An aminated mesoporous silica-natural polysaccharide co-encapsulated probiotic encapsulate, and a preparation method and application thereof

Abstract

This invention provides a probiotic encapsulation body co-encapsulated with aminated mesoporous silica and natural polysaccharides, its preparation method, and its application, belonging to the field of biomedical technology. The preparation method includes: preparing mesoporous silica using CTAB as a template and TEOS as a silicon source; modifying it with amination to obtain positively charged aminated mesoporous silica; modifying the natural polysaccharide with carboxymethylation to obtain negatively charged modified natural polysaccharides; and sequentially adding aminated mesoporous silica and carboxymethylated modified natural polysaccharides to a *Lactobacillus plantarum* suspension to obtain the probiotic encapsulation body. The probiotic encapsulation body prepared by this invention has an encapsulation efficiency as high as 93.5%, significantly improving the survival rate of probiotics in gastric and intestinal fluids, while also endowing the encapsulation body with pH-responsive intestinal targeted sustained-release properties. This probiotic encapsulation body can alleviate ulcerative colitis by upregulating VIP and activating the cAMP-PKA signaling pathway, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of functional food technology, specifically a probiotic encapsulation body co-encapsulated with aminated mesoporous silica and natural polysaccharides, its preparation method, and its application. Background Technology

[0002] Lactobacillus plantarum, a widely used probiotic, possesses various physiological activities, including regulating intestinal flora balance, enhancing immune function, and alleviating chronic metabolic diseases. However, Lactobacillus plantarum faces severe environmental challenges during oral delivery. During transport through the digestive tract, including the stomach and small intestine, free bacteria are highly susceptible to the combined attack of gastric acid, high concentrations of bile salts, and various digestive enzymes, leading to significant activity reduction or even death of many probiotics before reaching their intestinal sites of action. Existing research indicates that while physical barrier technologies such as microencapsulation and gel encapsulation can improve probiotic survival rates to some extent, the protective efficiency is limited due to material properties, with bacterial survival rates often falling below 30% under extreme conditions, severely restricting the practical application of probiotic formulations. Therefore, constructing a probiotic delivery system that combines efficient protection with precise release has become a core challenge urgently needing to be overcome in this field.

[0003] Ulcerative colitis (UC) is a disease characterized by chronic intestinal inflammation and recurrent attacks. Existing conventional chemical drug interventions often have limited efficacy, significant toxic side effects, and risks such as disrupting the intestinal microecological balance. Exogenous supplementation with highly active probiotic preparations to reshape the intestinal microecology and regulate the host immune response has become an important direction in the treatment of UC. However, currently reported probiotic encapsulation technologies still have many shortcomings: traditional encapsulation materials struggle to balance high protection with targeted release performance; single-layer encapsulation based on inorganic nanomaterials generally lacks effective sealing and controlled release methods; and most delivery systems cannot maintain structural stability in the acidic environment of the stomach, leading to premature exposure and inactivation of the bacteria. Therefore, there is an urgent need to develop an encapsulation system that can provide efficient physical protection for probiotics while possessing pH-responsive intestinal targeted release capabilities, in order to achieve precise intervention of probiotic preparations in the treatment of UC. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing probiotic encapsulations co-encapsulated with aminated mesoporous silica and natural polysaccharides. The probiotic encapsulations prepared by this method have the characteristics of high encapsulation rate, strong gastrointestinal tolerance, and the ability to achieve targeted sustained release in the intestine.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a probiotic encapsulation compound co-encapsulated with aminated mesoporous silica and natural polysaccharides, comprising the following steps: Using hexadecyltrimethylammonium bromide as a template and tetraethyl orthosilicate as a silicon source, a reaction was carried out under alkaline conditions. After removing the template agent, mesoporous silica was obtained. The mesoporous silica was then modified by amination to obtain amination-modified mesoporous silica with a positively charged surface. Natural polysaccharides were extracted and then modified by carboxymethylation to obtain carboxymethylated natural polysaccharides with negative charges. After activating Lactobacillus plantarum, a bacterial suspension was prepared. The aminated mesoporous silica was added to the bacterial suspension, so that the aminated mesoporous silica was electrostatically adsorbed and coated on the surface of Lactobacillus plantarum, thus obtaining a primary encapsulation product. The carboxymethylated modified natural polysaccharide is activated and then added to the primary encapsulation product, so that the carboxymethylated modified natural polysaccharide is electrostatically adsorbed and coated on the outer surface of the aminated mesoporous silica to obtain the probiotic encapsulation body. The mass ratio of the aminated mesoporous silica to the carboxymethylated modified natural polysaccharide is 1:2-2:1.

[0006] Preferably, the amount of hexadecyltrimethylammonium bromide added is 0.4-0.6 g; and the amount of tetraethyl orthosilicate added is 4-6 mL.

[0007] Preferably, the reaction temperature under alkaline conditions is 75-85℃, and the reaction time is 1.5-2.5h.

[0008] Preferably, the amination modification includes the following steps: dispersing the mesoporous silica in toluene, adding 3-aminopropyltriethoxysilane, refluxing at 75-85°C for 10-14 hours, and obtaining amination-modified mesoporous silica after washing and drying; the ratio of the mesoporous silica to the 3-aminopropyltriethoxysilane is 100 mg: 2-4 mL.

[0009] Preferably, the natural polysaccharide is chia seed gum or flaxseed gum; the extraction of the natural polysaccharide includes: adding the natural polysaccharide raw material to distilled water, stirring at 70-90℃ for 1.5-2.5h, and obtaining the natural polysaccharide after centrifugation, alcohol precipitation, and freeze drying.

[0010] Preferably, the carboxymethylation modification includes the following steps: dispersing the natural polysaccharide in an isopropanol aqueous solution, adding sodium hydroxide for pre-alkalization activation, then adding chloroacetic acid for carboxymethylation reaction, adjusting the pH to neutral after the reaction, and obtaining carboxymethylated modified natural polysaccharide by alcohol precipitation, washing, and freeze drying; the molar ratio of chloroacetic acid to the natural polysaccharide is 1:1-2:1.

[0011] Preferably, the activation treatment includes the following steps: dissolving the carboxymethylated modified natural polysaccharide in PBS buffer, adding EDC and NHS, and activating by stirring at room temperature for 1-3 hours.

[0012] Preferably, the final concentration of the aminated mesoporous silica in the bacterial suspension is 3-5 mg / mL; the primary encapsulation product is cultured at 35-37°C with shaking for 15-30 min; and the mass ratio of the aminated mesoporous silica to the carboxymethylated modified natural polysaccharide is 1:1.

[0013] The present invention also provides a probiotic encapsulation prepared by the preparation method described above.

[0014] The present invention also provides the use of the probiotic encapsulation in the preparation of medicaments for treating and / or alleviating ulcerative colitis.

[0015] Compared with the prior art, the present invention has the following advantages: This invention utilizes electrostatic adsorption to orderly self-assemble aminated mesoporous silica and carboxymethylated modified natural polysaccharides on the surface of *Lactobacillus plantarum*, constructing a probiotic encapsulation body with a double-core-shell structure. The aminated mesoporous silica, after grafting with APTES, carries a suitable amount of positive charge, generating strong electrostatic attraction with the negatively charged bacterial surface, achieving efficient and tight encapsulation at the single-cell level. The carboxymethylated chia seed gum carries a high density of negative charge, which tightly binds to the outer layer of the aminated mesoporous silica through electrostatic adsorption, forming a dense sealing layer. The two-step electrostatic self-assembly process is conducted under mild conditions, requiring no organic solvents or high-temperature treatment, effectively avoiding the loss of probiotic activity during preparation. The encapsulation body prepared by this invention achieves an encapsulation rate of up to 93.5%, with uniform particle size and good dispersibility. Furthermore, the preparation process is simple and easy to implement, facilitating large-scale production.

[0016] This invention utilizes the pH-responsive properties of carboxymethylated chia seed gum to endow the encapsulated body with pH-responsive intestinal-targeted release functionality. In the acidic environment of the stomach, the carboxyl groups undergo protonation, causing the polysaccharide chains to contract and form a dense protective layer, effectively resisting the erosion of gastric acid and pepsin. This results in a 90.50% survival rate of the encapsulated body after 2 hours of digestion in simulated gastric juice and an 87.01% survival rate after 4 hours of digestion in simulated intestinal juice. In the neutral intestinal environment, the carboxyl groups deprotonate, increasing electrostatic repulsion and causing the coating layer to loosen and degrade, achieving targeted sustained release of probiotics. The cumulative release rate in simulated intestinal juice reaches 85.1% after 5 hours, combining the advantages of sufficient release and long-lasting sustained release. Animal experiments have confirmed that this encapsulated body can efficiently deliver *Lactobacillus plantarum* to the mouse colon, significantly reducing the levels of pro-inflammatory factors IL-6 and TNF-α and increasing the level of anti-inflammatory factor IL-10 by upregulating vasoactive intestinal peptide (VIP) and activating the cAMP-PKA signaling pathway, effectively alleviating the pathological damage of ulcerative colitis. This invention provides an efficient and safe solution for the oral targeted delivery of probiotics and the biotherapy of inflammatory bowel disease. Attached Figure Description

[0017] Figure 1 The figures show the morphological characterization results of mesoporous silica and aminated mesoporous silica. In the figure, a and b are scanning electron microscope images of mesoporous silica and aminated mesoporous silica, respectively, with a scale bar of 500 nm; c and d are transmission electron microscope images of mesoporous silica and aminated mesoporous silica, respectively, with a scale bar of 50 nm.

[0018] Figure 2 Scanning electron microscope (SEM) images of *Lactobacillus plantarum* and the probiotic encapsulation prepared in Example 1 are shown. In the figures, a is a scanning electron microscope image of free *Lactobacillus plantarum*, with a scale bar of 500 nm; b is a scanning electron microscope image of the probiotic encapsulation prepared in Example 1, with a scale bar of 3 μm.

[0019] Figure 3 A comparison chart showing the amount of negatively charged groups in different natural polysaccharides.

[0020] Figure 4 A comparison diagram of the zeta potentials of different natural polysaccharides.

[0021] Figure 5 A bar chart comparing the survival rates of the probiotic encapsulated bacteria prepared in Example 1 and the control group at different digestion time points in simulated gastrointestinal fluid.

[0022] Figure 6 The cumulative release rate curves of the probiotic encapsulation prepared in Example 1 and the control group at different time points in simulated intestinal fluid.

[0023] Figure 7The images show H&E staining pathological images of colon tissue from mice in each group; the scale bars for the blank group, model group, positive group, and high-dose group are all 200 μm, while the scale bars for the free group and low-dose group are all 500 μm.

[0024] Figure 8 This is a graph showing the analysis of serum inflammatory cytokine levels.

[0025] Figure 9 Figure 1 shows the results of Western blot analysis of protein expression in mice with different treatment groups. Figure A shows the Western blot results and expression levels of Epac1 protein in the colon tissue of mice with different treatment groups. Figure B shows the Western blot results and expression levels of PKA protein in the colon tissue of mice with different treatment groups. Figure C shows the Western blot results and expression levels of p-PKA protein in the colon tissue of mice with different treatment groups. Figure D shows the quantitative analysis results of p-PKA / PKA in the colon tissue of mice with different treatment groups. Control is the blank group in Experiment 3, Model is the model group in Experiment 3, and MCL is the high-dose embedding group in Experiment 3. Detailed Implementation

[0026] This invention provides a method for preparing a probiotic encapsulation compound co-encapsulated with aminated mesoporous silica and natural polysaccharides, comprising the following steps: Using hexadecyltrimethylammonium bromide as a template and tetraethyl orthosilicate as a silicon source, a reaction was carried out under alkaline conditions. After removing the template agent, mesoporous silica was obtained. The mesoporous silica was then modified by amination to obtain amination-modified mesoporous silica with a positively charged surface. Natural polysaccharides were extracted and then modified by carboxymethylation to obtain carboxymethylated natural polysaccharides with negative charges. After activating Lactobacillus plantarum, a bacterial suspension was prepared. The aminated mesoporous silica was added to the bacterial suspension, so that the aminated mesoporous silica was electrostatically adsorbed and coated on the surface of Lactobacillus plantarum, thus obtaining a primary encapsulation product. The carboxymethylated modified natural polysaccharide is activated and then added to the primary encapsulation product, so that the carboxymethylated modified natural polysaccharide is electrostatically adsorbed and coated on the outer surface of the aminated mesoporous silica to obtain the probiotic encapsulation body. The mass ratio of the aminated mesoporous silica to the carboxymethylated modified natural polysaccharide is 1:2-2:1.

[0027] In this invention, the mesoporous silica is prepared as follows: Hexadecyltrimethylammonium bromide (CTAB) is dissolved in a deionized aqueous solution containing sodium hydroxide and stirred at 75-85°C until completely dissolved. Then, tetraethyl orthosilicate (TEOS) is added dropwise, wherein the final concentration of sodium hydroxide in the reaction system is 1.5-2.5 mol / L, the amount of CTAB added is 0.4-0.6 g, and the amount of TEOS added is 4-6 mL. The reaction is continued at 75-85°C for 1.5-2.5 h with stirring. After the reaction is complete, the product is centrifuged, washed alternately with deionized water and anhydrous ethanol, and then dried overnight at 75-85°C. The dried powder is placed in an acidified anhydrous ethanol solution (50 mL of anhydrous ethanol mixed with 5 mL of 37% hydrochloric acid) and refluxed for 10-14 h to remove the CTAB template. After washing, drying, and grinding again, mesoporous silica is obtained. The amination modification was performed as follows: The above-mentioned mesoporous silica was dispersed in toluene, and 3-aminopropyltriethoxysilane (APTES) was added at a ratio of 100 mg of mesoporous silica to 2-4 mL of APTES. The mixture was refluxed at 75-85 °C for 10-14 h. After the reaction was complete, the silica was thoroughly washed with anhydrous ethanol and deionized water, and then freeze-dried to obtain ammoniated mesoporous silica with a positively charged surface. Measurements showed that the obtained ammoniated mesoporous silica had a uniform and ordered mesoporous structure on its surface.

[0028] In this invention, the extraction and carboxymethylation modification steps of the natural polysaccharide are as follows: Chia seeds are added to distilled water at a material-to-liquid ratio of 1:15-1:25 (g / mL), and stirred at a constant speed at 70-90℃ for 1.5-2.5 h. The supernatant is collected by centrifugation, and anhydrous ethanol is added for alcohol precipitation (the volume ratio of chia seed gum to anhydrous ethanol is 1:3). The colloidal precipitate is collected by centrifugation again and then freeze-dried under vacuum to obtain chia seed gum. The above chia seed gum is dispersed in an isopropanol aqueous solution (the volume ratio of isopropanol to water is 7:3), and sodium hydroxide solution is added dropwise until the final concentration of the system is 4%-6% (w / v). The system is pre-alkalized and activated by stirring at 45-55℃ for 20-40 min to make the pH of the system greater than 13. Subsequently, chloroacetic acid solution was slowly added dropwise, with a molar ratio of chloroacetic acid to chia seed gum of 1:1-2:1. The reaction was carried out at 55-65℃ with stirring for 1.5-2.5 h, and the pH was controlled at 12.0-12.5 during the reaction. After the reaction was completed, the mixture was cooled to room temperature, and the pH was adjusted to neutral with glacial acetic acid. Anhydrous ethanol was added for alcohol precipitation, and the precipitate was collected after centrifugation. The precipitate was repeatedly washed with 80% ethanol solution, and the pH was adjusted to neutral again. After freeze-drying, carboxymethylated modified chia seed gum was obtained. The zeta potential of carboxymethylated modified chia seed gum was measured to be -24.1 mV, indicating that its surface carries a large number of negative charges.

[0029] In this invention, the activation of *Lactobacillus plantarum* and the preparation of the bacterial suspension are as follows: *Lactobacillus plantarum* JYLP002, stored in an ultra-low temperature freezer at -80℃, is inoculated into sterilized MRS liquid medium and cultured at 37℃ for 24 hours. This process is repeated 2-3 times to achieve full activation of the strain. The activated bacterial suspension is then transferred to fresh MRS liquid medium at an inoculation rate of 2% and cultured at 37℃ for another 24 hours until the late logarithmic growth phase. The culture is centrifuged at 4℃ to collect the bacterial sludge, the supernatant is discarded, and the bacterial cells are resuspended and washed 2-3 times with sterile physiological saline. Finally, the suspension is resuspended in sterile physiological saline, and the concentration of the bacterial suspension is adjusted to approximately 10. 9 CFU / mL was used to obtain a *Lactobacillus plantarum* bacterial suspension for later use. The above-mentioned aminated mesoporous silica was added to the *Lactobacillus plantarum* bacterial suspension under aseptic conditions, controlling the final concentration of aminated mesoporous silica in the bacterial suspension to be 3-5 mg / mL, preferably 4 mg / mL; the suspension was then incubated at 35-37℃ with shaking for 15-30 min, allowing the positively charged aminated mesoporous silica to bind tightly to the negatively charged *Lactobacillus plantarum* cells through electrostatic adsorption, forming a single-cell encapsulation structure with aminated mesoporous silica uniformly coating the cell surface, thus obtaining the primary encapsulation product. The entire process must be carried out in a sterile environment to ensure the microbiological safety of the final product. In this invention, the *Lactobacillus plantarum* content in the *Lactobacillus plantarum* JYLP-002 is ≥1.0 × 10⁻⁶. 10 CFU / g, powder, moisture <7%. This strain is commercially available and can be purchased through commercial channels.

[0030] In this invention, the activation and secondary encapsulation steps of the carboxymethylated modified natural polysaccharide are as follows: carboxymethylated modified chia seed gum is dissolved in PBS buffer (pH 6.8-7.2), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added for activation treatment. The amount of EDC and NHS added is 18-22 mg each. The mixture is stirred at room temperature for 1-3 h to obtain an activated carboxymethylated modified chia seed gum solution. The activated carboxymethylated chia seed gum solution was added to a system containing the primary encapsulation product. The mass ratio of aminated mesoporous silica to carboxymethylated chia seed gum was controlled at 1:2-2:1. The mixture was incubated at 35-37℃ with shaking for 15-30 min. This allowed the negatively charged activated carboxymethylated chia seed gum to uniformly coat the positively charged aminated mesoporous silica surface through electrostatic adsorption, forming a double-core-shell structure of an inorganic silica layer and a natural polysaccharide layer. The precipitate was collected by centrifugation to obtain the probiotic encapsulated body. Measurements showed that when the final concentration of aminated mesoporous silica in the bacterial suspension was 4 mg / mL and the mass ratio of aminated mesoporous silica to carboxymethylated chia seed gum was 1:1, the encapsulation efficiency reached a maximum of 93.5%. The resulting probiotic encapsulated bodies had uniform particle size and intact outer polysaccharide coating, exhibiting excellent bacterial protection in simulated gastrointestinal fluid.

[0031] This invention also provides a probiotic encapsulation body prepared by the aforementioned method. The probiotic encapsulation body prepared by this invention has a core-shell structure, comprising a *Lactobacillus plantarum* core, an aminated mesoporous silica layer covering the surface of the *Lactobacillus plantarum* core, and a carboxymethylated modified natural polysaccharide layer covering the outer surface of the aminated mesoporous silica layer. The aminated mesoporous silica layer carries a positive charge, and the carboxymethylated modified natural polysaccharide layer carries a negative charge; the two layers are tightly bound together by electrostatic adsorption. The resulting probiotic encapsulation rate can reach over 90%, the survival rate after 2 hours of digestion in simulated gastric juice is 90.50%, the survival rate after 4 hours of digestion in simulated intestinal juice is 87.01%, and the cumulative release rate after 5 hours in simulated intestinal juice is 80%-90%, achieving efficient encapsulation of probiotics, gastrointestinal protection, and targeted sustained release into the intestine.

[0032] This invention also provides the application of the aforementioned probiotic encapsulation in the preparation of drugs for treating and / or alleviating ulcerative colitis. In a specific embodiment of this invention, an acute ulcerative colitis model in mice was established using 2.5% sodium dextran sulfate (DSS) in drinking water. Simultaneously with model establishment, the probiotic encapsulation was administered via gavage at a dose equivalent to 10... 9 -10 10Lactobacillus plantarum at CFU / mL was administered once daily for 7 consecutive days. After administration, the disease activity index, colonic histopathological changes, and inflammatory cytokine levels were assessed. Results showed that the probiotic encapsulation significantly inhibited DSS-induced weight loss and disease activity index increases in mice, reduced inflammatory cell infiltration and crypt destruction in colonic tissue, decreased levels of pro-inflammatory cytokines IL-6 and TNF-α, and increased levels of anti-inflammatory cytokine IL-10. Mechanistic studies indicated that the probiotic encapsulation exerted its effect in alleviating ulcerative colitis by upregulating the expression of vasoactive intestinal peptide (VIP) in colonic tissue, specifically enhancing PKA phosphorylation activation, and regulating the cAMP-PKA signaling pathway.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0035] Example 1 A method for preparing a probiotic encapsulation compound co-encapsulated with aminated mesoporous silica and natural polysaccharides, the preparation method comprising the following steps; 1. Preparation and Aminoation Modification of Mesoporous Silica (MSN) (1) Preparation of mesoporous silica (MSN) Accurately weigh 4.0 g of sodium hydroxide and 50 mL of distilled water, stir until the solid is completely dissolved to obtain a 2 mol / L NaOH solution, and seal for later use. Take a beaker, add an appropriate amount of the prepared NaOH solution and deionized water, mix thoroughly, and heat. When the system temperature stabilizes at 80 °C, add 0.5 g of hexadecyltrimethylammonium bromide (CTAB), and continue stirring until CTAB is completely dissolved and the system becomes clear and transparent. Then, take 5 mL of tetraethyl orthosilicate (TEOS) and slowly add it dropwise along the beaker wall while continuously stirring with a magnetic stirrer to ensure that TEOS is uniformly dispersed in the reaction system. After the addition is complete, seal the beaker and continue stirring at 80 °C for 2 hours. The reaction solution containing the white colloid was transferred to a 100 mL centrifuge tube. The centrifuge was set to 9000 rpm and centrifuged for 5 minutes. The reaction solution separated into layers; the supernatant was poured off and discarded, retaining the white precipitate at the bottom. The precipitate was then washed three times alternately with anhydrous ethanol and deionized water to remove unreacted raw materials. The precipitate was then dried overnight in an 80°C oven. The powder was refluxed in an acidified anhydrous ethanol solution (50 mL anhydrous ethanol, 5 mL 37% HCl) for 12 hours to remove excess template agent CTAB at 80°C. The powder was separated, washed alternately with ethanol and distilled water, and dried overnight in an 80°C oven to obtain a white solid. This white powder was ground into a fine powder, which is the mesoporous silica.

[0036] (2) Preparation of aminated mesoporous silica (MSN-NH2) The prepared mesoporous silica was used as raw material and then modified by amination. Using toluene as solvent, 100 mg of mesoporous silica, 3 mL of 3-aminopropyltriethoxysilane, and 15 mL of toluene were refluxed at 80 °C for 12 h. After washing several times with anhydrous ethanol and deionized water, the amination-modified mesoporous silica was obtained by freeze-drying.

[0037] 2. Extraction and carboxymethyl modification of chia seed gum (CSG) (1) Extraction of chia seed gum Weigh chia seeds and add them to distilled water at a material-to-liquid ratio of 1:20 (g / mL). Stir at 80℃ for 2 hours. Centrifuge the solution at room temperature (10000 r / min, 15 min) and collect the intermediate layer. Add anhydrous ethanol to the intermediate layer (chia seed gum to anhydrous ethanol volume ratio of 1:3), centrifuge again (10000 r / min, 20 min), discard the supernatant, collect the colloidal precipitate, and freeze-dry under vacuum to obtain chia seed gum (CSG). Store in a sealed container at 4℃ for later use.

[0038] (2) Carboxymethyl modification of chia seed gum Mix 1g of chia seed gum with twice the volume of isopropanol solution (isopropanol to water volume ratio 7:3). Add 2mol / L NaOH solution dropwise to a final concentration of 5% (w / v), and activate by stirring continuously at 50℃ for 30 min for pre-alkalization treatment. The pH of the mixture after pre-alkalization should be greater than 13. Slowly add 40% chloroacetic acid solution dropwise to the alkalized colloidal suspension, with a molar ratio of chloroacetic acid to chia seed gum of 1:1. Stir continuously at 60℃ for 2 h, maintaining the pH between 12.0 and 12.5. After the reaction is complete, quickly cool to room temperature, adjust the pH to 7 with glacial acetic acid, add twice the volume of anhydrous ethanol, let stand for 30 min, centrifuge, discard the supernatant, wash several times with 80% ethanol solution, adjust the pH to neutral again, and finally freeze-dry and store at 4℃ to obtain carboxymethylated modified chia seed gum.

[0039] 3. Activation and preparation of bacterial suspension of Lactobacillus plantarum Lactobacillus plantarum (stored in an ultra-low temperature freezer) Lactobacillus plantarum JYLP002 was inoculated into sterile MRS liquid medium and cultured at 37°C for 24 hours. This process was repeated twice for activation. The activated strain was then added to MRS liquid medium at a 2% inoculation rate and cultured at 37°C for another 24 hours. After culturing, the culture was centrifuged at 4°C (10000 rpm, 10 min), the supernatant was discarded, and the bacterial sludge was collected and resuspended in sterile physiological saline to obtain a *Lactobacillus plantarum* suspension for later use.

[0040] 4. Preparation of the encapsulation Dissolve 10 mg of carboxymethylated chia seed gel in 20 mL of PBS buffer (pH 7.0), add 20 mg of EDC and 20 mg of NHS, and activate by magnetic stirring for 2 h. Prepare and use immediately.

[0041] Take 8 mg of aminated mesoporous silica (MSN-NH2) and sterilize it for later use, so that the final concentration of MSN-NH2 in the bacterial suspension is 4 mg / mL. Take 2 mL of Lactobacillus plantarum bacterial suspension, add 8 mg of MSN-NH2, and incubate in a shaker at 36 °C for 20 min. This allows the aminated mesoporous silica to coat the surface of Lactobacillus plantarum through electrostatic adsorption, resulting in a primary encapsulation product.

[0042] The activated carboxymethylated chia seed gum solution was added to the above primary encapsulation product (the mass ratio of aminated mesoporous silica to carboxymethylated chia seed gum was 1:1), and the mixture was shaken and incubated at 36°C for 20 min. This allowed the carboxymethylated chia seed gum to be electrostatically adsorbed and coated onto the outer surface of the aminated mesoporous silica, resulting in the secondary encapsulation product, namely the probiotic encapsulated body.

[0043] 5. Encapsulation rate determination Viable cell counts were performed using the dilution plating method. Bacterial suspensions before and after encapsulation were serially diluted and plated onto MRS solid culture plates. After incubation at 37°C for 48 hours, colony counts were performed. The encapsulation rate of *Lactobacillus plantarum* in the probiotic encapsulation body prepared in this example was determined to be 93.5%.

[0044] 6. Morphological and structural characterization The mesoporous silica (MSN) and aminated mesoporous silica (MSN-NH2) prepared in this embodiment were observed by scanning electron microscopy and transmission electron microscopy. Figure 1 As shown in the scanning electron microscope images of a and b, both MSN and MSN-NH2 exhibit relatively regular spherical morphologies, with uniform particle size and good dispersibility. Figure 1 As shown in the transmission electron microscopy images of c and d, the MSN surface has a distinct and orderly arranged mesoporous channel structure with uniform pore size; the amination-modified MSN-NH2 still maintains a clear mesoporous structure, and the mesoporous channels are not blocked or collapsed due to amination modification.

[0045] Further scanning electron microscopy was performed to observe *Lactobacillus plantarum* before and after encapsulation. Figure 2 As shown in Figure a, free *Lactobacillus plantarum* exhibits a typical short rod-like morphology with a smooth surface and clear outline. Figure 2 As shown in Figure b, the surface of *Lactobacillus plantarum* encapsulated by this invention becomes noticeably rougher, covered with a dense coating layer, and the cell outline is slightly blurred, indicating that the aminated mesoporous silica and carboxymethylated modified chia seed gum have been successfully encapsulated on the cell surface, forming a complete encapsulation structure.

[0046] Dynamic light scattering analysis showed that the average particle size of MSN-NH2 was 163.3 ± 5.6 nm, and the Zeta potential was +13.4 mV; the Zeta potential of carboxymethylated chia gum was -24.1 mV. This indicates that MSN-NH2 carries a positive surface charge, while carboxymethylated chia gum carries a strong negative surface charge, and the two can achieve layer-by-layer self-assembly through electrostatic adsorption.

[0047] Example 2: Effect of different MSN-NH2 concentrations on encapsulation efficiency The difference between this embodiment and Example 1 is that in Section 4 (Preparation of Encapsulation), the amount of aminated mesoporous silica (MSN-NH2) added is 2 mg, 4 mg, 6 mg, 8 mg, and 10 mg (corresponding to final concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL in the bacterial suspension, respectively). The remaining operations are the same as in Example 1. The encapsulation efficiency results are shown in Table 1.

[0048] Table 1. Encapsulation efficiency at different final MSN-NH2 concentrations

[0049] The results showed that the encapsulation efficiency was highest at a final concentration of 4 mg / mL in the bacterial suspension, reaching 93.5%. When the final concentration of MSN-NH2 was in the range of 1-4 mg / mL, the encapsulation efficiency increased with increasing concentration; however, when the concentration was too low (1 mg / mL) or too high (5 mg / mL), the encapsulation efficiency decreased.

[0050] Example 3: Effect of different mass ratios of MSN-NH2 to carboxymethylated chia seed gum on encapsulation efficiency The difference between this embodiment and Example 1 is that in Section 4 (Preparation of Encapsulation), the mass ratios of aminated mesoporous silica to carboxymethylated modified chia seed gum are 1:2, 1:1, and 2:1, respectively; the remaining operations are the same as in Example 1. The encapsulation efficiency results are shown in Table 2.

[0051] Table 2. Embedding efficiency at different mass ratios

[0052] The results showed that the encapsulation efficiency was the highest, reaching 93.5%, when the mass ratio of aminated mesoporous silica to carboxymethylated modified chia seed gum was 1:1.

[0053] Example 4 To screen suitable natural polysaccharides as sealing agents, 0.1 g each of chia seed gum, flaxseed gum, locust bean gum, guar gum, and chitosan were selected and dissolved in 50 mL of distilled water. The amount of negatively charged groups and zeta potential of each polysaccharide were measured for comparative analysis.

[0054] like Figure 3 and Figure 4 The results showed that, at the same mass concentration, chia seed gum had the highest amount of negatively charged groups (0.25 mmol), followed by flaxseed gum (0.23 mmol), locust bean gum (0.11 mmol), and the lowest amount was chitosan (only 0.02 mmol). There was no significant difference in the amount of negatively charged groups between chia seed gum and flaxseed gum. Zeta potential measurements showed that chitosan, being positively charged (+17.3 mV), could not bind to the equally positively charged MSN-NH2 via electrostatic adsorption; chia seed gum had the lowest Zeta potential (-24.1 mV), followed by flaxseed gum (-19.2 mV). Considering both the amount of negatively charged groups and the zeta potential, chia gum had the highest amount of negatively charged groups (0.25 mmol) and the lowest zeta potential (-24.1 mV), enabling it to generate the strongest electrostatic adsorption with positively charged MSN-NH2. Therefore, chia gum was ultimately selected as the sealing agent for aminated mesoporous silica in subsequent encapsulation experiments.

[0055] Experiment 1: Survival determination of encapsulated bodies in simulated gastrointestinal fluid To investigate the tolerance of the probiotic encapsulated organisms prepared in this invention to the gastrointestinal environment, simulated gastric and intestinal digestion experiments were conducted on free Lactobacillus plantarum (unencapsulated group), Lactobacillus plantarum encapsulated in a single layer of aminated mesoporous silica (MSN-NH2 group), and Lactobacillus plantarum encapsulated in a combination of aminated mesoporous silica and carboxymethylated chia seed gum (MSN-NH2-CSG group, i.e. the encapsulated organisms prepared in Example 1).

[0056] 1. Preparation of simulated gastric juice Take 3.2g of pepsin (1:3000), add 0.1mol / L hydrochloric acid solution to adjust the pH to 2.0, filter and sterilize with a 0.22μm aqueous filter membrane, and set aside for later use.

[0057] 2. Simulated gastric digestion experiment Take 1 mL of free *Lactobacillus plantarum* suspension (unencapsulated group), *Lactobacillus plantarum* culture encapsulated in MSN-NH2 monolayer, and *Lactobacillus plantarum* culture co-encapsulated in MSN-NH2-CSG prepared in Example 1 (each group has a viable count of approximately 10). 9 CFU / mL was added to 9 mL of simulated gastric fluid (pH 2.0, containing pepsin), and the mixture was placed in a constant-temperature shaking incubator (100 rpm, 37 °C) and shaken. Samples were taken at 0 h, 0.5 h, 1 h, 1.5 h, and 2 h. Viable bacteria were counted using the dilution plating method, and the survival rate of each group at different time points was calculated. The results are shown below. Figure 5 As shown.

[0058] 3. Preparation of simulated intestinal fluid The pH of the solution was adjusted to 7.0 using 0.1 mol / L NaOH, and 0.2% sodium chloride, 0.1% trypsin and 0.3% bile salts were added. The solution was then filtered and sterilized using a 0.22 μm aqueous phase filter membrane for later use.

[0059] 4. Simulated intestinal digestion experiment Take 1 mL of free *Lactobacillus plantarum* suspension (unencapsulated group), *Lactobacillus plantarum* culture encapsulated in MSN-NH2 monolayer, and *Lactobacillus plantarum* culture co-encapsulated in MSN-NH2-CSG prepared in Example 1 (each group initially had a viable count of approximately 10). 9 CFU / mL was added to 9 mL of simulated intestinal fluid (pH 7.0, containing 0.2% NaCl, 0.1% trypsin, and 0.3% bile salts), and the mixture was placed in a constant-temperature shaking incubator (100 rpm, 37°C) and shaken. Samples were taken from the simulated intestinal fluid at 0 h, 2 h, and 4 h after digestion. Viable bacteria were counted using the dilution plating method after sampling, and the survival rate of each group at different time points was calculated. Results Figure 5 As shown.

[0060] according to Figure 5 The results showed that the number of viable bacteria decreased in both the unencapsulated and encapsulated groups after 2 hours of simulated gastric fluid digestion. Specifically, the viable bacterial count in the unencapsulated group decreased from 10.01 to 5.74 log (CFU / mL), a reduction of 4.27 log values. In the MSN-NH2-CSG group, the viable bacterial count of *Lactobacillus plantarum* decreased from 9.68 to 8.76 log (CFU / mL), a reduction of only 0.92 log values, representing a relative increase in survival rate of 33.16%. After 4 hours of simulated intestinal fluid digestion, the viable bacterial survival rate in the encapsulated group was as high as 87.01 ± 0.14% (a decrease of 0.96 log CFU / mL), while the viable bacterial survival rate in the unencapsulated group was only 71.02% (a decrease of 2.9 log CFU / mL), representing a relative increase in survival rate of 15.99%. This indicates that MSN-NH2-CSG can significantly increase the viable bacterial count of *Lactobacillus plantarum* in simulated gastrointestinal fluid.

[0061] Experiment 2: Targeted release of the encapsulated body in simulated intestinal fluid To investigate the targeted release performance of the probiotic encapsulation prepared in this invention in the intestinal environment, fluorescein isothiocyanate (FITC) was used to label Lactobacillus plantarum. Simulated intestinal fluid release experiments were conducted on free Lactobacillus plantarum bacterial suspension (bacterial suspension group), Lactobacillus plantarum encapsulated in a monolayer of aminated mesoporous silica (MSN-NH2 group), and Lactobacillus plantarum encapsulated in a combination of aminated mesoporous silica and carboxymethylated chia seed gum (MSN-NH2-CSG group, i.e. the encapsulation prepared in Example 1).

[0062] 1. FITC-labeled Lactobacillus plantarum Prepare a 1 mg / mL stock solution using 1 mg fluorescein isothiocyanate (FITC) and 1 mL dimethyl sulfoxide (DMSO), vortex until completely dissolved, filter through a 0.22 μm aqueous filter membrane for sterilization, store at 4°C protected from light, and use immediately after preparation.

[0063] Take 1 mL of free Lactobacillus plantarum bacterial suspension, Lactobacillus plantarum bacterial suspension encapsulated with MSN-NH2 monolayer, and Lactobacillus plantarum bacterial suspension co-encapsulated with MSN-NH2-CSG prepared in Example 1 (the viable count of each group is approximately 10). 9 Add 10 μL of FITC stock solution to each sample (CFU / mL) and shake at 37°C for 1-2 h. Centrifuge to collect bacterial cells, wash 2-3 times with PBS buffer (pH 7.0) to remove excess FITC until the supernatant is free of fluorescence, and obtain the FITC-labeled samples for each group.

[0064] 2. Preparation of simulated intestinal fluid The pH of the solution was adjusted to 7.0 using 0.1 mol / L NaOH, and 0.2% sodium chloride, 0.1% trypsin and 0.3% bile salts were added. The solution was then filtered and sterilized using a 0.22 μm aqueous phase filter membrane for later use.

[0065] 3. Simulated intestinal fluid release experiment Take 1 mL of FITC-labeled free *Lactobacillus plantarum* bacterial suspension, MSN-NH2 monolayer-encapsulated *Lactobacillus plantarum* bacterial suspension, and MSN-NH2-CSG co-encapsulated *Lactobacillus plantarum* bacterial suspension prepared in Example 1, respectively, and add them to 9 mL of simulated intestinal fluid (pH 7.0, containing 0.2% NaCl, 0.1% trypsin, and 0.3% bile salts). Place the solutions in a constant temperature shaking incubator (100 r / min, 37℃) and shake. Take 1 mL samples at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h respectively, and measure the fluorescence intensity using a fluorescence spectrophotometer. Calculate the cumulative release rate for each group. The results are as follows: Figure 6 As shown. The cumulative release rate is calculated using the following formula: Cumulative release rate (%) = (FITC fluorescence intensity released into the supernatant at each time point / total fluorescence intensity) × 100%.

[0066] according to Figure 6 The results showed a positive correlation between fluorescence intensity and the number of bacteria released in the simulated intestinal fluid. Free *Lactobacillus plantarum* lacked protection in the simulated intestinal fluid and were directly exposed to the simulated intestinal environment, resulting in bacterial damage or inactivation and a gradual decrease in fluorescence signal. The MSN-NH2 group showed a release rate of only 16.45% at 0 h, significantly lower than the bacterial suspension group, indicating that mesoporous silica effectively encapsulated *Lactobacillus plantarum*, with the bacteria initially remaining encapsulated within the carrier and not released in large quantities. With prolonged exposure to the simulated intestinal fluid, the signal intensity showed a rapid initial increase followed by a slower increase, indicating that the MSN-NH2 carrier gradually degraded in the simulated intestinal fluid, achieving controlled release of the encapsulated *Lactobacillus plantarum*. The MSN-CSG group showed an initial release rate of only 8.23%, lower than the MSN-NH2 group, indicating that chia seed gum, as the sealing agent for MSN-NH2, enhanced the sealing of the encapsulated *Lactobacillus plantarum*, resulting in a lower initial release. With prolonged exposure to the simulated intestinal fluid, the release rate continued to increase, but the release rate was significantly slower than that of the MSN-NH2 group. At 5 hours, the release rate reached 85.1 ± 0.24%, comparable to that of the MSN-NH2 group. This result indicates that the cross-linking of CSG and MSN-NH2 forms a barrier on the bacterial cell surface, further slowing the release rate of *Lactobacillus plantarum*, achieving a more significant sustained-release effect. Furthermore, the total release at 5 hours is similar to that of the MSN-NH2 group, ensuring effective bacterial release while optimizing release kinetics and extending the release period.

[0067] Experiment 3: Treatment of a mouse model of ulcerative colitis with encapsulated bodies 1. Laboratory animals and grouping SPF-grade male C57BL / 6 mice, 6-8 weeks old, were used for the experiment after one week of acclimatization feeding. The mice were randomly divided into 7 groups (n=8 per group): Control group: Normal drinking water, and oral administration of physiological saline (0.2 mL / animal / day) Model group: Colitis induced by drinking 2.5% DSS, followed by gavage administration of physiological saline (0.2 mL / animal / day) Positive group: 2.5% DSS in drinking water, and 5-aminosalicylic acid (5mg / mL, 0.2mL / animal / day) by gavage. Free group: 2.5% DSS in drinking water, followed by gavage administration of free Lactobacillus plantarum bacterial solution (10... 9 CFU / mL, 0.2mL / animal / day) Low-dose encapsulation group: 2.5% DSS in drinking water, administered via gavage with the encapsulating body from Example 1 (equivalent to 10 9 (CFU / mL Lactobacillus plantarum, 0.2mL / animal / day) High-dose encapsulation group: 2.5% DSS in drinking water, administered by gavage with the encapsulating body from Example 1 (equivalent to 10 10 (CFU / mL Lactobacillus plantarum, 0.4 mL / animal / day) 2. Establishment of a colitis model and drug administration Except for the control group, all other groups were fed 2.5% DSS in drinking water to establish an acute colitis model. Administered medication by gavage began on day 8 and continued for 7 consecutive days.

[0068] 3. Histopathological analysis of colon tissue Mice were sacrificed after drug administration, and colon tissue was collected for H&E staining and pathological analysis. Results are as follows: Figure 7 The results showed that the colonic tissue in the model group exhibited significant inflammatory cell infiltration, crypt structure disruption, and mucosal damage. In the high-dose embedding group, inflammatory cell infiltration was significantly reduced, crypt structure remained largely intact, and mucosal damage was significantly improved, demonstrating effects comparable to the positive control group.

[0069] 4. Measurement of inflammatory factor levels Mouse colon tissue homogenate was collected, and the levels of pro-inflammatory cytokines IL-6, TNF-α, and anti-inflammatory cytokines IL-10 were measured by ELISA.

[0070] The results are as follows Figure 8As shown, the IL-6 level in the model group was significantly higher than that in the control group. Compared with the model group, the expression of IL-6 in the other groups was significantly reduced. The changes in serum TNF-α levels in DSS-induced UC mice were also roughly the same, with the gavage treatment groups treated with *Lactobacillus plantarum* significantly reducing TNF-α expression. Inflammatory factors TNF-α and IL-6 can induce T lymphocyte proliferation and activation by binding to intestinal immune cells; on the one hand, they overactivate the local mucosal immune response in the intestine, and on the other hand, they promote the infiltration and aggregation of inflammatory leukocytes by regulating intracellular inflammatory signaling pathways, thereby aggravating intestinal inflammatory damage. *Lactobacillus plantarum* can alleviate inflammation by reducing the expression of TNF-α and IL-6. As a key anti-inflammatory mediator, IL-10 plays an important regulatory role in the intervention and treatment of ulcerative colitis. The IL-10 level in the serum of DSS-induced UC mice in the intervention group was significantly higher than that in the model group, indicating that the treatment with *Lactobacillus plantarum* increased the expression of anti-inflammatory cytokines and played a good anti-inflammatory role. The IL-10 level in the high-dose group was close to that in the control group, indicating that the encapsulated *Lactobacillus plantarum* can be effectively released to alleviate the degree of inflammation in UC.

[0071] 5. Verification of the mechanism of action The expression levels of vasoactive intestinal peptide (VIP) and phosphorylated PKA (p-PKA) in mouse colon tissue were detected by Western blot.

[0072] The results are as follows Figure 9 As shown in Figure A, compared with the control group, Epac1 was significantly upregulated in the model group (P<0.01), and the levels of inflammatory factors TNF-α and IL-6 were increased in the model group, indicating that in the DSS-induced colitis model, the inflammatory response induced by immune cell infiltration may dominate, leading to the overall upregulation of Epac1 expression. The Epac1 content in the high-dose encapsulated group (MCL) was lower than that in the model group, inflammatory symptoms were alleviated, and intestinal damage was reduced, indicating that *Lactobacillus plantarum* treatment can alleviate the cAMP / Epac1 pathway imbalance. Figure 9 The BD analysis revealed that, compared to the Control group, the Model group showed significantly downregulated expression of PKA and p-PKA proteins, and a decreased p-PKA / PKA ratio, indicating that the cAMP-PKA signaling pathway was inhibited. After gavage intervention with *Lactobacillus plantarum*, there was no significant difference in PKA protein expression between the MCL and Model groups, but p-PKA protein expression was significantly upregulated in the MCL group. This suggests that *Lactobacillus plantarum* does not simply alleviate inflammation by increasing PKA protein synthesis, but rather specifically enhances PKA phosphorylation activation, restoring the signal transduction capacity of the cAMP pathway, thereby alleviating colitis in mice through the cAMP-PKA signaling pathway.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a probiotic encapsulation compound co-encapsulated with aminated mesoporous silica and natural polysaccharides, characterized in that, Includes the following steps: Using hexadecyltrimethylammonium bromide as a template and tetraethyl orthosilicate as a silicon source, a reaction was carried out under alkaline conditions. After removing the template agent, mesoporous silica was obtained. The mesoporous silica was then modified by amination to obtain amination-modified mesoporous silica with a positively charged surface. Natural polysaccharides were extracted and then modified by carboxymethylation to obtain carboxymethylated natural polysaccharides with negative charges. After activating Lactobacillus plantarum, a bacterial suspension was prepared. The aminated mesoporous silica was added to the bacterial suspension, so that the aminated mesoporous silica was electrostatically adsorbed and coated on the surface of Lactobacillus plantarum, thus obtaining a primary encapsulation product. The carboxymethylated modified natural polysaccharide is activated and then added to the primary encapsulation product, so that the carboxymethylated modified natural polysaccharide is electrostatically adsorbed and coated on the outer surface of the aminated mesoporous silica to obtain the probiotic encapsulation body. The mass ratio of the aminated mesoporous silica to the carboxymethylated modified natural polysaccharide is 1:2-2:

1.

2. The preparation method according to claim 1, characterized in that, The amount of hexadecyltrimethylammonium bromide added is 0.4-0.6 g; the amount of tetraethyl orthosilicate added is 4-6 mL.

3. The preparation method according to claim 1, characterized in that, The alkaline conditions are met at a reaction temperature of 75-85℃ and a reaction time of 1.5-2.5h.

4. The preparation method according to claim 1, characterized in that, The amination modification includes the following steps: dispersing the mesoporous silica in toluene, adding 3-aminopropyltriethoxysilane, refluxing at 75-85℃ for 10-14 h, and obtaining amination-modified mesoporous silica after washing and drying; the ratio of the mesoporous silica to the 3-aminopropyltriethoxysilane is 100 mg: 2-4 mL.

5. The preparation method according to claim 1, characterized in that, The natural polysaccharide is chia seed gum or flaxseed gum; the extraction of the natural polysaccharide includes: adding the natural polysaccharide raw material to distilled water, stirring at 70-90℃ for 1.5-2.5h, and obtaining the natural polysaccharide after centrifugation, alcohol precipitation, and freeze drying.

6. The preparation method according to claim 1, characterized in that, The carboxymethylation modification includes the following steps: dispersing the natural polysaccharide in an isopropanol aqueous solution, adding sodium hydroxide for pre-alkalization and activation, then adding chloroacetic acid for carboxymethylation reaction, adjusting the pH to neutral after the reaction, and obtaining carboxymethylated natural polysaccharide by alcohol precipitation, washing, and freeze drying; the molar ratio of chloroacetic acid to the natural polysaccharide is 1:1-2:

1.

7. The preparation method according to claim 1, characterized in that, The activation process includes the following steps: dissolving the carboxymethylated modified natural polysaccharide in PBS buffer, adding EDC and NHS, and activating by stirring at room temperature for 1-3 hours.

8. The preparation method according to claim 1, characterized in that, The final concentration of the aminated mesoporous silica in the bacterial suspension is 3-5 mg / mL; the primary encapsulation product is cultured at 35-37℃ with shaking for 15-30 min; the mass ratio of the aminated mesoporous silica to the carboxymethylated modified natural polysaccharide is 1:

1.

9. The probiotic encapsulation obtained by the preparation method according to any one of claims 1-8.

10. The use of the probiotic encapsulation of claim 9 in the preparation of a medicament for treating and / or alleviating ulcerative colitis.

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