Polysaccharide-encapsulated double-coating probiotics as well as preparation method and application thereof

Abstract

The invention discloses polysaccharide-encapsulated double-coating probiotics as well as a preparation method and application thereof, and relates to the technical field of biological preparations. Alpha, beta-unsaturated ester polysaccharide and thiolated polysaccharide are modified to the surfaces of probiotics to form nano-coatings, and the nano-coatings protect the probiotics and enhance the resistance of the probiotics to adverse environments, do not affect the activity and function of the probiotics, and can endow the probiotics with an exogenous function. The whole body has the advantages of gastrointestinal fluid resistance, heat resistance, ultraviolet resistance, superoxide radical removal, inflammation targeting and the like.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a polysaccharide-encapsulated double-coated probiotic, its preparation method, and its application. Background Technology

[0002] Probiotics are non-pathogenic live microorganisms that provide significant health benefits to the host and effectively regulate the composition and quantity of the human gut microbiota. Probiotic preparations provide beneficial functions to the host by altering the gut microbiota, producing metabolic entities, neutralizing dietary carcinogens, inducing cytokine synthesis, and controlling pathogens. The minimum concentration of probiotics required to reach the human gut and exert their effects is 10... 6 The CFU / g concentration of probiotics in the colon needs to reach at least 10. 8 To exert a clinical therapeutic effect, probiotics need to reach a concentration of CFU / g. However, due to their inherent instability and sensitivity, factors such as temperature, pH, water activity, osmotic pressure, light, oxygen, and mechanical force during food processing and storage, as well as the acidic environment, bile salts, digestive enzymes, and mechanical shear forces during digestion, all pose challenges to the effective delivery of probiotics. Furthermore, the adult microbiota possesses a "colonization barrier" protection mechanism, requiring exogenous probiotics to compete with existing gut bacteria, adhere to the intestinal mucus layer, and then colonize and proliferate to exert their physiological activity. Simultaneously, the survival and colonization of probiotics in the gut are also influenced by the pathological microenvironment of intestinal diseases (such as IBD).

[0003] Probiotic encapsulation strategies are a promising approach to protect probiotics for successful oral delivery to target sites. The most common method for protecting probiotics is multi-cell encapsulation technology, a coarse encapsulation method that considers billions of individual probiotics as a whole. This involves both forming a protective shell structure to allow the probiotics to embed, and embedding the probiotics within its network structure. Common encapsulation materials include emulsions, liposomes, nanoparticles, microcapsules, microspheres, hydrogels, and microgels. Common encapsulation materials are based on food-grade polymers, primarily derived from polysaccharides, proteins, and lipids. The combination of biocompatibility, biodegradability, low cost, and availability of polysaccharides makes them one of the most commonly used materials for probiotic encapsulation. However, in traditional bulk packaging technology, polysaccharides form gels through ionic cross-linking or combine with other polysaccharides and proteins through electrostatic and hydrophobic interactions to form a packaging matrix. However, such a matrix has high porosity and instability, lacks control over particle size, is prone to cell leakage, has limited in vivo colonization, and low bioavailability. Such microcapsule structures can only provide temporary protection for probiotics and lack precise and effective targeting. Summary of the Invention

[0004] In view of this, the purpose of this invention is to propose a method for single-cell encapsulation of probiotics by forming a polysaccharide network coating through Michael addition reaction-mediated covalent coupling between polysaccharides and the surface of probiotics. These nano-coatings not only protect probiotics and enhance their resistance to adverse environments, but also do not affect their activity and function, and can even endow probiotics with exogenous functions. First, polysaccharides are grafted and modified using methacrylic anhydride, etc., to obtain Michael addition acceptor polysaccharides containing α,β-unsaturated esters. Then, utilizing the naturally occurring reactive groups on the surface of probiotics, such as thiol, hydroxyl, carboxyl, and amino groups containing active hydrogen, a Michael addition reaction occurs with the α,β-unsaturated esters on the modified polysaccharide under physiological conditions, forming a robust protective layer on the surface of the probiotics, resulting in probiotics encapsulated with a polysaccharide coating. Finally, a low molecular weight thiolated polysaccharide is added to rapidly strengthen the coating. Under ultraviolet light irradiation, the -SH of the thiolated polysaccharide will react with the remaining unsaturated bonds on the Michael receptor polysaccharide to form a thiol-ene reaction, thereby achieving dense polysaccharide coating of probiotics and providing individualized protection for each probiotic cell.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A method for preparing polysaccharide-encapsulated double-coated probiotics includes the following steps:

[0007] (1) Polysaccharide A was prepared into a modified polysaccharide containing α,β-unsaturated esters; polysaccharide B was prepared into a thiolized polysaccharide;

[0008] (2) Add the activated probiotic suspension to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution, vortex, and obtain a dispersed probiotic suspension;

[0009] (3) Mix the probiotic suspension dispersed by vortex in step (2) with the modified polysaccharide obtained in step (1), vortex, and incubate by shaking; after incubation, wash and resuspend to obtain a probiotic suspension mixed with modified polysaccharide.

[0010] (4) The probiotic suspension containing modified polysaccharides obtained in step (3) is mixed with the thiolized polysaccharides obtained in step (1), vortexed thoroughly, and reacted under ultraviolet light. After the reaction is completed, the mixture is washed and resuspended.

[0011] Further, in step (1), the preparation of the modified polysaccharide containing α,β-unsaturated esters includes: dissolving polysaccharide A in dimethyl sulfoxide, adding triethylamine and methacrylic anhydride, stirring overnight, dialysis, vacuum concentration, and drying to obtain the polysaccharide containing α,β-unsaturated esters.

[0012] Furthermore, in step (1),

[0013] The polysaccharide A includes at least one of dextran, hyaluronic acid, tamarind polysaccharide, and amylopectin, and the molecular weight of the polysaccharide A is 5000-40000 Da, preferably 10000 Da;

[0014] The molar ratio of polysaccharide A to methacrylic anhydride is 1:0.2-1;

[0015] The molar ratio of triethylamine to methacrylic anhydride is 1:1 to 10;

[0016] The amount of polysaccharide and dimethyl sulfoxide added is 1g:5-15mL, preferably 1g:10mL;

[0017] When stirring overnight, the temperature is 20-25°C and the time is 10-15 hours, preferably 25°C and 12 hours.

[0018] During dialysis, deionized water is used, and the dialysis time is 48–96 hours, preferably 72 hours.

[0019] In this invention, dextran is preferentially selected as the encapsulation material for probiotics in polysaccharide A. Dextran is a water-soluble polysaccharide, abundant in sources, non-toxic, biocompatible, and biodegradable. It is a large molecular polymer composed of glucose molecules linked by 1,6-α-D-glucopyranose bonds. Bacteria in the colon synthesize glucanase, which hydrolyzes the α-D-(1→6)-glucosidic bonds in dextran, releasing probiotics in the colon and simultaneously producing oligosaccharides, which are beneficial to the intestinal microbiota and human health.

[0020] Furthermore, in step (1), the vacuum concentration temperature is 60°C and the rotation speed is 50 rpm / min.

[0021] Furthermore, in step (1), the drying is spray drying or freeze drying, preferably freeze drying.

[0022] Of course, the preparation of polysaccharides containing α,β-unsaturated esters can also refer to other existing technologies, and will not be limited in too much here.

[0023] Further, in step (1), the preparation of the thiolated polysaccharide includes: dissolving polysaccharide B in distilled water, adding mercaptoacetic acid and HCl solution, reacting at 75-85°C for 150-200 min; after the reaction is completed, pouring into methanol to precipitate the thiolated polysaccharide, washing with methanol, air-drying naturally, and then freeze-drying to obtain the thiolated polysaccharide.

[0024] Furthermore, the polysaccharide B includes at least one of inulin, dextran, hyaluronic acid, and tamarind polysaccharide, and the molecular weight of the polysaccharide B is 3000-20000 Da, preferably 4500 Da;

[0025] The ratio of polysaccharide B: mercaptoacetic acid: hydrochloric acid is 2-4 g: 1-3 mL: 0.5-1.5 mL, and the concentration of the hydrochloric acid solution is 6-8 M.

[0026] In this invention, polysaccharide B preferentially uses inulin as the encapsulation material for probiotics. Inulin has a linear chain containing 2 to 60 fructose units linked by β-(2,1)-fructosyl-fructo bonds. As a prebiotic, it improves host health by selectively stimulating the growth and activity of certain microorganisms in the colon. It is generally considered safe by the FDA and is widely used in the food and pharmaceutical industries.

[0027] Specifically, the preparation of thiolated polysaccharides includes: dissolving 3g of inulin in 20mL of distilled water, adding 2mL of mercaptoacetic acid and 1mL of 7M HCl to the above solution, and reacting at 80℃ for 180min. The reaction mixture is then poured into 200mL of methanol to precipitate the thiolated polysaccharides. The resulting milky white precipitate is washed three times with methanol, and after being air-dried in a fume hood for 12h, it is freeze-dried again for later use.

[0028] Similarly, the preparation of thiolated polysaccharides can also refer to other existing technologies, and will not be limited in too much here.

[0029] Furthermore, in step (2),

[0030] The pH of the HEPES buffer at 37°C is 8.0–8.5, preferably 8.2;

[0031] The probiotics include at least one of Enterobacter, Streptococcus thermophilus, Lactobacillus, Bifidobacterium, Actinomycetes, and yeast;

[0032] Without HEPES buffer, the concentration of probiotics in the probiotic suspension is 1×10⁻⁶. 6 ~1×10 10 CFU / mL, preferably 1×10⁻⁶ 9 CFU / mL;

[0033] After mixing with HEPES buffer, the concentration of probiotics in HEPES buffer is 2 × 10⁻⁶. 4 ~2×10 8 CFU / mL, preferably 2×10⁻⁶ 7 CFU / mL.

[0034] The preparation of HEPES buffer solution is as follows: Weigh 1.1915 g of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid into a beaker, add 450 mL of deionized water to dissolve it, adjust the pH to 8.2 with NaOH, and then make up the volume to 500 mL to prepare a 10 mmol / L HEPES solution. Then autoclave the solution and store it at room temperature.

[0035] The activated probiotic suspension was prepared as follows: 0.4 mL of probiotics was placed in 40 mL of LB medium and cultured overnight (12 h) on a shaker at 37 °C and 200 rpm / min. The bacterial suspension was centrifuged at 4000 rpm / min for 10 min, washed twice with physiological saline, and resuspended in 1 mL of physiological saline.

[0036] In this invention, Escherichia coli Nissle1917 is the preferred probiotic. Escherichia coli Nissle1917 (EcN) provides health benefits by improving gut microbiota balance and inhibiting the expression of pro-inflammatory cytokines and chemokines, and has been used as a typical probiotic to inhibit pathogen invasion, restore the intestinal epithelial barrier, and enhance immune regulation.

[0037] Furthermore, in step (2), the vortex time is 1 to 5 seconds, preferably 3 seconds.

[0038] Furthermore, in step (3),

[0039] The modified polysaccharide used is a modified polysaccharide solution prepared with physiological saline.

[0040] During oscillation incubation, the speed is 500-1000 rpm and the incubation time is 30-60 min, preferably 800 rpm and 45 min.

[0041] In the probiotic suspension obtained in step (3), the concentration of modified polysaccharide is 0.5-10 mg / mL, preferably 1 mg / mL.

[0042] In step (3), the vortex time is 1 to 5 seconds, preferably 3 seconds.

[0043] Further, in step (3), the washing specifically involves washing twice with physiological saline, and resuspending with physiological saline during resuspension. In some other methods, PBS with a pH of 6.0 to 7.0 may also be used; however, physiological saline is preferred in this embodiment of the invention.

[0044] In this invention, the modified polysaccharide containing α,β-unsaturated esters has α,β-unsaturated ester functional groups formed by the conjugation of olefinic bonds and electron-withdrawing groups, which are good acceptors for Michael addition reactions.

[0045] Further, in step (4), the thiolated polysaccharide is a thiolated polysaccharide solution prepared with physiological saline; in the probiotic suspension of step (4), the final concentration of the thiolated polysaccharide is 1-5 mg / mL, preferably 2 mg / mL;

[0046] The ultraviolet light wavelength is 350–370 nm, and the ultraviolet light irradiation time is 5–30 s, preferably 15 s under 360 nm ultraviolet light.

[0047] Furthermore, in step (4), the vortex time is 1 to 10 seconds, preferably 5 seconds.

[0048] Further, in step (4), after the reaction is completed, the sample is collected by centrifugation at 4000 rpm / min, washed twice with physiological saline, and then resuspended in physiological saline.

[0049] The second objective of this invention is to provide polysaccharide-encapsulated double-coated probiotics prepared by any of the above-mentioned preparation methods.

[0050] The present invention also aims to provide the application of the above-mentioned polysaccharide-encapsulated double-coated probiotics in the preparation of formulations that alter intestinal flora, generate metabolic entities, neutralize dietary carcinogens, induce cytokine synthesis, and control pathogens.

[0051] The beneficial effects of this invention include at least the following:

[0052] (1) The method for preparing probiotics by covalent coupling via Michael addition reaction and UV-mediated thiol-ene click reaction proposed in this invention first involves grafting methacrylic anhydride onto a pyranose ring, linking multiple methacrylates to the polysaccharide. The modified polysaccharide, acting as a Michael reaction acceptor, contains α,β-unsaturated esters, which are functional groups formed by the conjugation of olefin bonds and electron-withdrawing groups, and can undergo Michael addition reactions with nucleophiles. Furthermore, the bacterial outer membrane is composed of various substances such as peptidoglycan, lipopolysaccharide, lipids, and proteins, which provide functional groups such as thiol, hydroxyl, carboxyl, and amino groups. These functional groups are nucleophiles containing active hydrogen, which lose active hydrogen under alkaline conditions to generate carbanions. Then, the carbanions undergo 1,4-conjugative addition with α,β-unsaturated esters on the modified polysaccharide chains. The adduct abstracts a proton from the solvent to form an enol. Enol tautomerism yields the final product, ultimately enabling the modified polysaccharide to covalently couple with the probiotic surface, thus forming a stable and robust protective layer on the probiotic surface. Adding thiol-containing polysaccharides triggers the generation of thiol free radicals under ultraviolet light, which add to electron-rich double bonds. Utilizing the remaining olefin bonds of the original polysaccharide coating to couple with the probiotic surface, rapid and efficient coupling with the original polysaccharide coating can be achieved, thereby strengthening the polysaccharide coating that embeds the probiotics. By binding to naturally occurring and exposed groups on the peptidoglycan surface of probiotics, the activity and function of natural bacteria can be maintained. At the same time, the dense polysaccharide structure on the surface of probiotics effectively improves the efficiency of probiotics in intestinal transport and colonization.

[0053] (2) The modified probiotics prepared by the method provided in this invention have the advantages of being resistant to intestinal fluid, heat, and ultraviolet light, scavenging superoxide free radicals, and targeting inflammation. Attached Figure Description

[0054] Figure 1 For the activity test of unmodified and modified probiotics, ECN: unencapsulated probiotics; ECN-Dex-MA: single-coated probiotics encapsulated with polysaccharides; ECN-Dex-MA-Inu-SH: double-coated probiotics modified with thiolized polysaccharides.

[0055] Figure 2 This is a TEM image of the unmodified probiotic ECN.

[0056] Figure 3 TEM image of polysaccharide-encapsulated probiotic ECN-Dex-MA.

[0057] Figure 4 TEM image of ECN-Dex-MA-Inu-SH, a double-coated probiotic modified with alcoholized polysaccharides.

[0058] Figure 5 To assess the tolerance of ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH to artificially simulated gastrointestinal fluid.

[0059] Figure 6 The results show the storage stability test results for ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH.

[0060] Figure 7 Thermal stability testing for ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH.

[0061] Figure 8 UV stability testing for ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH.

[0062] Figure 9 Antioxidant capacity testing for ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH.

[0063] Figure 10 Validation targeting capability testing for ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH. Detailed Implementation

[0064] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0065] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0066] The following specific embodiments illustrate the solution proposed in this invention:

[0067] Example 1: Single-cell encapsulation

[0068] (1) Preparation of α,β-unsaturated ester polysaccharide: 5 kDa dextran (2 g, 12.4 mmol pyranose ring) was dissolved in 30 mL DMSO, and then 450 μL triethylamine (3.1 mmol) was slowly added. 1000 μL methacrylic anhydride (0.5 equiv / agu, 6.2 mmol) was slowly added dropwise. The mixture was stirred at 600 rpm at 50 °C for 12 h with magnetic stirring. After stirring, the mixture was dialyzed for 72 h with a dialysis bag with a molecular weight cutoff of 3500 Da to remove organic reagents. The mixture was then concentrated under vacuum, pre-frozen at -80 °C for 6 h, and freeze-dried in a freeze dryer for 72 h to obtain modified dextran.

[0069] (2) Preparation of activated probiotics: Take 0.4 mL of Escherichia coli Nissle1917 in 40 mL of LB medium and incubate overnight (12 h) on a shaker at 37 °C and 200 rpm / min. Centrifuge the bacterial suspension at 4000 rpm for 10 min, wash twice with physiological saline, and resuspend in 1 mL of physiological saline. Then, add 0.1 mL of the bacterial suspension to 4.4 mL of HEPES buffer (10 mmol / L, pH 8.2) and gently vortex for 3 s.

[0070] (3) Covalent coupling and encapsulation of modified polysaccharide and probiotics: Take the modified dextran prepared in step (1), prepare it with physiological saline, add it to the probiotic suspension in the HEPES buffer in step (2), so that the concentration of the modified dextran system is 1 mg / mL, gently vortex for 3s, and incubate at 37°C and 800 rpm for 45 min.

[0071] (4) Preparation of polysaccharide-encapsulated single-coat probiotic suspension: Centrifuge the solution after the reaction in step (3) at 4000 rpm, wash twice with physiological saline, resuspend in physiological saline, and wash away excess polysaccharide wall material that has not been bound to probiotics to facilitate photocoupling in the subsequent coating reinforcement step.

[0072] (5) Preparation of thiolized inulin polysaccharide: Dissolve 3g of inulin in 20mL of distilled water, then add 2mL of mercaptoacetic acid and 1mL of 7M HCl, and react at 80℃ for 180min. Pour the resulting reaction mixture into 200mL of methanol to precipitate the thiolized polysaccharide. Wash the resulting milky white precipitate three times with methanol, and after the methanol is naturally air-dried in a fume hood for 12h, freeze-dry it again to obtain the thiolized inulin polysaccharide for later use.

[0073] (6) UV-mediated thiolized polysaccharide-modified double-coated probiotics: Add the thiolized inulin polysaccharide prepared in step (5) to the probiotic suspension in step (4) so ​​that the system concentration of thiolized polysaccharide is 2 mg / mL. After vortexing, react under 360 nm UV light for 30 s, and immediately remove and vortex for 10 s.

[0074] (7) Preparation of UV-mediated thiolized polysaccharide-modified double-coated probiotic suspension: The solution after the reaction in step (6) was centrifuged at 4000 rpm / min, washed twice with physiological saline, and resuspended in physiological saline.

[0075] Example 2 Experimental Test

[0076] 1. Probiotic conjugation activity test

[0077] To verify whether the chemical coupling between polysaccharides and the surface of probiotics inhibits the activity of probiotics and affects bacterial cell division, the unencapsulated probiotic sample (ECN) from Example 1, the polysaccharide-encapsulated single-coated probiotic (ECN-Dex-MA) obtained in step (4) of Example 1, and the thiol-modified double-coated probiotic (ECN-Dex-MA-Inu-SH) suspension obtained in step (7) of Example 1 were respectively inoculated into LB medium and incubated with shaking at 37°C for 2 days to evaluate the optical density at 600 nm during probiotic growth.

[0078] The results are as follows Figure 1 As shown, EcN can naturally destroy the polysaccharide shell after detecting favorable conditions for replication. Unlike the original system, the probiotics modified with thiolized polysaccharides experienced a 4-hour growth rate delay, exhibiting almost complete inhibition and insignificant growth within 0-4 hours, but by 16 hours, their absorbance was close to that of normal EcN. This cell inhibition may be caused by the dense polysaccharide coating, which acts as a physical barrier, reducing nutrient uptake. The probiotics encapsulated via polysaccharide Michael reaction, polysaccharide Michael reaction coupling, and polysaccharide photocoupling showed similar growth curves to normal probiotics, indicating that the Michael reaction and UV photocoupling on the probiotic surface do not affect probiotic viability.

[0079] 2. Morphological characterization of probiotics encapsulated with polysaccharide coating

[0080] To observe whether the UV-mediated covalent coupling between thiolized polysaccharides and the surface of probiotics was successful, and whether it was still encapsulation by a single probiotic cell, ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH were examined using transmission electron microscopy (TEM) to observe the nanoscale coating structure. The results for unencapsulated ECN showed... Figure 2 In the middle, the results of ECN-Dex-MA are as follows Figure 3 As shown, ECN-Dex-MA-Inu-SH Figure 4 As shown.

[0081] The results showed that, compared to the smooth edges of EcN, the polysaccharide-encapsulated ECN-Dex-MA had a significantly rougher outer shell, and a dense nanoscale polysaccharide coating was observed. The surface of the thiol-modified polysaccharide-coated probiotic ECN-Dex-MA-Inu-SH was even rougher, with a thicker polysaccharide network. The probiotics encapsulated with polysaccharides maintained normal morphology, and each probiotic cell was individually encapsulated, demonstrating that the thiol-modified polysaccharides, through UV-mediated thiol-ene click reaction, modified the probiotic surface to form a coating network that was then solidified, without affecting the morphology of the probiotics.

[0082] 3. Tolerance of polysaccharide-coated probiotics to simulated gastrointestinal fluid.

[0083] ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH were added to 4.9 mL of simulated gastric fluid, respectively, and mixed thoroughly with constant shaking. At 0, 0.5, 1.0, 1.5, and 2 h of culture, 0.1 mL of the solution was immediately added to 1 mL of phosphate buffer solution, and serially diluted. The resulting solutions were then inoculated into LB culturing plates for counting. After 2 h of simulated gastric digestion, 5 mL of simulated intestinal fluid was added and mixed thoroughly. At 3.0, 4.0, 5.0, and 6.0 h of culture, 0.1 mL of the solution was immediately added to 1 mL of phosphate buffer solution, and serially diluted. The resulting solutions were then inoculated into LB culturing plates for counting. Results are as follows: Figure 5 As shown.

[0084] from Figure 5As can be seen, the unencapsulated probiotic ECN died quickly in simulated gastric juice, decreasing by 3.2 Log after 2 hours of in vitro digestion. The survival rate of the polysaccharide-encapsulated single-coated probiotic ECN-Dex-MA decreased by 0.46 Log, while the survival rate of the thiol-modified double-coated probiotic ECN-Dex-MA-Inu-SH decreased by only 0.25 Log. This indicates that the thiol-modified double-coated probiotic prepared by the method of this invention has excellent gastric acid resistance. During the simulated intestinal digestion stage, the unencapsulated probiotic ECN decreased by 0.7 Log, indicating near-total death and inability to reach the target intestinal tract. The polysaccharide-encapsulated single-coated probiotic ECN-Dex-MA and the thiol-polysaccharide-modified double-coated probiotic ECN-Dex-MA-Inu-SH decreased by 0.09 Log CFU / g and 0.05 Log CFU / g, respectively, after 4 hours of in vitro simulated intestinal digestion. The survival rate of all samples was significantly higher than that of free probiotics. The thiol-polysaccharide-modified double-coated probiotic prepared by the method of this invention still achieved a survival rate of 10% after simulated gastrointestinal digestion. 8 The concentration of CFU / ml or higher indicates that the structure of covalent coupling between polysaccharides and probiotics, and covalent binding between polysaccharides, is more effective in protecting probiotics. It has good acid and bile salt resistance properties. It overcomes the defects of traditional wall material structures that are loose and not compact, and achieves the effect of targeted release and colonization of probiotics, which are not released in the stomach but released in the intestine.

[0085] 4. Storage stability test of probiotics encapsulated with polysaccharide coating

[0086] Suspensions of ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH were collected in sterile glass bottles and stored at 4°C for 5 weeks. Each week, 0.1 mL of the suspension was weighed into a sterile EP tube, and 0.9 mL of 0.2 M phosphate buffer was added. The viable count was then calculated. The survival rates of the probiotics after different storage times are shown below. Figure 6 As shown.

[0087] from Figure 6 As can be seen, the unencapsulated probiotic ECN loses nearly 3 Log CFU / g of activity. Although the survival rate of the single-coated probiotic ECN-Dex-MA encapsulated with polysaccharides is improved, it is still much lower than the initial concentration. The double-coated probiotic ECN-Dex-MA-Inu-SH modified with thiol polysaccharides in this invention has significantly improved storage stability, and its survival rate can still reach 9.7 Log CFU / mL after being stored at 4°C for 5 weeks.

[0088] 5. Determination of the thermal stability of probiotics encapsulated with polysaccharide coating

[0089] To evaluate the tolerance of free and encapsulated probiotics under humid and hot conditions, 0.1 mL of ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH suspensions were first added to 0.9 mL of sterile water at 65°C. The mixture was then placed in a water bath, and 50 mL centrifuge tubes were removed at 4 min, 8 min, and 12 min, respectively, and cooled with 10 mL of PBS (4°C) to prevent further bacterial damage. After serial dilution, the mixture was inoculated into LB agar plates for counting.

[0090] Thicker coatings can extend the heat transfer path from the environmental medium to the interior, and are considered a method to improve the thermal stability of probiotics. The survival rates of free and encapsulated probiotics are as follows: Figure 7 As shown in the figure, for the free probiotic sample ECN, treatment at 65℃ for 8 min resulted in a decrease in viable cell count of 5.72 Log CFU / g. With prolonged heat treatment, the viability of the free probiotic ECN rapidly decreased, and no viable cells were detected after 12 min. The survival rate of the polysaccharide-encapsulated single-coat probiotic ECN-Dex-MA was significantly higher than that of the free probiotic, but the viability loss also increased with increasing heating time, decreasing by 2.1 Log CFU / g after 12 min. The thiol-modified polysaccharide-modified double-coat probiotic ECN-Dex-MA-Inu-SH exhibited stronger thermal stability, with a loss of only 1.04 Log CFU / g. This may be because the structure of the two polysaccharide layers, after UV coupling, is more dense, prolonging the diffusion path of hot water and delaying the contact between the probiotic and hot water. These results indicate that thiol-modified polysaccharide modification plays an important role in improving the structure and performance of the encapsulated polysaccharide network coating.

[0091] 6. UV stability analysis of probiotics encapsulated with polysaccharide coating

[0092] To assess the tolerance of free and encapsulated probiotics to ultraviolet radiation, suspensions of ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH were added to sterile tubes and exposed to 235.7 nm ultraviolet light for 30 min, followed by 1 h of light protection. After the reaction, the suspensions were transferred to sterile tubes containing 0.9 mL of phosphate-buffered saline and serially diluted before being inoculated into LB broth plates for counting.

[0093] Figure 8The effects of UV irradiation on the survival of free and encapsulated probiotics were shown. After 30 min of UV treatment, no viable bacteria were detected in the free probiotic ECN sample. However, the activity of the polysaccharide-encapsulated single-coated probiotic ECN-Dex-MA decreased by only 0.23 Log CFU / g, reaching a loss of 1.15 Log CFU / g after 60 min of treatment. This indicates that the polysaccharide coating can effectively improve the UV resistance of probiotics. The thiol-modified polysaccharide-modified double-coated probiotic ECN-Dex-MA-Inu-SH showed almost no decrease after 60 min of UV irradiation, possibly because the thiol-modified polysaccharide reacted more fully with the remaining α,β-unsaturated ester bonds on the polysaccharide coating under UV irradiation, forming a denser protective layer.

[0094] 7. Determination of the antioxidant capacity of probiotics encapsulated with polysaccharide coating

[0095] To evaluate whether polysaccharide coating can endow probiotics with multiple exogenous functions, the physiological state of pathological sites often changes, and the level of reactive oxygen species (ROS) increases, which is not conducive to the colonization of probiotics in the intestine. Therefore, we determined whether the ROS scavenging ability of probiotics after polysaccharide encapsulation is enhanced.

[0096] Using the scavenging of superoxide anion free radicals as an indicator, the procedure was as follows: 4 mL of 0.05 mol / L Tris-HCl buffer (pH 8.2) was placed in a test tube and incubated at 25°C for 20 min. Then, 1 mL of ECN, ECN-Dex-MA, and ECN-Dex-MA-Inu-SH suspensions were added, with an equal volume of deionized water as a negative control and vitamin C as a positive control. Next, 20 μL of 80 mM pyrogallol solution was added to each tube, mixed thoroughly, and incubated at 25°C for 5 min. The reaction was then terminated by rapidly adding 200 μL of 10 M HCl. The absorbance of the supernatant was measured at 325 nm, and the scavenging rate was calculated. The results are shown below. Figure 9 The superoxide anion radical scavenging rate is as follows:

[0097]

[0098] In the formula, A1 is Tris-HCl + pyrogallol + sample;

[0099] A2 – Tris-HCl + equal volume of distilled water + sample;

[0100] A0 — Tris-HCl + pyrogallol + equal volume of distilled water.

[0101] Figure 9The results showed that the superoxide radical scavenging rate of the unencapsulated probiotic group (ECN) was 53.21%, the superoxide radical scavenging rate of the polysaccharide-encapsulated single-coated probiotic ECN-Dex-MA was 84.36%, and the superoxide radical scavenging rate of the thiol-modified double-coated probiotic ECN-Dex-MA-Inu-SH was 93.21%. This indicates that the polysaccharide coating has antioxidant capacity and scavenges free radicals. The modification of thiol-modified polysaccharides enhances the antioxidant capacity of the probiotic encapsulation system, thus better protecting probiotics from damage by reactive oxygen species. This also shows that encapsulated probiotics can reduce oxidative damage to cells in the inflamed area during IBD treatment, clear excess ROS in vivo, regulate the redox environment in the colon, and reduce tissue oxidative damage.

[0102] 8. Determination of the inflammation-targeting ability of thiol-modified double-coated probiotics

[0103] To verify that polysaccharide coating endows probiotics with inflammatory targeting functions, the physiological state of pathological sites often changes, leading to the in situ accumulation of positively charged proteins on the surface and microenvironment of inflammatory intestinal epithelial cells, including increased transferrin and eosinophilic cationic proteins. The study determined whether polysaccharide-encapsulated probiotics exhibited enhanced adhesion and targeting at the intestinal mucosa. Normal epithelial cells were simulated using mucin, and inflammatory sites were simulated using recombinant human transferrin. Mucin (2 mg / mL) or transferrin (0.5 mg / mL) was dissolved in PBS and added to 24-well polystyrene plates, which were incubated overnight at 37°C. After washing with PBS, 0.2 mL of ECN, ECN-Dex-MA, or ECN-Dex-MA-Inu-SH suspensions were added to the plates, and incubation was performed at room temperature for 1 h. The plates were then washed three times with PBS, and plate counts were performed.

[0104] Figure 10The results showed that the adhesion rates of the unencapsulated ECN group on empty plates, mucin, and transferrin were 1.79%, 1.9%, and 2.29%, respectively. The adhesion rates of the polysaccharide-encapsulated single-coat probiotic ECN-Dex-MA group on empty plates, mucin, and transferrin were 53.21%, 78.95%, and 86.93%, respectively. The adhesion rates of the thiol-modified polysaccharide-modified double-coat probiotic ECN-Dex-MA-Inu-SH group on empty plates, mucin, and transferrin were 58.36%, 88.21%, and 88.21%, respectively. The percentages of 93.95% and 93.95% are attributed to the hydrophobic nature of methacrylated polysaccharides, which enhances the adhesion of single-coated probiotics. Furthermore, methacrylated polysaccharides can undergo a Michael addition reaction with cysteine ​​(-SH) residues on mucin, leading to increased adhesion of coated probiotics to the mucin. The surface of the double-coated probiotics, modified with thiol-modified polysaccharides, is rich in thiol groups, which can undergo disulfide exchange reactions with the thiol groups on the mucin surface, further binding to the mucin and enhancing adhesion to the intestinal mucosa, thus promoting intestinal colonization. Additionally, coated probiotics exhibit a tendency to preferentially adhere to surfaces of positively charged proteins, and since the intestinal tract is positively charged, this makes it easier to target inflamed areas.

[0105] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0106] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0107] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing polysaccharide-encapsulated double-coated probiotics, characterized in that, Includes the following steps: (1) Polysaccharide A was prepared into a modified polysaccharide containing α,β-unsaturated esters; Polysaccharide B was prepared into thiolized polysaccharide; (2) Add the activated probiotic suspension to HEPES buffer and vortex to obtain a dispersed probiotic suspension. (3) Mix the probiotic suspension dispersed by vortex in step (2) with the modified polysaccharide obtained in step (1), vortex, and incubate by shaking. After incubation, the bacteria were washed and resuspended to obtain a probiotic suspension mixed with modified polysaccharides. (4) The probiotic suspension containing modified polysaccharides obtained in step (3) is mixed with the thiolized polysaccharides obtained in step (1), vortexed thoroughly, and reacted under ultraviolet light. After the reaction is completed, the mixture is washed and resuspended.

2. The preparation method according to claim 1, characterized in that, In step (1), the preparation of the modified polysaccharide containing α,β-unsaturated esters includes: dissolving polysaccharide A in dimethyl sulfoxide, adding triethylamine and methacrylic anhydride, stirring overnight, dialysis, vacuum concentration, and drying to obtain the polysaccharide containing α,β-unsaturated esters.

3. The preparation method according to claim 2, characterized in that, In step (1), The polysaccharide A includes at least one of dextran, hyaluronic acid, tamarind polysaccharide, and amylopectin, and the molecular weight of the polysaccharide A is 5000-40000 Da, preferably 10000 Da; The molar ratio of polysaccharide A to methacrylic anhydride is 1:0.2-1; The molar ratio of triethylamine to methacrylic anhydride is 1:1 to 10; The amount of polysaccharide and dimethyl sulfoxide added is 1g: 5-15mL; When stirring overnight, the temperature should be 20–25°C and the time should be 10–15 hours. During dialysis, deionized water is used, and the dialysis time is 48–96 hours.

4. The preparation method according to claim 1, characterized in that, In step (1), the preparation of the thiolated polysaccharide includes: dissolving polysaccharide B in distilled water, adding mercaptoacetic acid and HCl solution, reacting at 75-85℃ for 150-200 min; after the reaction is completed, pouring into methanol to precipitate the thiolated polysaccharide, washing with methanol, air-drying naturally, and then freeze-drying to obtain the thiolated polysaccharide.

5. The preparation method according to claim 4, characterized in that, In step (1), The polysaccharide B includes at least one of inulin, dextran, hyaluronic acid, and tamarind polysaccharide, and the molecular weight of the polysaccharide B is 3000-20000 Da, preferably 4500 Da; The ratio of polysaccharide B: mercaptoacetic acid: hydrochloric acid is 2-4 g: 1-3 mL: 0.5-1.5 mL, and the concentration of the hydrochloric acid solution is 6-8 M.

6. The preparation method according to claim 1, characterized in that In step (2), The pH of the HEPES buffer is 8.0–8.5; The probiotics include at least one of Enterobacter, Streptococcus thermophilus, Lactobacillus, Bifidobacterium, Actinomycetes, and yeast; Without HEPES buffer, the concentration of probiotics in the probiotic suspension is 1×10⁻⁶. 6 ~1×10 10 CFU / mL; After mixing with HEPES buffer, the concentration of probiotics in HEPES buffer is 2 × 10⁻⁶. 4 ~2×10 8 CFU / mL.

7. The preparation method according to claim 1, characterized in that, In step (3), The modified polysaccharide used is a modified polysaccharide solution prepared with physiological saline. In the probiotic suspension finally obtained in step (3), the concentration of modified polysaccharide is 0.5-10 mg / mL. During shaking incubation, the speed is 500-1000 rpm and the incubation time is 30-60 min.

8. The preparation method according to claim 1, characterized in that, In step (4), the thiolated polysaccharide is a thiolated polysaccharide solution prepared with physiological saline, and the final concentration of the thiolated polysaccharide is 1-5 mg / mL; The ultraviolet light wavelength is 350–370 nm, and the ultraviolet light irradiation time is 5–30 s.

9. The polysaccharide-encapsulated double-coated probiotic prepared by any of the preparation methods described in claims 1 to 8.

10. The use of the polysaccharide-encapsulated double-coated probiotic of claim 9 in the preparation of formulations that alter intestinal flora, generate metabolic entities, neutralize dietary carcinogens, induce cytokine synthesis, and control pathogens.

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