Preparation of functional probiotics and application of functional probiotics in treatment of ulcerative colitis

Abstract

The invention relates to the technical field of probiotics, in particular to preparation of functionalized probiotics and application of the functionalized probiotics in treatment of ulcerative colitis, and the preparation method comprises the following steps: A, adding a buffer agent into a probiotic aqueous solution, adjusting the pH value to be alkaline, then adding a tannic acid solution and a metal ion solution, incubating, and then purifying to obtain a first reaction bacterium solution; b, adding enzyme into the first reaction bacterium solution, incubating, and purifying to obtain a second reaction bacterium solution; and C, adding medicinal resin and an acid solution into the second reaction bacterium solution, reacting, and purifying to obtain the functional probiotics. The research result can effectively solve the bottleneck problem of the probiotics in practical application, and the treatment effect of the probiotics on UC is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to the preparation of functionalized probiotics and application thereof in the treatment of ulcerative colitis. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic disease that causes inflammation of the colon and small intestine, including ulcerative colitis (UC) and Crohn's disease (CD). Its etiology and pathogenesis have not yet been fully clarified, but many studies have shown that the occurrence and development of IBD are related to impaired 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 themselves and the uncertainty of colonization, it also faces threats to the digestive system. For example, the strong acidity of gastric juice, the strong digestibility of pepsin, and the oxidative properties of reactive oxygen in the inflammatory area will lyse cells at a very fast rate, reducing the activity and therapeutic concentration of probiotics, limiting the clinical application and therapeutic effect of probiotics. The present invention provides a single-cell encapsulation method for the multi-pronged treatment of UC, with the probiotic E. coli Nissle 1917 as the encapsulation object, plant polyphenols as the encapsulation material, adsorbed antioxidant enzymes to further enhance the antioxidant capacity, and supplemented with a pH-responsive enteric coating, thereby simultaneously achieving multiple therapeutic functions such as improving intestinal ecology, removing ROS, and targeted intestinal treatment. Patent CN202311158530 proposes a probiotic microcapsule delivery system, a preparation method, and an application thereof. The patent uses methacrylate (ester) copolymer to encapsulate metal-polyphenols and verifies its potential for industrial production and application in terms of in vitro resistance and freeze-drying loss rate. However, the patented technology does not explain the structural changes of each layer of wall material during the double-layer wall material encapsulation process, and the ability to protect probiotics from gastric acid and intestinal fluid damage is limited. No animal experiments have been conducted to evaluate the in vivo therapeutic potential of probiotics, based on the fact that probiotics are mainly colonized in the intestinal tract and the number of live bacteria is 10 6 The best effect can only be achieved when the CFU is above 100. This technology may not meet the needs of live bacteria delivery. Therefore, how to actively develop bio-friendly live bacteria delivery technology is the key and difficulty in the field of effective treatment of enteritis. Summary of the invention

[0003] The purpose of the present invention is to address the many challenges currently faced by probiotics in the treatment of IBD. The present invention provides a single-cell encapsulation method that can not only protect probiotics from the threat of gastrointestinal digestive juices and smoothly reach the intestines, but also eliminate active oxygen in the inflamed intestines. In addition, the released probiotics can also effectively improve the intestinal flora, thereby achieving a multi-pronged treatment effect for ulcerative colitis.

[0004] The invention discloses a packaging technology for constructing enzyme catalytic functional probiotics based on a metal-polyphenol self-assembly framework, that is, 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 min and then washed twice with water. Catalase and superoxide dismutase solution were added, adsorbed for 10 min 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. The volume ratio of ice-bath calcium phosphate: L100-55: HCl was 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. adding a buffer to the probiotic aqueous solution to adjust the pH to alkaline, then adding a tannic acid solution and a metal ion solution, incubating, and then purifying 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, and then purifying to obtain functionalized probiotics.

[0010] How to functionalize probiotics so that they can successfully adsorb enzymes and produce a synergistic enhancement effect with the enzymes is the focus and difficulty of the research. Through long-term research, the present invention unexpectedly found that after the tannic acid coating is first formed, the enzyme can be successfully adsorbed, and finally encapsulated with a medicinal resin, a functionalized probiotic that produces a synergistic enhancement effect with the enzyme can be obtained, which significantly enhances 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, among the enzymes, 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 min; in step B, the incubation operation includes: incubating at 37°C on a shaker at 100-150 rpm / min for 10 min; 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 oscillation or shaking conditions.

[0015] Preferably, in step A, the purification operation includes: centrifugation, resuspending with ultrapure water to obtain the first reaction bacterial solution; in step B, the purification operation includes: washing with water, resuspending with a phosphate solution under ice bath conditions to obtain the second reaction bacterial solution; 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, the use of MOPS buffer can not only adjust the pH to alkaline, but MOPS is also one of the necessary components of many buffered culture media, which can stabilize the protein structure and provide a more stable and safe environment for bacteria during the encapsulation process. In addition, MOPS lacks the ability to react with most metal ions and is suitable for non-coordinating buffers 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 preparation method of the functionalized probiotic.

[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 nano-coating is formed on the surface of a single probiotic through a complex reaction to improve the survival ability of probiotics in various stress environments during the treatment of diseases and their colonization ability in the intestine. Then, the strong adsorption capacity of the nano-coating is used to adsorb catalase (CAT) and superoxide dismutase (SOD) to enhance its antioxidant activity and the ability to remove ROS in inflammatory sites. The enteric coating is further wrapped on its outer layer through the pH conversion method. L100-55 improves the tolerance of probiotics to the gastric environment and enables them to be released at specific points in the intestine. This research result can effectively solve the bottleneck problem in the practical application of probiotics and significantly improve 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] Fig. 9 This is a diagram showing the resistance of EcN in different treatment groups to simulated intestinal fluid.

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

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

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

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

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

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

[0039] Fig.16 Figure 2 is the colon length of mice in each group.

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

[0041] Fig.18 This is a comparison chart 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 further described in 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 TA to Fe was 2:1:0.5:0.5. After incubation for 15 minutes, centrifugation was performed and the bacterial solution was resuspended in ultrapure water to remove uncomplexed TA and Fe. 3+, repeated twice. Add 210U catalase and 100U superoxide dismutase solution, incubate for 10min and wash twice to remove unadsorbed enzymes. Resuspend the bacterial precipitate with ice-bath calcium phosphate solution (12.5mM), add L100-55 (1mg / mL) and hydrochloric acid solution (0.1M), with a volume ratio of ice-bath calcium phosphate: L100-55: HCl of 90:50:3, and collect by centrifugation to obtain encapsulated probiotics.

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

[0047] 4.8 ml of bacterial aqueous solution was taken, and 100 μL of epigallocatechin-3-oxide solution with a concentration of 20 mg / ml was added dropwise to the bacterial suspension under vortexing conditions, and then allowed to stand for 2 min. Under vortexing conditions, 100 μL of ferric chloride solution with a concentration of 5 mg / ml was added dropwise to the bacterial suspension after standing, and after the addition was completed, vortexing was continued for 3 min. 5 ml of phosphate buffer with a pH of 7.6 was added, and after being fully mixed, it was allowed to stand for 5 min, and then centrifuged at 5000 rpm for 3 min, the supernatant was removed, and this step was repeated with phosphate buffer with a pH of 7.6 and centrifuged and washed twice. 210 U of catalase and 100 U of superoxide dismutase solution were added, incubated for 10 min, and centrifuged and washed twice with phosphate buffer with a pH of 7.6 to remove the unadsorbed enzyme. 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 under vortexing, mix thoroughly, adjust the pH of the solution to 5.0, and continue vortexing for 5 min. Add 5 ml of pH 5.0 phosphate buffer, mix thoroughly, let stand for 5 min, then centrifuge at 5000 rpm for 3 min, remove the supernatant, and repeat this step with pH 5.0 phosphate buffer for 2 more centrifugation washes.

[0048] 3) Particle size determination

[0049] After the three groups of EcN, EcN@T, and EcN@TL were encapsulated, they were resuspended in ultrapure water and diluted 10 times for later use. The particle size was detected using MarvernZetasizer pro. The name was changed, the dissolving medium was water, the number of repeated tests was set, the thermal equilibrium time was 120s, and the interval time was 30s. The test results are shown in the attached Figure 1 shown.

[0050] 4) Zeta potential detection

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

[0052] 5) Laser confocal

[0053] Wash 1 ml of bacterial suspension twice, resuspend in 1 mL PBS, add 2 μL CFDA fluorescent dye, vortex for 5 min, incubate at 37 °C for 15 min, and wash twice to remove excess fluorescent dye. Add MOPS, tannic acid and Fe 3+ The solution was incubated for 15 min and then washed twice with water. 50 μL of Cy5.5 fluorescent dye was added, vortexed for 4 min and incubated at 37°C for 16 min. 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. Pipette 80μl of the treated bacterial aqueous solution into a centrifuge tube, add 27μl of glutaraldehyde solution (30%) (just enough to cover the main copper mesh) for fixation (to make the final concentration 25%), and the fixation time is 30 seconds. Add 20μl of the fixed bacteria onto a 300-mesh copper mesh carbon support membrane, wait for 3 minutes, wash off the excess liquid with filter paper, and then stain with 2% sodium phosphotungstate solution for 30 seconds. Use filter paper to absorb the excess liquid, place it in a culture dish lined with filter paper, and dry it in an oven for 2 hours. Finally, observe it 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 out the best 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 placed in an incubator for incubation for 1 hour. Finally, after washing with PBS twice, the corresponding dilution was plated and counted. The number of surviving bacteria was shown in the attached figure. Figure 5 shown.

[0058] 8) CAT and SOD enzyme activity detection

[0059] After the three groups of EcN, EcN@T, and EcN@Te were encapsulated, they were resuspended in ultrapure water for later use. CAT activity was detected by the ammonium molybdate method, and SOD activity was detected by the WST-8 method. Briefly, EcN@Te was mixed with H2O2 or xanthine oxidase / WST-8 solution, and ammonium molybdate was added after incubation at 37°C. The absorbance was detected using a UV spectrophotometer. EcN@T and enzyme solution were used 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 for later use. An equal amount of prepared simulated gastric juice (pH = 2, 10 g / L) was added and placed in an incubator for incubation for different periods of time. The excess gastric juice was then washed off by centrifugation. The cells were washed twice with PBS and the plates were diluted to the corresponding multiples 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 less than 5 for later use. 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 off by centrifugation. The cells were washed twice with PBS and the plates were diluted to the corresponding multiples for counting. The number of surviving bacteria was shown in the attached figure. Fig. 9 shown.

[0064] 11) Digestive system resistance experiment simulating inflammation

[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 value of less than 5 for later use. An equal amount of prepared simulated gastric fluid (pH = 2, 10 g / L) was added and placed in an incubator for incubation for 1 hour. After centrifugation, the excess gastric fluid was washed away and washed with PBS twice, each time for 5 minutes. Subsequently, an equal amount of 30 mM H2O2 solution diluted with simulated intestinal fluid (pH = 6.8, 10 g / L) was added and placed in an incubator for incubation for 1 hour. Finally, after washing twice with PBS, the corresponding multiples were diluted and plated for counting. The number of surviving bacteria is shown in the attached figure. Fig.10 shown.

[0066] 12) Simulated digestive fluid resistance comparison experiment

[0067] The experimental steps were carried out according to the "Gastrointestinal Digestion Test" steps in patent CN202311158530. The three groups of encapsulated probiotics, EcN, EcN@TL, and Example 3 in patent CN202311158530, were resuspended in ultrapure water with a pH value of <5 for later use. 9 times the volume of simulated gastric fluid (0.32% pepsin, pH 2.5) or simulated intestinal fluid (1% trypsin, 0.3% bile salt, pH 6.8) was added, and incubated in a shaker at 37°C and 150 r / min for 60 min. After that, 1 mL was removed from the mixture for decapsulation, resuspended in PBS, and then the number of viable bacteria was detected by the plate colony count method. The number of surviving bacteria was as shown in the attached figure. Fig.11 , 12 shown.

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

[0069] In order to explore the performance of probiotics in the in vivo experiment, the present invention used 3% dextran sulfate sodium salt (DSS) to continuously feed mice for 7 days to create an ulcerative colitis model. The control group was given the same volume of distilled water, and after the model was successfully established, 5×10 7 CFUs of probiotics (EcN, EcN@L, EcN@TL, EcN@TeL) and 5-aminosalicylic acid for 5 days. The weight of mice was recorded every day, and the mice were dissected on the 12th day. The colon tissue of the mice was separated, and the length was measured and the intestinal segment of 0.5-1 cm near the anus was cut and immersed in 4% paraformaldehyde for fixation. The relevant data are shown in the attached Figure 13-16 shown.

[0070] 14) Histopathological analysis

[0071] The extent of damage was assessed by hematoxylin-eosin (H&E) staining of fixed colon tissue. Briefly, the isolated colon tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with HE. The cellular structure was scanned using a Leica microscope. The relevant data are shown in the attached Fig.17 , 18 shown.

[0072] Effect Example 1

[0073] The experimental results of Example 1 are summarized and the results are shown in the figure.

[0074] Among them, unless otherwise specified, the group codes in the examples are abbreviations, which are specifically 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 10-20nm and 200nm, respectively, to 564.9nm and 867.2nm, 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 coated 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 coating with L100, indicating the successful encapsulation of TA coating and enteric-coated L100.

[0077] like Figure 3 As shown, EcN added CFDA dye to show green fluorescence, and TA nanoshell added Cy5.5 dye to show 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 in Figure 2, the EcN bacteria are oval and smooth, while the EcN@T surface has a layer of lighter Fe 3+ -TA sawtooth coating, with a thickness of about 10-20nm, further proves the successful encapsulation of TA coating. After encapsulating L100, the cell size of EcN@TL increased by about 200nm, and the surface became smooth again, which is similar to the particle size change results above, indicating the successful encapsulation of L100.

[0079] like Figure 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 is added in the example, and "e" represents the addition of antioxidant enzymes to the solution or node. After preparation, the bacteria are added to 30mM H2O2 and incubated for 60 minutes. The feasibility of the adsorption method is 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 in L100 and then unshelled, indicating that the antioxidant enzymes have been tightly adsorbed in the tannic acid coating. And the adsorption effect of adding antioxidant enzymes after L100 encapsulation is 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 remove reactive oxygen in the body.

[0080] like Figure 6 , Figure 7 As shown in the figure, the TA coating itself has a certain antioxidant capacity and exhibits CAT and SOD enzyme activities. The main antioxidant activity may come from CAT enzyme activity. After adsorbing antioxidant enzymes, the enzyme activity of EcN@Te is significantly improved. Compared with the enzyme solution, the average adsorption efficiency of CAT on EcN@Te is 75.71% and that of SOD is 76.11%.

[0081] like Figure 8 As shown in the figure, all EcN bacteria died after 60 minutes of simulated digestion, and about 90% of EcN@T bacteria died after 120 minutes of simulated digestion. The reason why the survival rate of the EcN@T group decreased may be that the long-term strong acid environment triggered the depolymerization of the TA coating. The EcN@TL group can still maintain almost 100% survival rate. This shows that L100 can effectively protect probiotics from degradation by simulated gastric fluid.

[0082] In the intestine, the physiological activity and on-demand release of EcN are crucial to its efficacy in treating IBD, such as Fig. 9 As shown, the activity of the EcN group did not decrease significantly, and the neutral pH triggered the depolymerization of the L100 coating, the TA coating fell off, and the probiotics were slowly released and recovered and began to proliferate, and the proliferation activity was significantly higher than that of the unencapsulated group. This shows that EcN@TL can be reactivated on demand in the intestine and respond to the surrounding environment more quickly.

[0083] The present invention uses simulated gastric juice, simulated intestinal juice and 30mM H2O2 to simulate a more realistic digestive system of ulcerative colitis. After 1 hour of simulated digestion of probiotics by simulated gastric juice, the L100 coating is depolymerized, and then treated with simulated intestinal juice and 30mM H2O2 for 30 minutes to simulate the high-concentration active oxygen microenvironment of the intestinal inflammation site, so as to evaluate the stability of the adsorbed enzyme and its effect on the antioxidant performance of armored EcN. Fig.10 It can be seen that the EcN, EcN@T and EcN@Te groups without L100 coating all died after being treated with simulated gastric juice and 30mM H2O2. The reason may be that the pepsin in the simulated gastric juice inactivated the adsorbed CAT enzyme and SOD enzyme, reducing their resistance to H2O2. However, EcN@TL and EcN@TeL can still maintain a high survival rate, and the colony count of the EcN@TeL group is more than 10 times higher than that of the EcN@TL group, indicating that L100 can effectively protect the adsorbed enzyme from degradation by pepsin. At the same time, after L100 is depolymerized in the intestine, the CAT enzyme and SOD enzyme improve the resistance of EcN@Te to H2O2.

[0084] In addition, the present invention found that the specific materials and operation sequence will have a significant impact on the protective effect of the encapsulation coating. For example, the armored probiotics prepared according to the preparation method of the reference document-CN202311158530 and the preparation method of the present invention have obvious differences in resistance to gastric juice and intestinal juice. Therefore, the present invention designed a comparative experiment of simulated gastric juice and intestinal juice digestion to evaluate the advancement of the present invention and the superiority of the preparation method.

[0085] like Fig.11 , Fig.12As shown, the survival of the armored probiotics prepared by the present invention is always better than that of Example 3+enzyme in patent CN202311158530 in comparative digestion experiments, including gastric juice digestion experiments and intestinal juice digestion experiments. The survival rate of Example 3+enzyme in the gastric juice digestion experiment was significantly higher than that of the control group EcN, but slightly weaker than EcN@TeL, but the survival in intestinal juice digestion was not significantly different from that of the unencapsulated EcN, which proved the advancement and superiority of the preparation method in the present invention. According to the comparison of the experimental process, the reasons for such a difference may be: 1. MOPS is added at the beginning of the encapsulation process of this patent. In addition to buffering pH, it is also an important component of bacterial buffer culture 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 at a high concentration. Combined with the antibacterial properties of natural polyphenols, sudden contact of bacteria in a short period of time will affect the activity; 3. The ratio of calcium chloride:Eudragit S100 added in the comparative document is 24:1, while in this patent, the ratio of calcium chloride:Eudragit L100 is 9:5. The ratio of enteric coating may also affect the protective effect of the coating, thus causing the difference in experimental results.

[0086] like Fig.13 As shown in the figure, in order to further explore the therapeutic effect of probiotics in in vivo experiments, the present invention used 3% dextran sulfate sodium salt (DSS) to continuously feed mice for 7 days to create an ulcerative colitis model. The control group was given the same volume of distilled water, and after the model was successfully established, the mice were gavaged with probiotics and 5-aminosalicylic acid with different treatments for 5 days (EcN: 5*10 7 CFU / d, 5-ASA: 250mg / kg).

[0087] One of the symptoms of ulcerative colitis is weight loss. Changes in weight are crucial to the severity of UC and can also reflect the effectiveness of drugs in treating UC. Fig.14 As shown in the figure, except for the EcN group, all groups can alleviate the weight loss caused by ulcerative colitis, but the treatment effect is unsatisfactory. However, the EcN@TeL group can significantly improve the weight loss, almost reaching 100% of the initial weight, and effectively treat UC.

[0088] The disease activity index (DAI) is a comprehensive score of weight change rate, stool viscosity, and fecal occult blood level, which can reflect the development trend and severity of UC. Fig.15As shown in the figure, as the experimental time prolonged, the DAI of each group gradually increased and reached the highest on the seventh day. After DSS was removed on the eighth day, the DAI of the DSS group remained unchanged 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, and the DAI of the EcN@TeL group was the lowest, close to that of the control group, proving that EcN@TeL can significantly alleviate UC symptoms.

[0089] Colon length Fig.16 As shown in the figure, 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 group and EcN@L group 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 it increased significantly compared with other groups, and 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 increased significantly.

[0090] like Fig.17 , Fig.18 As shown, the present invention embeds the mouse colon in paraffin and slices it, and then uses eosin-hematoxylin (H&E) staining to observe the changes at the microscopic level, and scores and counts the pathological changes of the colon. The more severe the lesion, the higher the score. The DSS group showed pathological changes such as crypt deformation or disappearance, goblet cell loss, ulcer production, and inflammatory cell infiltration in the colon, while EcN@TeL can significantly reverse the above symptoms and lower the pathological score, which is not significantly different from the healthy group and the 5-ASA group.

[0091] The above disclosure is only the preferred embodiment of the present invention, which 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 functionalized probiotics, characterized in that: The following steps are involved: A. adding a buffer to the probiotic aqueous solution to adjust the pH to alkaline, then adding a tannic acid solution and a metal ion solution, incubating, and then purifying to obtain a first reaction bacterial solution; B. adding an enzyme to the first reaction bacterial solution, incubating, and then purifying to obtain a second reaction bacterial solution; C. Adding a medicinal resin and an acidic solution to the second reaction bacterial solution, reacting, and then purifying to obtain functionalized probiotics.

2. The method for preparing the functionalized probiotics according to claim 1, characterized in that: In step B, the enzyme includes one or more of catalase and superoxide dismutase.

3. The method for preparing the functionalized probiotics according to claim 2, characterized in that: In step B, among the enzymes, the enzyme activity ratio of the catalase to the superoxide dismutase is 21U:10U.

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

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

6. The method for preparing functionalized probiotics according to claim 5, characterized in that: 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 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 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.

7. The method for preparing functionalized probiotics according to claim 5, characterized in that: 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.

8. A functionalized probiotic obtained according to the method for preparing the functionalized probiotic according to claim 1.

9. An application of the functionalized probiotics according to claim 8, characterized in that: Formulations for the preparation of targeted delivery of ROS.

10. The use of the functionalized probiotics according to claim 9, characterized in that: Used for preparing preparations for treating ulcerative colitis.

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