Michael addition reaction mediated polysaccharide and probiotic surface covalent coupling single cell encapsulation method

Abstract

The invention relates to the technical field of biological agents, in particular to a Michael addition reaction mediated polysaccharide and probiotic surface covalent coupling single cell encapsulation method. The method comprises the following steps: adding polysaccharide into dimethyl sulfoxide for dissolving, adding triethylamine and methacrylic anhydride to obtain modified polysaccharide containing alpha, beta-unsaturated ester, adding activated probiotic suspension into an HEPES buffer solution, adding a modified polysaccharide solution containing alpha, beta-unsaturated ester into the vortex-dispersed probiotic suspension, oscillating and incubating to obtain the probiotic suspension containing alpha, beta-unsaturated ester. Preparing a probiotic suspension of the nano coating encapsulated by the polysaccharide; according to the method, a stable and firm protective layer is formed on the surface of the probiotics, the activity and functions of natural bacteria are kept, meanwhile, the conveying and colonization survival efficiency of the probiotics in the intestinal tract is effectively improved through a compact polysaccharide structure on the surface of the probiotics, and the commercial application value of the probiotics in the fields of food and medicine is improved.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a single-cell encapsulation method for covalent coupling of polysaccharides with the surface of probiotics mediated by Michael addition reaction. 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] To address the shortcomings of the existing technology, this invention aims to provide a single-cell encapsulation method for covalently coupling polysaccharides to the surface of probiotics via Michael addition reaction, thereby protecting probiotics, enhancing their resistance, and endowing them with exogenous functions.

[0005] To solve the above problems, the present invention adopts the following technical solution:

[0006] By grafting polysaccharides with methacrylic anhydride and other materials, Michael addition receptor polysaccharides containing α,β-unsaturated esters are obtained. Natural reactive groups present on the surface of probiotics, such as thiol, hydroxyl, carboxyl, and amino groups containing active hydrogen, undergo Michael addition reactions with the α,β-unsaturated esters on the modified polysaccharides under physiological conditions, forming a robust protective layer on the surface of the probiotics. This results in probiotics encapsulated with a polysaccharide coating, providing individualized protection for each probiotic cell.

[0007] In a first aspect, the present invention provides a method for encapsulating single cells by covalently coupling polysaccharides to the surface of probiotics via Michael addition reaction, comprising:

[0008] (1) Dissolve the polysaccharide in dimethyl sulfoxide, add triethylamine and methacrylic anhydride, stir, dialyze, concentrate under vacuum, and freeze dry to obtain a modified polysaccharide containing α,β-unsaturated esters.

[0009] (2) Add the activated probiotic suspension to HEPES buffer and vortex;

[0010] (3) Add a modified polysaccharide solution containing α,β-unsaturated esters to the probiotic suspension dispersed by vortexing in (2), vortex, and incubate at 37°C to covalently encapsulate the modified polysaccharide containing α,β-unsaturated esters with the probiotics.

[0011] (4) Wash the covalently coupled encapsulated solution with physiological saline, resuspend it in physiological saline, and prepare a polysaccharide-encapsulated nano-coated probiotic suspension.

[0012] Furthermore, the polysaccharide is at least one of dextran, hyaluronic acid, tamarind polysaccharide, and amylopectin; the molecular weight of the polysaccharide is 5000 Da to 40000 Da.

[0013] Furthermore, the molar ratio of the polysaccharide to methacrylic anhydride is 1:0.2-1; the molar ratio of the methacrylic anhydride to triethylamine is 1:1-10; and the ratio of the polysaccharide to dimethyl sulfoxide is 1g:10mL.

[0014] Furthermore, the probiotics include at least one of Enterobacter, Streptococcus thermophilus, Lactobacillus, Bifidobacterium, Actinomycetes, and yeast.

[0015] Furthermore, the concentration of probiotics in the probiotic suspension is 1×10⁻⁶. 6 ~1×10 10 CFU / mL; the concentration of the probiotics in HEPES buffer is 2 × 10⁻⁶. 4 ~2×10 8 CFU / mL.

[0016] Furthermore, the concentration of the modified polysaccharide solution containing α,β-unsaturated esters is 0.5–10 mg / mL; the shaking speed is 500–1000 rpm; and the incubation time is 30–60 min.

[0017] Furthermore, the preparation method of the polysaccharide-encapsulated nano-coated probiotic suspension includes:

[0018] The covalently coupled encapsulated solution was centrifuged at 4000 rpm / min, washed twice with physiological saline, and then resuspended in physiological saline.

[0019] Furthermore, the vortex duration is 1–5 seconds.

[0020] Furthermore, the preparation of the activated probiotic suspension includes: taking 0.4 mL of probiotics in 40 mL of LB medium, placing it on a shaker and culturing it overnight at a temperature of 37°C and a shaking speed of 200 rpm / min; centrifuging the bacterial suspension at 4000 rpm / min for 10 min, washing it twice with physiological saline, and resuspending it in 1 mL of physiological saline.

[0021] Secondly, the present invention provides a nano-coated probiotic suspension encapsulated by the single-cell encapsulation method of covalently coupling polysaccharides to the surface of probiotics mediated by the Michael addition reaction.

[0022] The beneficial effects of this invention are as follows: methacrylic anhydride is grafted onto the pyranose ring, so that the polysaccharide is linked with multiple methacrylates; the modified polysaccharide, as a Michael reaction acceptor, contains α,β-unsaturated esters, which are functional groups formed by the conjugation of alkene 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, providing 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-conjugated 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. This binding with naturally occurring and exposed groups on the peptidoglycan surface of the probiotics maintains the activity and function of the natural bacteria. At the same time, the dense polysaccharide structure on the probiotic surface effectively improves the efficiency of probiotic transport and colonization in the intestine, enhancing its commercial application value in the food and pharmaceutical fields. Attached Figure Description

[0023] Figure 1 This is a photometric density diagram at 600 nm during the growth of probiotics.

[0024] Figure 2 This is a TEM morphology image of unencapsulated probiotics.

[0025] Figure 3 This is a TEM image of the encapsulated probiotics.

[0026] Figure 4 A comparison chart showing the decline rate of probiotic survival in artificially simulated gastrointestinal fluid.

[0027] Figure 5 A comparison chart showing the storage stability of probiotics.

[0028] Figure 6 This is a comparison chart of the thermal stability of probiotics.

[0029] Figure 7 This is a comparison chart of the UV stability of probiotics.

[0030] Figure 8 This is a comparison chart of the antioxidant capacity of probiotics.

[0031] Figure 9 A comparison chart of the inflammation-targeting capabilities of probiotics. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments.

[0033] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0034] Example 1

[0035] A single-cell encapsulation method for covalent coupling of polysaccharides to the surface of probiotics via Michael addition reaction is disclosed. The probiotics coated with this method exhibit superior resistance to gastrointestinal conditions and significant ROS scavenging ability. In particular, the polysaccharide coating enables the probiotics to target inflammatory sites through various interactions. This method includes the following steps:

[0036] (1) Dissolve the polysaccharide in dimethyl sulfoxide, add triethylamine and methacrylic anhydride, stir, dialyze, concentrate under vacuum, and freeze dry to obtain a modified polysaccharide containing α,β-unsaturated esters.

[0037] (2) Add the activated probiotic suspension to HEPES buffer and vortex;

[0038] (3) Add a modified polysaccharide solution containing α,β-unsaturated esters to the probiotic suspension dispersed by vortexing in (2), vortex, and incubate at 37°C to covalently encapsulate the modified polysaccharide containing α,β-unsaturated esters with the probiotics.

[0039] (4) Wash the covalently coupled encapsulated solution with physiological saline, resuspend it in physiological saline, and prepare a polysaccharide-encapsulated nano-coated probiotic suspension.

[0040] In this embodiment of the invention, in step (1), the modified polysaccharide containing α,β-unsaturated esters is prepared by the following steps: dissolving the polysaccharide in dimethyl sulfoxide, then slowly adding triethylamine and methacrylic anhydride, and stirring the solution overnight at 25°C. Subsequently, the solution is dialyzed with deionized water and concentrated under vacuum. Finally, the modified polysaccharide is dried.

[0041] Specifically, polysaccharides include at least one of dextran, hyaluronic acid, tamarind polysaccharide, and amylopectin. Dextran is a water-soluble polysaccharide, abundant, 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 dextranase, which hydrolyzes the α-D-(1→6)-glucosidic bonds in dextran, releasing beneficial bacteria in the colon and simultaneously producing oligosaccharides, which are beneficial to the gut microbiota and human health.

[0042] Specifically, the molecular weight of the polysaccharide can be selected from 5000 Da to 40000 Da, with 10000 Da being preferred.

[0043] Specifically, 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.

[0044] Specifically, in step (1), the molar ratio of polysaccharide to methacrylic anhydride is 1:0.2 to 1. The catalyst is triethylamine, and the molar ratio of triethylamine to methacrylic anhydride is 1:1 to 10.

[0045] Specifically, in step (1), the stirring time is 12 hours.

[0046] Specifically, in step (1), the amount of polysaccharide and dimethyl sulfoxide added is 1g:10mL.

[0047] Specifically, in step (1), the dialysis time is 48 to 96 hours, preferably 72 hours.

[0048] Specifically, in step (1), the vacuum concentration temperature is 60°C and the rotation speed is 50 rpm / min.

[0049] Specifically, polysaccharide drying can be achieved through spray drying or freeze drying, with freeze drying being the preferred method in this example.

[0050] In this embodiment of the invention, in step (2), the pH range of the HEPES buffer at 37°C is 8.0 to 8.5, preferably pH = 8.2. The buffer solution is prepared by weighing 1.1915 g of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid into a beaker, adding 450 mL of deionized water to dissolve it, adjusting the pH to 8.2 with NaOH, and then making up the volume to 500 mL to prepare a 10 mmol / L HEPES solution. The solution is then sterilized by high temperature and high pressure and stored at room temperature.

[0051] In this embodiment of the invention, in step (2), the activated probiotic suspension is prepared as follows: 0.4 mL of probiotics is placed in 40 mL of LB medium and cultured overnight (12 h) on a shaker at a temperature of 37 °C and a shaking speed of 200 rpm / min; the bacterial suspension is centrifuged at 4000 rpm / min for 10 min, washed twice with physiological saline, and resuspended in 1 mL of physiological saline.

[0052] Specifically, the probiotics include at least one of Enterobacteriaceae, Streptococcus thermophilus, Lactobacillus, Bifidobacterium, Actinomycetes, and yeast. 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.

[0053] Specifically, in step (2), the concentration of probiotics in the probiotic suspension is 1×10⁻⁶. 6 ~1×1010 CFU / mL, preferably 1×10⁻⁶ 9 CFU / mL.

[0054] Specifically, in step (2), the concentration of probiotics in the HEPES buffer is 2 × 10⁻⁶. 4 ~2×10 8 CFU / mL, preferably 2×10⁻⁶ 7 CFU / mL.

[0055] In this embodiment of the invention, the modified polysaccharide containing α,β-unsaturated ester in step (3) has α,β-unsaturated ester functional groups formed by the conjugation of olefinic bonds and electron-withdrawing groups, and is a good Michael addition reaction acceptor.

[0056] In this embodiment of the invention, in step (3), a modified polysaccharide solution containing α,β-unsaturated esters is prepared. 10 mg of the modified polysaccharide containing α,β-unsaturated esters is added to 0.5 mL of saline solution to obtain a modified polysaccharide solution of 20 mg / mL. All of the modified polysaccharide solution is added.

[0057] Specifically, in step (3), the concentration of the modified polysaccharide solution containing α,β-unsaturated esters is 0.5 to 10 mg / mL, preferably 1 mg / mL.

[0058] Specifically, the gentle vortexing time is 1 to 5 seconds, preferably 3 seconds.

[0059] Specifically, in step (3), the shaking table oscillates at 500-1000 rpm, preferably 800 rpm.

[0060] Specifically, in step (3), the incubation time is 30 to 60 minutes, preferably 45 minutes.

[0061] In this embodiment of the invention, in step (4), after the covalent coupling encapsulation is completed, the sample is collected by centrifugation at 4000 rpm / min, washed twice with physiological saline, and then resuspended in physiological saline.

[0062] Specifically, in step (4), PBS or physiological saline with a pH of 6.0 to 7.0 can be used.

[0063] Example 2

[0064] Preparation of modified polysaccharides containing α,β-unsaturated esters: 5 kDa dextran (2 g, 12.4 mmol pyranose ring) was dissolved in 30 mL DMSO, then 450 μL triethylamine (3.1 mmol) was slowly added, followed by the slow dropwise addition of 1000 μL methacrylic anhydride (0.5 equiv / agu, 6.2 mmol). The mixture was stirred at 50 °C with magnetic stirring at 600 rpm for 12 hours. After stirring, the mixture was dialyzed for 72 hours using 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 hours, and finally freeze-dried in a freeze dryer for 72 hours.

[0065] Preparation of activated probiotics: 0.4 mL of *Escherichia coli* Nissle 1917 was placed in 40 mL of LB medium and incubated 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. Then, 0.1 mL of the bacterial suspension was added to 4.4 mL of HEPES buffer (10 mmol / L, pH 8.2) and gently vortexed for 3 s.

[0066] Covalently encapsulating modified polysaccharides containing α,β-unsaturated esters with probiotics: Take 0.5 mL of 10 mg / mL modified dextran containing α,β-unsaturated esters and add it to the probiotic suspension in HEPES buffer to make the concentration of the modified dextran solution containing α,β-unsaturated esters 1 mg / mL. Gently vortex for 3 s and incubate at 37°C and 800 rpm / min for 45 min.

[0067] Preparation of polysaccharide-encapsulated nano-coated probiotic suspension: The solution after the reaction was completed was centrifuged at 4000 rpm / min, washed twice with physiological saline, and finally suspended in 0.1 mL of physiological saline to prepare a polysaccharide-encapsulated nano-coated probiotic suspension.

[0068] In Example 2 of this invention, Escherichia coli Nissle 1917 (EcN) was preferentially selected, and dextran was preferentially selected as the encapsulation material for probiotics. The above-mentioned steps were performed, and the following test was conducted.

[0069] Experiment 1: Probiotic conjugation activity test

[0070] To verify whether the covalent coupling of polysaccharides to the surface of probiotics inhibited probiotic activity and affected bacterial cell division, unencapsulated probiotic samples and the probiotic suspension obtained in Example 2 after polysaccharide Michael reaction coupling encapsulation were inoculated into LB medium and incubated with shaking at 37°C for 24 h. The optical density at 600 nm during probiotic growth was evaluated. The results showed that... Figure 1 .

[0071] Tests showed that EcN could naturally break its polysaccharide coat after detecting favorable conditions for replication. Similar to the original system, the probiotics that underwent polysaccharide coupling exhibited a similar growth trend to EcN, and their absorbance tended to stabilize with that of normal EcN after 16 hours. Figure 1 As shown, the polysaccharide coating acts as a physical barrier, but can still maintain the function and vitality of probiotics in vitro and in vivo. The growth curves of probiotics after polysaccharide covalent coupling are similar to those of normal probiotics, indicating that the occurrence of Mike addition reaction on the surface of probiotics does not affect the growth and vitality of probiotics.

[0072] Experiment 2 Morphological characterization of probiotics encapsulated with polysaccharide coating

[0073] To observe whether the covalent coupling between the polysaccharide and the probiotic surface was successful, and whether the encapsulation was performed by a single probiotic cell, unencapsulated probiotic samples and the probiotic suspension encapsulated with the polysaccharide coating obtained in Example 2 were examined using transmission electron microscopy (TEM). The nanoscale coating structure could be observed. The results for the unencapsulated EcN are as follows: Figure 2 The result of the encapsulated EcN is as follows Figure 3 .

[0074] Tests showed that, compared to the smooth edges of EcN, the polysaccharide-coated network had a significantly rougher coating shell after encapsulation, and a dense nanoscale polysaccharide coating was observed. The probiotics encapsulated with polysaccharides exhibited normal morphology, and each probiotic cell was individually encapsulated, demonstrating that the polysaccharides were successfully modified onto the probiotic surface via the Michael addition reaction for single-cell encapsulation without affecting the probiotic morphology.

[0075] Experiment 3: Tolerance of probiotics encapsulated with polysaccharide coating to simulated gastrointestinal fluid.

[0076] Unencapsulated probiotic samples and the probiotic suspension obtained in Example 2 after polysaccharide Michael reaction coupling encapsulation were added to 4.9 mL of simulated gastric fluid and continuously shaken to mix. At incubation times of 0, 0.5, 1.0, 1.5, and 2 h, 0.1 mL of the solution was immediately added to 1 mL of phosphate buffer solution and serially diluted. The solution was then inoculated into LB 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, 0.1 mL of the solution was immediately added to 1 mL of phosphate buffer solution and serially diluted. The solution was then inoculated into LB plates for counting. The results are as follows: Figure 4 .

[0077] from Figure 4 As can be seen, unencapsulated probiotics quickly died in simulated gastric juice, decreasing by 3.2 Logs after 2 hours of in vitro digestion. The survival rate of probiotics encapsulated with polysaccharide-Michael reaction decreased by 0.46 Logs, indicating that the polysaccharide-coated probiotics prepared by the method of this invention have excellent gastric acid resistance. In the simulated intestinal juice digestion stage, the unencapsulated probiotics decreased again by 0.7 Logs, almost completely dying and unable to reach the designated intestinal site. The probiotics encapsulated with polysaccharide-Michael reaction and those encapsulated with polysaccharide-Michael reaction decreased again by 0.09 Log CFU / g after 4 hours of in vitro simulated intestinal juice digestion. The survival rate of these samples was significantly higher than that of free probiotics, indicating that this covalent coupling of polysaccharide and probiotics is more effective in protecting probiotics and has good acid and bile salt resistance. It overcomes the defects of traditional loose and non-firm wall materials, achieving the targeted release and colonization effect of probiotics not being released in the stomach but only in the intestine.

[0078] Experiment 4: Storage stability test of probiotics encapsulated with polysaccharide coating

[0079] Unencapsulated probiotic samples and the probiotic suspension obtained in Example 2 after polysaccharide Michael reaction coupling encapsulation 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 probiotic survival rate after different storage times is shown below. Figure 5 As shown.

[0080] from Figure 5As can be seen, the viability loss of unencapsulated probiotics is close to 3 Log CFU / g. The survival rate of probiotics encapsulated by polysaccharide Michael reaction is improved. The storage stability of probiotics prepared by combining polysaccharide with the surface of probiotics in this invention is significantly improved. After being stored at 4°C for 10 weeks, the survival rate can still reach 8.9 Log CFU / mL.

[0081] Experiment 5: Determination of the thermal stability of probiotics encapsulated with polysaccharide coating

[0082] To evaluate the tolerance of free and encapsulated probiotics under humid and hot conditions, 0.1 mL of the unencapsulated probiotic sample and 0.1 mL of the probiotic suspension obtained in Example 2 after polysaccharide Michael reaction coupling encapsulation were 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. Results are shown below. Figure 6 .

[0083] Coatings can prolong the heat transfer pathway 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 6 As shown in the figure, for free probiotic samples, 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 free probiotics rapidly declined, and no viable bacteria were detected after 12 min. After polysaccharide-coated probiotics, the survival rate was significantly higher than that of free probiotics, decreasing by 2.1 Log CFU / g after 12 min. The probiotics encapsulated via polysaccharide Michael reaction coupling exhibited strong thermal stability. These results indicate that the polysaccharide coating plays an important role in improving the structure and performance of the encapsulation network.

[0084] Experiment 6: UV stability analysis of probiotics encapsulated with polysaccharide coating

[0085] To assess the tolerance of free and encapsulated probiotics to ultraviolet (UV) radiation, unencapsulated probiotic samples and the probiotic suspension obtained in Example 2 after polysaccharide Michael reaction coupling encapsulation were added to sterile tubes. Both were exposed to UV light at 235.7 nm for 30 min and then protected from light for 1 h. After the reaction, the samples were transferred to sterile tubes containing 0.9 mL of phosphate-buffered saline and serially diluted. The diluted solutions were then inoculated into LB plates for counting. Results are shown below. Figure 7 .

[0086] Figure 7The effects of UV irradiation on the survival of free and encapsulated probiotics were shown. No viable bacteria were detected in the free probiotic samples after UV treatment, while the viability of probiotics encapsulated via the Michael reaction with polysaccharides decreased by only 0.23 LogCFU / g. This indicates that the polysaccharide coating can effectively improve the UV resistance of probiotics.

[0087] Experiment 7: Determination of the antioxidant capacity of probiotics encapsulated with polysaccharide coating

[0088] To evaluate the various exogenous functions that polysaccharide coating can impart to probiotics, and considering that the physiological state of pathological sites often changes, leading to increased reactive oxygen species (ROS) levels which are unfavorable for probiotic colonization in the intestine, this study determined whether the ROS scavenging ability of probiotics encapsulated with polysaccharides was enhanced. Using superoxide anion radical scavenging as an indicator, the procedure was as follows: 4 mL of 0.05 mol / L Tris-HCl buffer (pH 8.2) was added to a test tube and incubated at 25°C for 20 min. 1 mL of unencapsulated probiotic sample and 1 mL of the probiotic suspension obtained in Example 2 after polysaccharide Michael reaction coupling encapsulation were added to each tube, with an equal volume of deionized water as a negative control and vitamin C as a positive control. Then, 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 in [Figure number missing]. Figure 8 The superoxide anion radical scavenging rate is as follows:

[0089]

[0090] In the formula, A1 is Tris-HCl + pyrogallol + sample;

[0091] A2 – Tris-HCl + equal volume of distilled water + sample;

[0092] A0 — Tris-HCl + pyrogallol + equal volume of distilled water.

[0093] Figure 8 The results showed that the superoxide radical scavenging rate of the unencapsulated probiotic group was 53.21%, while that of the probiotics after polysaccharide encapsulation was 84.36%. This is because the polysaccharide coating has antioxidant capacity and scavenges free radicals, thus better protecting the probiotics from damage by reactive oxygen species. This also indicates that encapsulated probiotics can reduce oxidative damage to cells in the inflamed area during IBD treatment, and can remove excess ROS in vivo, regulate the redox environment in the colon, and reduce tissue oxidative damage.

[0094] Experiment 8: Determination of the inflammation-targeting ability of probiotics encapsulated with polysaccharide coating

[0095] To verify that polysaccharide coating endows probiotics with inflammatory targeting function, 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 the unencapsulated probiotic sample and the probiotic suspension obtained in Example 2 after polysaccharide Michael reaction coupling encapsulation were added to the plate, and the plate was incubated at room temperature for 1 h. The plate was then washed three times with PBS, and plate counts were performed. The results are shown in [Figure number missing]. Figure 9 .

[0096] Figure 9 The 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, while the adhesion rates of the polysaccharide-encapsulated coated probiotic group on empty plates, mucin, and transferrin were 53.21%, 78.95%, and 86.93%, respectively. This is because the hydrophobicity of the methacrylated polysaccharide makes the coated probiotics inherently highly adhesive. Furthermore, the methacrylated polysaccharide can undergo a Michael addition reaction with cysteine-SH on mucin, resulting in more coated probiotics adhering to the mucin. Additionally, the coated probiotics exhibited 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 inflammatory sites.

[0097] Finally, it should be noted that 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 with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for encapsulating single cells by covalent coupling of polysaccharides to the surface of probiotics via Michael addition reaction, characterized in that, include: (1) Dissolve the polysaccharide in dimethyl sulfoxide, add triethylamine and methacrylic anhydride, stir, dialyze, concentrate under vacuum, and freeze dry to obtain a modified polysaccharide containing α,β-unsaturated esters. (2) Add the activated probiotic suspension to HEPES buffer and vortex; (3) Add a modified polysaccharide solution containing α,β-unsaturated esters to the probiotic suspension dispersed by vortexing in (2), vortex, and incubate at 37°C to covalently encapsulate the modified polysaccharide containing α,β-unsaturated esters with the probiotics. (4) Wash the covalently coupled encapsulated solution with physiological saline, resuspend it in physiological saline, and prepare a polysaccharide-encapsulated nano-coated probiotic suspension.

2. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 1, characterized in that, The polysaccharide is at least one of dextran, hyaluronic acid, tamarind polysaccharide, and amylopectin; the molecular weight of the polysaccharide is 5000 Da to 40000 Da.

3. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 2, characterized in that, The molar ratio of the polysaccharide to methacrylic anhydride is 1:0.2-1; the molar ratio of the methacrylic anhydride to triethylamine is 1:1-10; and the ratio of the polysaccharide to dimethyl sulfoxide is 1g:10mL.

4. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 1, characterized in that, The probiotics include at least one of Enterobacter, Streptococcus thermophilus, Lactobacillus, Bifidobacterium, Actinomycetes, and yeast.

5. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 4, characterized in that, The concentration of probiotics in the probiotic suspension is 1×10⁻⁶. 6 ~1×10 10 CFU / mL; the concentration of the probiotics in HEPES buffer is 2 × 10⁻⁶. 4 ~2×10 8 CFU / mL.

6. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 1, characterized in that, The concentration of the modified polysaccharide solution containing α,β-unsaturated esters is 0.5–10 mg / mL; the shaking speed is 500–1000 rpm; and the incubation time is 30–60 min.

7. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 1, characterized in that, The preparation method of the polysaccharide-encapsulated nano-coated probiotic suspension includes: The covalently coupled encapsulated solution was centrifuged at 4000 rpm / min, washed twice with physiological saline, and then resuspended in physiological saline.

8. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 1, characterized in that, The vortex lasts for 1 to 5 seconds.

9. The single-cell encapsulation method for covalent coupling of polysaccharides to probiotic surfaces mediated by Michael addition reaction according to claim 1, characterized in that, The preparation of the activated probiotic suspension includes: taking 0.4 mL of probiotics in 40 mL of LB medium, placing it on a shaker and culturing it overnight at 37°C and shaking at 200 rpm / min; centrifuging the bacterial suspension at 4000 rpm / min for 10 min, washing it twice with physiological saline, and resuspending it in 1 mL of physiological saline.

10. The nano-coated probiotic suspension encapsulated by the single-cell encapsulation method of Michael addition reaction-mediated covalent coupling of polysaccharides to the surface of probiotics as described in any one of claims 1-9.

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