acid method, and the absorbance was detected at 490 nm. An elution curve was plotted to obtain the purified crude extracellular polysaccharide sample.

[0036] 3. Purification using Sepharose CL-6B molecular sieve Sepharose CL-6B packing material was loaded into a Column XK 16 / 100 chromatography column (height: 100 cm, inner diameter: 16 mm). Packing was stopped when the packing material was 10 cm from the top of the column. The column was equilibrated with ultrapure water and Tris-HCl solution (55 mM, pH 7.8) at a flow rate of 2 mL / min, eluting for 3 column volumes. 50 mg of the crude extracellular polysaccharide sample purified by DEAE column was weighed, dissolved thoroughly in 5 mL of Tris-HCl, filtered through a 0.22 μm filter membrane, and loaded onto the column. The mobile phase was eluted with Tris-HCl elution buffer at a flow rate of 2 mL / min, collecting 6 mL per tube. The polysaccharide content was determined by the phenol-sulfuric acid method, and the elution curve was plotted. After lyophilization, the extracellular polysaccharide LCEPS was obtained.

[0037] 4. LCEPS Production Detection The extracellular polysaccharide LCEPS yield in this example was determined to be 18.6 g / L by the sulfuric acid phenol method.

[0038] Structural analysis and physicochemical characterization of the polysaccharide LCEPS are as follows: Example 2 1. Structural characterization of the extracellular polysaccharide LCEPS (1) Monosaccharide composition of extracellular polysaccharide LCEPS Chromatographic analysis showed that all monosaccharide standard samples were effectively separated without peak overlap, and the retention times showed regular changes, indicating that the monosaccharide standards were qualified. LCEPS mainly consisted of fructose and glucose, with a molar ratio of fructose to glucose of 29.67:1.

[0039] The results are as follows Figure 1 .

[0040] (2) Molecular weight of extracellular polysaccharide LCEPS The scattering intensity of light by extracellular polysaccharide LCEPS molecules in the sample solution was measured by static light scattering (SLS), and the average molecular weight (Mw) of LCEPS was calculated to be 3.12 kDa–5.88 kDa. The results are shown in [Figure number missing]. Figure 2 .

[0041] (3) Structural characteristics of extracellular polysaccharide ELCEPS Fourier transform infrared spectroscopy (FI-IR) The major functional groups and glycosidic bond characteristics of the extracellular polysaccharide LCEPS were analyzed by Fourier transform infrared spectroscopy (FI-IR). At 3383 cm⁻¹ - The broad and strong absorption band at ¹ can be attributed to the stretching vibration of the hydroxyl group (O–H), indicating the presence of a large number of hydroxyl groups in the molecule and strong hydrogen bonding; 2925 cm⁻¹ - ¹ and 2879 cm - The absorption peak near ¹ corresponds to the C–H stretching vibration. 1624 cm⁻¹ - The absorption peaks near ¹ are usually related to the bending vibrations of adsorbed water, and the presence of acidic groups cannot be ruled out. 1234–1010 cm⁻¹ - The presence of distinct absorption peaks in region ¹, primarily attributable to C–O–C and C–O stretching vibrations, are typical characteristic peaks of the polysaccharide ring backbone and glycosidic bonds, indicating that the sample possesses polysaccharide structural features. Results are shown below. Figure 3 .

[0042] (4) Experimental analysis of the triple helix structure of extracellular polysaccharide LCEPS Congo red The spatial conformation of LCEPS was analyzed using the Congo red assay. The triple-helix polysaccharide formed a complex with Congo red, causing a redshift in the maximum absorption wavelength. In the LCEPS-Congo red complex system, the maximum absorption wavelength gradually decreased with increasing NaOH concentration at different NaOH concentrations, without exhibiting an overall trend of first increasing and then decreasing. Compared with the Congo red control group, the LCEPS group did not show a significant redshift, indicating that LCEPS may not possess a typical triple-helix structure. Results are shown below. Figure 4 .

[0043] (5) Structure of extracellular polysaccharide LCEPS The molecular formula of extracellular polysaccharide LCEPS is as follows: Figure 5As shown, LCEPS is mainly composed of the main chain formed by interconnected →6)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→), and the side chains are mainly composed of β-D-Fruf-(2→ linked to the O-1 position of the sugar residue →1,6)-β-D-Fruf-(2→), which are linked by t-Fru(f), t-Glc(p), 6-Fru(f) and 1,6-Fru(f) glycosidic bonds, with a glycosidic bond molar ratio of 9.54:1.89:83.65:4.92.

[0044] 2. Physicochemical characterization of extracellular polysaccharide LCEPS (1) Extracellular polysaccharide LCEPS DLS particle size and zeta potential DLS particle size and Zeta potential analyses were performed on LCEPS using ZS XPLOR software. DLS particle size analysis showed that the average particle size of LCEPS was 77 ± 5.16 nm, with a good particle size distribution, indicating that LCEPS in aqueous solution may not exist as single molecules, but rather as nanoscale aggregates due to hydration and intermolecular interactions. The Zeta potential was -21.29 mV, indicating that the LCEPS particles carry a certain negative charge on their surface, and there is electrostatic repulsion between the particles, suggesting that LCEPS aqueous solution exhibits a certain degree of colloidal stability. (See attached figures). Figure 6 .

[0045] (2) SEM and TEM analysis of extracellular polysaccharides LCEPS Further observation of the microstructure and aggregation state of LCEPS was conducted using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM results showed that LCEPS exhibited distinct strip-like, rod-like, and granular morphologies, indicating an irregular surface morphology, which may be related to the entanglement, aggregation, and stacking structure formed during the drying process of the polysaccharide molecular chains. TEM results showed that LCEPS mainly existed in the form of dispersed particles with a particle size of 20-30 nm, suggesting that this polysaccharide can form nanoparticle structures in a dispersed state. (See results below.) Figure 7 .

[0046] Example 3 The study on the activation and training of immune responses by extracellular polysaccharides is detailed below: 1. LCEPS stimulates the release of TNF-α and IL-6 from BMDM by training the immune response. Mouse femoral and tibial bone marrow cells were isolated and cultured in macrophage induction medium (1640 medium, 10% FBS, 1% penicillin-dextrose antibody, 1% non-essential amino acids, 20 ng / mL M-CSF) at 37 ℃ for 7 days in a 5% CO2 incubator. Cell growth and morphological changes were observed periodically under an inverted microscope to induce mature BMDM. BMDM cells were then cultured at a ratio of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1000 mcg / well in 96-well plates and incubated overnight at 37 °C in a 5% CO2 incubator to allow for full cell adhesion and stable cell set. Once the cells were stable, the original culture medium was discarded, and complete culture medium containing LCEPS was added to perform the first stimulation treatment on BMDM cells to construct an in vitro training immune model.

[0047] A blank control group (PBS) and a positive training control group (β-glucan) were set up. Cells in each group were cultured at 37 ℃ and 5% CO2 for 24 h to allow the stimulant to fully bind to the cells and induce training-related functional reprogramming of BMDM. After the first stimulation, the LCEPS medium was discarded, and the cells were gently washed with pre-warmed PBS to remove residual stimulant. Fresh complete medium was then added for further culture to allow the cells to enter a resting recovery phase. This phase simulates the transition of cells from a short-term activated state after initial stimulation to a relatively stable reprogrammed state during training-based immunity. During culture, the medium was replaced with fresh medium as needed based on cell growth to maintain cell stability. After 6 days of rest, the medium was discarded, and complete medium containing the restimulant was added to re-stimulate the BMDM in each group. Each group was added 200 μL of complete medium containing IFN-γ and cultured for 24 h, then replaced with complete medium containing Pam3CSK4 and cultured for another 24 h. The cell culture supernatant was collected, and the cytokines TNF-α and IL-6 were detected by ELISA. Results are shown below. Figure 8 The levels of TNF-α and IL-6 in the cell supernatant stimulated by LCEPS were not significantly different from those in the β-glucan group, but were significantly higher than those in the blank control group.

[0048] 2. LCEPS trains the immune response to resist... P. aeruginosa Infect The experimental group received an intraperitoneal injection of 4 mg LCEPS per mouse, the positive control group received an injection of 4 mg β-glucan, and the negative control group received an injection of the same volume of PBS. LCEPS was injected on days 0 and 7, and 5 × 10⁻⁶ mg per mouse was administered 24 h post-injection. 7 CFU P. aeruginosa(Purchased from Beina Biotechnology, ATCC27853) for respiratory tract infection. Mice were observed continuously for 48 hours, and their survival was recorded. Lungs, spleens, and inguinal lymph nodes were harvested 24 hours post-infection for statistical analysis. P. aeruginosa The load was determined by histopathological examination of lung tissue sections, and the results were as follows: Figure 9 As shown, the survival rate of mice in the LCEPS immunization group (50%) was significantly higher than that in the β-glucan immunization group (20%) and the PBS group (0%). Mice in the LCEPS and β-glucan immunization groups were infected with... P. aeruginosa The bacterial load in the posterior lungs, spleen, and inguinal lymph nodes was significantly lower in the mice immunized with LCEPS and β-glucan than in the PBS group. P. aeruginosa The lung tissue morphology of the mice in the posterior lung group was better, and the alveoli and connective tissue structure were more intact, while the lung damage of the mice in the PBS group was more severe.

[0049] 3. LCEPS trains the immune response to resist... S. aureus Infect The experimental group received an intraperitoneal injection of 4 mg LCEPS per mouse, the positive control group received an injection of 4 mg β-glucan, and the negative control group received an injection of the same volume of PBS. LCEPS was injected on days 0 and 7, and 5 × 10⁻⁶ mg per mouse was administered 24 h post-injection. 7 CFU S. aureusa (Purchased from Beina Biotechnology, USA300 TCH 1516) for respiratory infection. Mice were observed continuously for 48 hours, and their survival was recorded. Lungs, spleens, and inguinal lymph nodes were harvested 24 hours post-infection for statistical analysis. S. aureus The load was determined by histopathological examination of lung tissue sections, and the results were as follows: Figure 10 As shown, the survival rate of mice in the LCEPS immunization group (40%) was significantly higher than that in the β-glucan immunization group (20%) and the PBS group (0%). Mice in the LCEPS and β-glucan immunization groups were infected with... S. aureus The bacterial load in the posterior lungs, spleen, and inguinal lymph nodes was significantly lower in the mice immunized with LCEPS and β-glucan than in the PBS group. S. aureus The lung tissue morphology of the mice in the posterior lung group was better, and the alveoli and connective tissue structure were more intact, while the lung damage of the mice in the PBS group was more severe.

[0050] 4. LCEPS trains the immune response to resist H1N1 influenza virus A / PR / 8 strain infection. The experimental group received an intraperitoneal injection of 4 mg LCEPS per mouse, the positive control group received an injection of 4 mg β-glucan, and the negative control group received an injection of the same volume of PBS. LCEPS was injected on days 0 and 7, and 1 × 10⁻⁶ mg was administered 24 h post-injection. 6 PFU was administered intranasally as a 50 μL dose to each mouse to inoculate them with H1N1 influenza virus strain A / PR / 8 (a gift from the R&D Department of Jinyu Baoling Biopharmaceutical Co., Ltd.). The mice were observed daily for 14 days after challenge, and their weight and survival status were recorded. Results are as follows: Figure 11 As shown in the figure. Both LCEPS and β-glucan significantly improved the survival rate of mice, and after 14 days, the weight of the mice recovered to more than 95% of the pre-infection level. Lung tissue was aseptically harvested from mice on day 14 post-infection for histopathological observation, and the results are as follows. Figure 12 As shown, the lungs of mice in the PBS group exhibited congestion and hemorrhage, with severe lung swelling. In contrast, the small lungs of mice immunized with LCEPS and β-glucan showed clear alveolar structures and only mild histopathological changes. This demonstrates that LCEPS and β-glucan can significantly reduce inflammatory cell infiltration and lung tissue morphological changes.

[0051] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0052] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An extracellular polysaccharide, characterized in that, The structural formula is as follows: 。 2. The extracellular polysaccharide as described in claim 1, characterized in that, The average molecular weight is 3.12kDa-5.88kDa.

3. A bacterium that secretes the extracellular polysaccharide of claim 1, characterized in that, The bacteria mentioned are: Haloxysporum resistant to Bacillus (… Bacillus halotolerans LC-5; accession number: CGMCC No.39583.

4. A method for extracting and purifying extracellular polysaccharides as described in claim 1 or 2, characterized in that, Includes the following steps: (1) The bacterial culture was obtained by fermentation using the strain described in claim 3; the supernatant was collected by centrifugation of the bacterial culture, trichloroacetic acid was added, the culture was allowed to stand, centrifuged again, and the supernatant was collected; the culture was precipitated with alcohol and dialyzed to obtain crude extracellular polysaccharide; (2) Extracellular crude polysaccharide LCEPS was obtained by purification of extracellular polysaccharide using DEAE-Sepharose Fast Flow ion exchange column and Sepharose CL-6B molecular sieve.

5. The use of the extracellular polysaccharide of claim 1 or 2 or the strain of claim 3 in the preparation of training immune inducers.

6. The use of the extracellular polysaccharide of claim 1 or 2 or the strain of claim 3 in the preparation of a drug for the prevention and / or treatment of bacterial respiratory infections or viral influenza.

7. A pharmaceutical composition, characterized in that, It comprises the extracellular polysaccharide as described in claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.

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