Preparation of functionalized probiotic bacteria and their use in the treatment of ulcerative colitis

Abstract

The present application relates to the technical field of probiotics, and particularly relates to preparation of functionalized probiotics and application of the functionalized probiotics in treatment of ulcerative colitis, comprising the following steps: A, a buffer is added into a water solution of probiotics, pH is adjusted to be alkaline, then a tannic acid solution and a metal ion solution are added, incubation is carried out, then purification is carried out, and a first reaction bacterial solution is obtained; B, an enzyme is added into the first reaction bacterial solution, incubation is carried out, then purification is carried out, and a second reaction bacterial solution is obtained; C, a medicinal resin and an acid solution are added into the second reaction bacterial solution, reaction is carried out, then purification is carried out, and functionalized probiotics are obtained. The research result can effectively solve the bottleneck problem of probiotics in practical application, and significantly improve the treatment effect of probiotics on UC.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics, in particular to a preparation of functionalized probiotics and its application in the treatment of ulcerative colitis. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is a chronic disease that can cause inflammation of the colon and small intestine, including ulcerative colitis (UC) and Crohn's disease (CD), the etiology and pathogenesis of which are not fully understood, but numerous studies have shown that the occurrence and development of IBD are related to the damage of intestinal mucosal barrier function and intestinal microecological imbalance. Oral probiotics have become a common method for treating IBD due to their convenience and patient compliance. However, probiotic therapy also faces many challenges, in addition to the fragility of probiotics and the uncertainty of colonization, it also faces the threat of the digestive system, such as the strong acidity of gastric juice, the strong digestibility of pepsin and the oxidation of active oxygen in the inflammatory area, which can rapidly lyse cells, reduce the activity and therapeutic concentration of probiotics, and limit the clinical application and therapeutic effect of probiotics. The present application provides a single-cell encapsulation method for treating UC, using probiotic E. coli Nissle 1917 as the encapsulation object, plant polyphenols as the encapsulation material, and adsorbing antioxidant enzymes to further enhance the antioxidant capacity, supplemented by pH-responsive enteric coating, thereby achieving multiple therapeutic functions such as improving intestinal ecology, removing ROS, and targeting intestinal therapy. Patent CN202311158530 proposes a probiotic microcapsule delivery system and its preparation method and application. The patent uses methacrylic acid (ester) copolymer to wrap metal-polyphenol and verifies its potential for industrial production and application in terms of in vitro resistance and freeze-drying loss rate. However, this patent technology does not clarify the structural changes of each layer of wall material during the encapsulation process, and the ability to protect probiotics from damage by gastric acid and intestinal fluid is limited, and animal experiments are not conducted to evaluate the in vivo therapeutic potential of probiotics. Based on the fact that probiotics mainly colonize in the intestinal tract, and the number of viable bacteria must be more than 10 6 CFU to achieve the best effect, this technology may not meet the demand for live bacteria delivery. Therefore, how to actively develop a biologically friendly live bacteria delivery technology is a key and difficult point in the field of effective treatment of intestinal inflammation. SUMMARY

[0003] The purpose of the present application is to address the many challenges faced by probiotic therapy for IBD. The present application provides a single-cell encapsulation method that not only protects probiotics from the threat of gastrointestinal digestive fluids and successfully reaches the intestinal tract, but also eliminates active oxygen in the inflammatory intestinal tract. In addition, the released probiotics can effectively improve the intestinal flora, thereby achieving the effect of treating ulcerative colitis in multiple ways.

[0004] The present invention discloses a packaging technology for constructing enzyme catalytic functional probiotics based on a metal-polyphenol self-assembly framework, wherein MOPS buffer, tannic acid solution and Fe 3+ The aqueous solution was added to the bacterial aqueous solution with a volume ratio of MOPS: probiotics: TA: Fe 3+ The mixture was incubated for 15 minutes, washed twice with water, and then catalase and superoxide dismutase solutions were added. The mixture was adsorbed for 10 minutes and washed twice with water. The mixture was resuspended in an ice-bath calcium phosphate solution, and L100-55 and hydrochloric acid solution were added at a volume ratio of ice-bath calcium phosphate: L100-55: HCl of 90:50:3. The encapsulated probiotics were collected by centrifugation.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for preparing functional probiotics comprises the following steps:

[0007] A. Add a buffer to the probiotic aqueous solution to adjust the pH to alkaline, then add a tannic acid solution and a metal ion solution, incubate, and then purify to obtain a first reaction bacterial solution;

[0008] B. adding an enzyme to the first reaction bacterial solution, incubating, and then purifying to obtain a second reaction bacterial solution;

[0009] C. Adding a medicinal resin and an acidic solution to the second reaction bacterial solution, reacting the solution, and then purifying the solution to obtain functionalized probiotics.

[0010] The key and challenge of this research is how to functionalize probiotics so that they can successfully adsorb enzymes and achieve a synergistic effect with them. Through long-term research, the present invention unexpectedly discovered that by first forming a tannic acid coating, the enzyme can be successfully adsorbed, and then encapsulated with a pharmaceutical resin, a functionalized probiotic with a synergistic effect can be obtained, significantly enhancing the therapeutic effect on enteritis.

[0011] Preferably, in step B, the enzyme includes one or more of catalase and superoxide dismutase.

[0012] Preferably, in step B, the enzyme activity ratio of the catalase to the superoxide dismutase is 21U:10U.

[0013] Preferably, in step A, the incubation operation includes: incubating at 37°C on a shaker at 100-150 rpm / min for 15 minutes; in step B, the incubation operation includes: incubating at 37°C on a shaker at 100-150 rpm / min for 10 minutes; in step C, the reaction conditions include: reacting under continuous shaking for 10 seconds to 3 minutes.

[0014] Specifically, the reaction conditions of step C include: reaction for 10 to 30 seconds under vortex conditions, and reaction for 1 to 3 minutes under manual shaking or agitation conditions.

[0015] Preferably, in step A, the purification operation includes: centrifugation and resuspension with ultrapure water to obtain the first reaction bacterial liquid; in step B, the purification operation includes: washing with water and resuspension with phosphate solution under ice bath conditions to obtain the second reaction bacterial liquid; in step C, the purification operation includes: centrifugation to obtain the functionalized probiotics.

[0016] Preferably, the buffer comprises 3-morpholinepropanesulfonic acid buffer; the probiotic aqueous solution comprises intestinal probiotic E.coli Nissle 1917 solution; the metal ion solution comprises Fe 3+ solution; the medicinal resin includes L100-55 resin; the acidic solution includes hydrochloric acid solution; the phosphate solution includes calcium phosphate solution; the volume ratio of the buffer, the probiotic aqueous solution, the tannic acid solution, and the metal ion solution includes 2:1:0.5:0.5; the volume ratio of the phosphate solution, the medicinal resin, and the hydrochloric acid solution includes 20-200:10-20:1; the strain concentration of the probiotic aqueous solution includes 10 8 -10 9 CFU / mL; the strain concentration of the first reaction solution includes 10 8 -10 9 CFU / mL; the strain concentration of the second reaction solution includes 10 8 -10 9 CFU / mL.

[0017] In Step A, MOPS buffer not only adjusts the pH to alkaline, but is also an essential component of many buffered media, stabilizing protein structure and providing a more stable and safe environment for bacteria during encapsulation. Furthermore, MOPS lacks reactivity with most metal ions, making it suitable for use as a non-coordinating buffer in metal ion solutions.

[0018] Preferably, the concentration of the buffer includes 20 to 50 mM; the concentration of the tannic acid solution includes 1 to 10 mM; the concentration of the metal ion solution includes 1 to 10 mM; the concentration of the medicinal resin includes 0.5 to 5 mg / mL; the concentration of the acidic solution includes 0.1 to 0.5 M; and the concentration of the phosphate solution includes 5 to 20 mM.

[0019] A functionalized probiotic obtained by the above-mentioned preparation method of functionalized probiotics.

[0020] An application of the functionalized probiotics is used to prepare a preparation for targeted delivery of ROS.

[0021] Preferably, it is used for preparing a preparation for treating ulcerative colitis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This project uses the intestinal probiotic E. coli Nissle 1917 as the research object. Based on the metal-polyphenol self-assembly framework, a nanocoating is formed on the surface of a single probiotic through a complex reaction, which improves the survival ability of probiotics in various stress environments during the treatment of diseases and their ability to colonize in the intestine. The strong adsorption capacity of the nanocoating is then used to adsorb catalase (CAT) and superoxide dismutase (SOD) to enhance its antioxidant properties and ability to scavenge ROS in inflammatory sites. The enteric coating is further wrapped on the outer layer through a pH conversion method. L100-55 improves the probiotic's tolerance to the gastric environment and enables targeted release in the intestine. This research finding can effectively address the bottleneck problem in the practical application of probiotics and significantly enhance the therapeutic effect of probiotics on UC. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the particle size change diagram of EcN after encapsulation.

[0025] Figure 2 This is the zeta potential change diagram of EcN after encapsulation.

[0026] Figure 3 Laser confocal images of EcN in different treatment groups.

[0027] Figure 4 Transmission electron microscopy images of EcN in different treatment groups.

[0028] Figure 5 The resistance effect of EcN to H2O2 after adsorbing antioxidant enzymes in different treatment groups

[0029] Figure 6 This is a comparison chart of CAT enzyme activity after EcN@T adsorbed antioxidant enzymes.

[0030] Figure 7 This is a comparison chart of SOD enzyme activity after EcN@T adsorbed antioxidant enzymes.

[0031] Figure 8 This is a diagram showing the resistance of EcN in different treatment groups to simulated gastric fluid.

[0032] Figure 9 This is a diagram showing the resistance of EcN in different treatment groups to simulated intestinal fluid.

[0033] Figure 10 This is a diagram showing the resistance of EcN in different treatment groups to the simulated gastrointestinal inflammatory environment.

[0034] Figure 11 This is a comparison of the resistance of EcN in different treatment groups to simulated gastric juice digestion.

[0035] Figure 12 This is a comparison of the resistance of EcN in different treatment groups to simulated intestinal fluid digestion.

[0036] Figure 13 This is a time flow chart for animal experiments.

[0037] Figure 14 The figure is a line graph of the weight changes of mice in each group.

[0038] Figure 15 This is a line graph showing the changes in the disease activity index (DAI) of each group of mice.

[0039] Figure 16 Figure 3 is the colon length of mice in each group.

[0040] Figure 17 H&E staining images of colon sections of mice in each group.

[0041] Figure 18 Comparison of colon injury pathological scores of mice in each group. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0043] Example 1

[0044] 1) Preparation of encapsulation coating

[0045] 3-Morpholinepropanesulfonic acid (MOPS) buffer (20 mM, pH = 7.4) was added to the bacterial aqueous solution, followed by tannic acid solution (5 mM) and Fe 3+ Solution (5mM), volume ratio MOPS: probiotics: TA: Fe 3+ The ratio of the mixture was 2:1:0.5:0.5. After incubation for 15 minutes, the mixture was centrifuged and the bacteria were resuspended in ultrapure water to remove uncomplexed TA and Fe. 3+Repeat twice. Add 210 U of catalase and 100 U of superoxide dismutase solution, incubate for 10 minutes, and wash twice with water to remove unadsorbed enzymes. Resuspend the bacterial pellet with ice-cold calcium phosphate solution (12.5 mM), add L100-55 (1 mg / mL) and hydrochloric acid solution (0.1 M) at a volume ratio of ice-cold calcium phosphate: L100-55: HCl of 90:50:3, and collect by centrifugation to obtain the encapsulated probiotics.

[0046] 2) Reference Document - Example 3 - Preparation of Enzyme

[0047] Pipette 4.8ml of the bacterial aqueous solution and add 100μL of a 20mg / ml epigallocatechin gallate solution dropwise to the bacterial suspension while vortexing. The suspension is then allowed to stand for 2 minutes. Add 100μL of a 5mg / ml ferric chloride solution dropwise to the suspension while vortexing. Continue vortexing for 3 minutes. Add 5ml of pH 7.6 phosphate buffer, mix thoroughly, and let stand for 5 minutes. Centrifuge at 5000 rpm for 3 minutes, discard the supernatant, and repeat this centrifugation and washing twice with pH 7.6 phosphate buffer. Add 210U of catalase and 100U of superoxide dismutase solution, incubate for 10 minutes, and wash twice with pH 7.6 phosphate buffer to remove any unadsorbed enzymes. Resuspend the washed cells in 4.8 ml of neutral phosphate buffer containing 1.5 mg / ml calcium chloride. Add 200 μL of 3 mg / ml Eudragit S100 solution while vortexing. Mix thoroughly, adjust the pH of the solution to 5.0, and continue vortexing for 5 minutes. Add 5 ml of pH 5.0 phosphate buffer, mix thoroughly, let stand for 5 minutes, and then centrifuge at 5000 rpm for 3 minutes. Remove the supernatant and repeat this centrifugation and washing step twice using pH 5.0 phosphate buffer.

[0048] 3) Particle size determination

[0049] After the EcN, EcN@T, and EcN@TL groups were encapsulated, they were resuspended in ultrapure water and diluted 10-fold for later use. Particle size was measured using a Marvern Zetasizer pro. The name was changed, the dissolution medium was water, the number of repeated tests was set, the thermal equilibrium time was 120 seconds, and the interval time was 30 seconds. The test results are shown in the attached figure. Figure 1 shown.

[0050] 4) Zeta potential detection

[0051] After the EcN, EcN@T, and EcN@TL groups were encapsulated, they were resuspended in ultrapure water and diluted 10-fold for later use. Zeta potential detection was performed using a Marvern Zetasizer pro. The name was changed, the potential sample cell was adjusted to DTS1070, the dissolution medium was water, the number of repeated detections was set, the thermal equilibrium time was 120 seconds, and the interval time was 30 seconds. The test results are shown in the attached figure. Figure 2 shown.

[0052] 5) Laser confocal microscopy

[0053] Wash 1 ml of bacterial suspension twice with water, resuspend in 1 mL of PBS, add 2 μL of CFDA fluorescent dye, vortex for 5 minutes, incubate at 37°C for 15 minutes, and wash twice with water to remove excess fluorescent dye. 3+ The solution was incubated for 15 minutes and then washed twice with water. 50 μL of Cy5.5 fluorescent dye was added, vortexed for 4 minutes, and incubated at 37°C for 16 minutes. After washing, fluorescence was observed using the Zeiss LSM800 fluorescence imaging system. The imaging results are shown in the attached figure. Figure 3 shown.

[0054] 6) Transmission electron microscopy

[0055] After the three groups of EcN, EcN@T, and EcN@TL are encapsulated, they are resuspended in ultrapure water for later use. 80μl of the treated bacterial aqueous solution is drawn into a centrifuge tube, and 27μl of glutaraldehyde solution (30%) (just enough to cover the main copper mesh) is added for fixation (making the final concentration 25%), and the fixation time is 30 seconds. 20μl of the fixed bacteria is added dropwise to a 300-mesh copper mesh carbon support film, wait for 3 minutes, wash off the excess liquid with filter paper, and then stain with 2% sodium phosphotungstate solution for 30 seconds. The excess liquid is absorbed with filter paper, and placed in a culture dish lined with filter paper and dried in an oven for 2 hours. Finally, it is observed under TEM. The imaging results are shown in the attached figure. Figure 4 shown.

[0056] 7) Screening of the best enzyme adsorption method

[0057] In order to screen the optimal adsorption step for antioxidant enzymes, the bacterial suspensions of the nine examples were prepared and resuspended in PBS for later use. An equal amount of 30 mM H2O2 solution diluted with PBS was added and incubated in an incubator for 1 hour. Finally, after washing with PBS twice, the solution was diluted to the corresponding multiple and plated for counting. The number of surviving bacteria was shown in the attached figure. Figure 5 shown.

[0058] 8) CAT and SOD enzyme activity detection

[0059] After encapsulation, the three groups of EcN, EcN@T, and EcN@Te were resuspended in ultrapure water for later use. CAT activity was determined using the ammonium molybdate method, and SOD activity was determined using the WST-8 method. Briefly, EcN@Te was mixed with H₂O₂ or a xanthine oxidase / WST-8 solution, incubated at 37°C, and then ammonium molybdate was added. The absorbance was measured using a UV spectrophotometer. EcN@T and the enzyme solution served as negative and positive controls to calculate the adsorption rate. The test results are shown in the attached figure. Figure 6 、 7 shown.

[0060] 9) Simulated gastric juice digestion experiment

[0061] After the EcN, EcN@T, and EcN@TL groups were encapsulated, they were resuspended in ultrapure water with a pH value of <5 and set aside. An equal amount of simulated gastric fluid (pH = 2, 10 g / L) was added and placed in an incubator for incubation for different periods of time. The excess gastric fluid was then washed off by centrifugation and washed twice with PBS. The cells were diluted to the corresponding multiples and plated for counting. The number of surviving bacteria was shown in the attached figure. Figure 8 shown.

[0062] 10) Simulated intestinal digestion experiment

[0063] After the EcN, EcN@T, and EcN@TL groups were encapsulated, they were resuspended in ultrapure water with a pH value of <5 and set aside. An equal amount of prepared simulated intestinal fluid (pH = 6.8, 10 g / L) was added and placed in an incubator for incubation for different periods of time. The excess intestinal fluid was then washed away by centrifugation and washed twice with PBS. The cells were diluted to the corresponding multiples and plated for counting. The number of surviving bacteria was shown in the attached figure. Figure 9 shown.

[0064] 11) Simulated inflammation digestive system resistance experiment

[0065] After the five groups of EcN, EcN@T, EcN@Te, EcN@TL, and EcN@TeL were encapsulated, they were resuspended in ultrapure water with a pH of <5 and set aside. An equal amount of prepared simulated gastric fluid (pH = 2, 10g / L) was added and placed in an incubator for incubation for 1 hour. After centrifugation, the excess gastric fluid was washed away and the cells were washed twice with PBS for 5 minutes each. Subsequently, an equal amount of 30mM H2O2 solution diluted with simulated intestinal fluid (pH = 6.8, 10g / L) was added and the cells were incubated in an incubator for 1 hour. Finally, after washing twice with PBS, the cells were diluted to the corresponding multiples and plated for counting. The number of surviving bacteria is shown in the attached figure. Figure 10 shown.

[0066] 12) Comparative experiment on resistance to simulated digestive fluid

[0067] The experimental steps were carried out according to the "Gastrointestinal Digestion Test" steps in the patent CN202311158530. EcN, EcN@TL, and the encapsulated probiotics in Example 3 of the patent CN202311158530 were resuspended in pH <5 ultrapure water for use. 9 times the volume of simulated gastric juice (0.32% pepsin, pH 2.5) or simulated intestinal juice (1% trypsin, 0.3% cholate, pH 6.8) was added, and after incubation at 37°C, 150 r / min on a shaking table for 60 min, 1 mL was removed for de-capsulation. Resuspend in PBS, then use plate colony counting method to detect the number of viable bacteria. The number of bacteria surviving is shown in FIGS. 1-3. Figure 11 、 12

[0068] 13) Therapeutic effect of encapsulated probiotics on ulcerative colitis

[0069] To explore the performance of probiotics in in vivo experiments, the present application uses 3% dextran sulfate sodium salt (DSS) to continuously feed mice for 7 days to create an ulcerative colitis model. The control group is given the same volume of distilled water, and after the model is successfully created, 5x10 7 CFUs of probiotics (EcN, EcN@L, EcN@TL, EcN@TeL) and 5-aminosalicylic acid are administered by gavage for 5 days. The body weight of the mice is recorded daily, and on the 12th day, the mice are dissected, the colon tissue is isolated, the length is measured, and then a 0.5-1 cm section of the intestine near the anus is cut and immersed in 4% paraformaldehyde for fixation. The relevant data are shown in FIGS. 4-6. Figure 13-16

[0070] 14) Histopathological analysis

[0071] The fixed colon tissue is subjected to hematoxylin-eosin staining (H&E) to evaluate the degree of damage. Simply put, the isolated colon tissue is fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and HE-stained, and then scanned at the cellular level using a Leica microscope. The relevant data are shown in FIGS. 7-9. Figure 17 、 18

[0072] Example 1

[0073] The results of each experiment in Example 1 are summarized in the following table.

[0074] ​​​Unless otherwise specified, the group codes in the examples are abbreviations, which are described as follows: control: blank control group; DSS: DSS modeling group; 5-ASA: 5-aminosalicylic acid group; EcN: E. coli Nissle 1917 group; EcN@L: EcN encapsulated L100 group; EcN@T: EcN encapsulated TA-Fe 3+ Group; EcN@Te:EcN encapsulated TA-Fe 3+ + antioxidant enzyme group; EcN@TL: EcN encapsulated TA-Fe 3+ +L100 group; EcN@TeL: EcN encapsulated TA-Fe 3+ +antioxidant enzyme+L100 group.

[0075] The present invention uses dynamic light scattering (DLS) to characterize the cell size of probiotics before and after encapsulation. Figure 1 As shown, the original average diameter of bacteria in EcN was 581.6, and the TA coating and L100-55 increased the size of probiotics by about 10-20 nm and 200 nm, respectively, to 564.9 nm and 867.2 nm, proving the successful encapsulation of the coating.

[0076] The present invention uses dynamic light scattering (DLS) to characterize the cell size of probiotics before and after encapsulation. Figure 2 As shown, the zeta potential of EcN is -39.70±0.47mV, and the surface is covered with Fe 3+ After TA coating, the zeta potential decreased to -47.17 ± 0.72 mV, and then increased to -33.37 ± 0.34 mV after L100 coating, indicating the successful encapsulation of TA coating and enteric-coated L100.

[0077] like Figure 3 As shown, EcN added CFDA dye and showed green fluorescence, and TA nanoshell added Cy5.5 dye and showed red fluorescence. From the merge diagram, it can be seen that each bacterial surface is encapsulated with a shell, indicating the successful encapsulation of TA. At the same time, all bacteria have green fluorescence, which further proves that the nanocoating has no effect on the activity of probiotics.

[0078] like Figure 4 As shown, the EcN bacteria are oval and smooth, while the EcN@T surface has a layer of lighter Fe 3+ The TA coating is serrated, with a thickness of approximately 10-20 nm, further demonstrating the successful encapsulation of the TA coating. After encapsulating L100, the EcN@TL cell size increased by approximately 200 nm, and the surface became smooth again, similar to the particle size change results above, indicating the successful encapsulation of L100.

[0079] likeFigure 5 As shown, in order to screen out the best adsorption method of antioxidant enzymes, the present invention prepared 9 groups of example bacteria by adding antioxidant enzymes to different nodes or solutions in the encapsulation step. The "+, -" symbols below each group of example numbers correspond to whether the solution was added in the example, and "e" represents that antioxidant enzymes were added to the solution or node. After preparation, the bacteria were added to 30mM H2O2 and incubated for 60 minutes. The feasibility of the adsorption method was judged by the final bacterial survival rate. In order to remove the interference caused by the protection of L100, the present invention will remove the L100 shell before H2O2 treatment. As can be seen from the figure, all the bacteria of EcN (Example 1), EcN@T (Example 2), and EcN@TL (Example 3) died after being treated with H2O2. In addition, the enzyme was added to Fe 3+ The adsorption effect in TA and MOPS solutions was not ideal. The bacterial survival rate of Examples 4, 5, and 6 was only 10 3 ~10 4 CFU / mL. In Examples 7 and 8, the adsorption method of adding antioxidant enzymes after the tannic acid coating is formed greatly improves the resistance to H2O2. And in Example 8, the survival rate of the bacteria is not affected after the bacteria are encapsulated with L100 and then uncapped, indicating that the antioxidant enzymes have been tightly adsorbed in the tannic acid coating. The adsorption effect of adding antioxidant enzymes after L100 encapsulation is also not ideal. In summary, the present invention explores the best adsorption method of antioxidant enzymes through the comparison of survival rates of different embodiments, that is, adding antioxidant enzymes after the tannic acid coating is formed. This method can greatly improve the antioxidant capacity of armored probiotics and enhance the ability of armored probiotics to scavenge active oxygen in the body.

[0080] like Figure 6 、 Figure 7 As shown, the TA coating itself possesses a certain degree of antioxidant capacity and exhibits CAT and SOD enzyme activity, with the primary antioxidant activity likely derived from CAT. Adsorption of antioxidant enzymes significantly enhanced the enzymatic activity of EcN@Te. Compared to the enzyme solution, the average adsorption efficiency of CAT on EcN@Te was 75.71% for CAT and 76.11% for SOD.

[0081] like Figure 8 As shown, all EcN bacteria died after 60 minutes of simulated digestion, while approximately 90% of EcN@T bacteria died after 120 minutes of simulated digestion. The decreased survival rate in the EcN@T group may be due to the depolymerization of the TA coating caused by the prolonged strong acid environment. In contrast, the EcN@TL group maintained a nearly 100% survival rate, demonstrating that L100 effectively protects probiotics from degradation in simulated gastric fluid.

[0082] In the intestine, the physiological activity and on-demand release of EcN are crucial for its efficacy in treating IBD, such as Figure 9 As shown, the activity of the EcN group did not decrease significantly. When the neutral pH triggers the depolymerization of the L100 coating, the TA coating falls off, and the probiotics are slowly released, then they regain activity and begin to proliferate. The proliferative activity is significantly higher than that of the unencapsulated group. This indicates that EcN@TL can be reactivated on demand in the intestine and respond more quickly to the surrounding environment.

[0083] The present invention uses simulated gastric fluid, simulated intestinal fluid and 30mM H2O2 to simulate a more realistic digestive system of ulcerative colitis. After 1 hour of simulated digestion of probiotics in simulated gastric fluid, the L100 coating is depolymerized and then treated with simulated intestinal fluid and 30mM H2O2 for 30 minutes to simulate the high concentration of active oxygen microenvironment in the intestinal inflammation site. This is used to evaluate the stability of the adsorbed enzyme and its effect on the antioxidant performance of armored EcN. Figure 10 As can be seen, the EcN, EcN@T, and EcN@Te groups without L100 coating all died after being treated with simulated gastric fluid and 30mM H2O2. This may be because the pepsin in the simulated gastric fluid inactivated the adsorbed CAT and SOD enzymes, reducing their resistance to H2O2. However, EcN@TL and EcN@TeL maintained high survival rates, and the colony count of the EcN@TeL group was over 10 times higher than that of the EcN@TL group, indicating that L100 effectively protected the adsorbed enzymes from degradation by pepsin. Furthermore, after L100 depolymerization in the intestine, the CAT and SOD enzymes enhanced the EcN@Te's resistance to H2O2.

[0084] Furthermore, the present inventors discovered that differences in the specific materials used and the sequence of operations significantly impacted the protective effectiveness of the encapsulation coating. For example, armored probiotics prepared using the preparation method of reference document CN202311158530 and the present invention exhibited significant differences in their resistance to gastric and intestinal fluids. Therefore, the present inventors designed comparative digestion experiments using simulated gastric and intestinal fluids to evaluate the advanced nature of the present invention and the superiority of its preparation method.

[0085] like Figure 11 、 Figure 12As shown, the survival of the armoring probiotics prepared by the present application is always better than that of Example 3 + enzyme in patent CN202311158530 in the comparative digestion experiment, including gastric juice digestion experiment and intestinal juice digestion experiment. The survival rate of Example 3 + enzyme in the gastric juice digestion experiment is significantly higher than that of the control group EcN, but slightly weaker than EcN@TeL, but there is no significant difference in the survival in the intestinal juice digestion compared with EcN without encapsulation, which proves the advancement and superiority of the preparation method in the present application. According to the comparison of the experimental process, the reasons for such difference may be: 1. MOPS is added at the beginning of the encapsulation process in the present patent, which can not only buffer the pH, but also is an important component of bacterial buffer medium. Compared with phosphate buffer, MOPS can buffer various changes in bacterial suspension to a greater extent and maintain bacterial activity. 2. The preparation process of probiotic microcapsules in patent CN202311158530 shows that gallic acid catechin is first added to the bacterial solution with a relatively high concentration, and combined with the antibacterial properties of natural polyphenols, the sudden contact of bacteria in a short time will affect the activity; 3. The comparison file adds calcium chloride: Eudragit S100 is 24:1, and the ratio of calcium chloride: Eudragit L100 in the present patent is 9:5, and the ratio of enteric coating may also affect the protection effect of the coating, thus causing the difference in experimental results.

[0086] As Figure 13 shown, in order to further explore the therapeutic effect of probiotics in in vivo experiment, the present application uses 3% dextran sulfate sodium (DSS) to continuously feed mice for 7 days to manufacture an ulcerative colitis model, the control group is given the same volume of distilled water, and after the modeling is successful, different treated probiotics and 5-aminosalicylic acid are given by gavage for 5 days (EcN: 5*10 7 CFU / d, 5-ASA: 250mg / kg).

[0087] One of the symptoms of ulcerative colitis is weight loss, and the change in body weight is crucial for the evaluation of the severity of UC, and can also reflect the therapeutic effect of drugs on UC. As Figure 14 shown, except for the EcN group, each group can alleviate the weight loss caused by ulcerative colitis, but the therapeutic effect is not satisfactory. The EcN@TeL group can significantly improve the weight loss, almost reaching 100% of the initial weight, effectively treating UC.

[0088] Disease activity index (DAI) is a comprehensive score of body weight change rate, fecal consistency and fecal occult blood level, which can reflect the development trend and severity of UC. As Figure 15As shown, the DAI of each group gradually increased over the experimental period, reaching its highest level on day 7. After DSS removal on day 8, the DAI of the DSS group remained constant within a certain range, while the DAI of each treatment group decreased to varying degrees. Compared with the DSS group, the DAI of the EcN@TL, 5-ASA, and EcN@TeL groups decreased significantly, with the EcN@TeL group achieving the lowest DAI, close to that of the control group, demonstrating that EcN@TeL can significantly alleviate UC symptoms.

[0089] Colon length Figure 16 As shown in the data, the colon length of the model group was significantly shortened after DSS treatment, and there was no significant improvement in colon length after EcN and EcN@TL treatment. The 5-ASA and EcN@L groups had better treatment effects, and the colon length was significantly restored. It is worth noting that the EcN@TeL group had the best treatment effect compared with all treatment groups, and the colon length was close to or even exceeded that of the control group, and was significantly increased compared with other groups. In addition, due to the addition of CAT and SOD enzymes, the EcN@TeL group had a better treatment effect than the EcN@TL group, and the colon length was significantly increased.

[0090] like Figure 17 、 Figure 18 As shown, the present invention embedded the mouse colon in paraffin and sectioned it, then used eosin-hematoxylin (H&E) staining to observe microscopic changes. Pathological changes in the colon were scored and statistically analyzed, with more severe lesions receiving higher scores. The DSS group showed pathological changes such as crypt deformation or disappearance, goblet cell loss, ulcer formation, and inflammatory cell infiltration. EcN@TeL significantly reversed these symptoms and lowered the pathological score, with no significant differences compared to the healthy group or the 5-ASA group.

[0091] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing functional probiotics, characterized in that: The following steps are involved: A. Add a buffer to the probiotic aqueous solution to adjust the pH to alkaline, then add a tannic acid solution and a metal ion solution, incubate, and then purify to obtain a first reaction bacterial solution; the buffer is a 3-morpholinepropanesulfonic acid buffer; the probiotic aqueous solution is an intestinal probiotic E. coli Nissle 1917 solution; the metal ion solution is Fe 3+ solution; B. adding an enzyme to the first reaction bacterial solution, incubating, and then purifying to obtain a second reaction bacterial solution; the enzyme is one or more of catalase and superoxide dismutase; C. Adding a medicinal resin and an acidic solution to the second reaction bacterial solution, reacting, and then purifying to obtain functionalized probiotics; the medicinal resin is L100-55 resin.

2. The method for preparing the functionalized probiotics according to claim 1, wherein: In step B, the enzyme activity ratio of the catalase to the superoxide dismutase is 21U:10U.

3. The method for preparing the functionalized probiotics according to claim 1, wherein: In step A, the incubation operation includes: incubation at 37°C on a shaker at 100-150 rpm / min for 15 min; in step B, the incubation operation includes: incubation at 37°C on a shaker at 100-150 rpm / min for 10 min; in step C, the reaction conditions include: reaction under continuous shaking for 10 seconds to 3 minutes.

4. The method for preparing functionalized probiotics according to claim 1, characterized in that: In step A, the purification operation includes: centrifugation and resuspension with ultrapure water to obtain the first reaction bacterial liquid; in step B, the purification operation includes: washing with water and resuspension with phosphate solution under ice bath conditions to obtain the second reaction bacterial liquid; in step C, the purification operation includes: centrifugation to obtain the functionalized probiotics.

5. The method for preparing the functionalized probiotics according to claim 4, wherein: The acidic solution includes a hydrochloric acid solution; the phosphate solution includes a calcium phosphate solution; the volume ratio of the buffer, the probiotic aqueous solution, the tannic acid solution, and the metal ion solution is 2:1:0.5:0.5; the volume ratio of the phosphate solution, the medicinal resin, and the hydrochloric acid solution is 20-200:10-20:1; the strain concentration of the probiotic aqueous solution is 10 8 -10 9 CFU / mL; the strain concentration of the first reaction solution was 10 8 -10 9 CFU / mL; the strain concentration of the second reaction solution was 10 8 -10 9 CFU / mL.

6. The method for preparing the functionalized probiotics according to claim 4, wherein: The concentration of the buffer is 20-50 mM; the concentration of the tannic acid solution is 1-10 mM; the concentration of the metal ion solution is 1-10 mM; the concentration of the medicinal resin is 0.5-5 mg / mL; the concentration of the acidic solution is 0.1-0.5 M; and the concentration of the phosphate solution is 5-20 mM.

7. A functionalized probiotic obtained according to the preparation method of the functionalized probiotic according to claim 1.

8. A use of the functionalized probiotics according to claim 7, characterized in that: Used for preparing preparations for treating ulcerative colitis.

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