A probiotic sustained-release microsphere for gastrointestinal and a preparation method thereof

Abstract

This invention discloses a probiotic sustained-release microsphere for the gastrointestinal tract and its preparation method, belonging to the field of microbial preparation technology. The microsphere has a multi-layered structure, consisting of, from the inside out: a core layer containing probiotics, juglone-7-O-β-D-glucuronide, and γ-polyglutamic acid; an inner wall layer composed of calcium alginate-low methoxy pectin gel; and an outer adhesion layer formed by alternating assembly of cysteine-modified chitosan and fucoidan. In preparation, a core material mixture and a composite wall material solution are first mixed to form an embedding solution, which is then electrostatically sprayed to solidify into primary gel microspheres. These microspheres are then sequentially immersed and coated with cysteine-modified chitosan solution and fucoidan solution, and dried to obtain the final product. The microspheres of this invention effectively protect probiotics from damage caused by gastric acid and a simulated digestive environment, achieving sustained-release, significantly improving the freeze-dried survival rate of probiotics, and contributing to enhanced intestinal adhesion potential and local retention capacity. They are suitable for products regulating gastrointestinal function.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a probiotic sustained-release microsphere for the gastrointestinal tract and its preparation method. Background Technology

[0002] Probiotic preparations have broad application prospects in the adjunctive treatment of gastrointestinal diseases and the regulation of intestinal microecology. However, during the entire process of preparation, storage, and oral administration to the gastrointestinal tract, probiotics are highly susceptible to multiple adverse factors such as gastric acid, bile salts, digestive enzymes, and dehydration stress, leading to a significant decrease in the number of live bacteria and thus weakening their actual efficacy. To improve the survival rate and targeted delivery efficiency of probiotics, researchers have developed various encapsulation technologies. Among them, gel microsphere systems, represented by alginate, pectin, and their complexes, have attracted widespread attention due to their good biocompatibility and controllable release characteristics.

[0003] In existing technologies, probiotic microspheres prepared using materials such as calcium alginate gel and pectin-algin composite gel can improve the gastrointestinal tolerance of bacteria to some extent, but still have many shortcomings. For example, single calcium alginate microspheres are prone to swelling and structural relaxation in simulated gastric juice, leading to gastric acid penetration and premature leakage of probiotics. While conventional alginate-pectin composite systems can improve gel density, they often experience rapid erosion or disintegration in simulated intestinal juice, making it difficult to achieve true colon-targeted sustained release. In addition, some microspheres are not adequately protected in gastric juice, release too quickly in intestinal juice, and lack the ability to actively adhere to the intestinal mucus layer, resulting in a short local residence time of the microspheres in the intestine. Probiotics are excreted before they can fully colonize, significantly limiting their practical application effectiveness.

[0004] On the other hand, current probiotic microsphere designs often focus on the physical encapsulation of the bacteria themselves, neglecting the crucial influence of the intestinal microenvironment on probiotic colonization and function. In pathological states such as inflammatory bowel disease and irritable bowel syndrome, the intestinal mucosal barrier is damaged, oxidative stress and inflammatory factor levels are elevated, creating an inflammatory microenvironment that is highly unfavorable for the survival and competitive colonization of probiotics. Furthermore, existing microsphere systems generally lack functional components that actively regulate intestinal barrier function or improve the local inflammatory environment, making it difficult to synergistically protect the bacteria and repair the microenvironment. Therefore, developing a multilayer probiotic sustained-release microsphere that combines gastric acid barrier function, intestinal segment sustained release, mucus adhesion, and microenvironment regulation has significant clinical implications and application value. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a probiotic sustained-release microsphere for the gastrointestinal tract and its preparation method.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing probiotic sustained-release microspheres for the gastrointestinal tract includes the following steps: S1. Preparation of core material mixture: Mix probiotic freeze-dried powder, jujube extract-7-O-β-D-glucuronide and γ-polyglutamic acid evenly in sterile buffer to obtain core material mixture; S2. Preparation of embedding solution: Sodium alginate and low-methoxyl pectin are dissolved in sterile water to obtain a composite wall material solution. Then, the core material mixture obtained in S1 is added to the composite wall material solution, mixed evenly, and allowed to stand to remove bubbles to obtain the embedding solution. S3. Preparation of primary gel microspheres: The embedding solution obtained in S2 is added to the curing solution by electrostatic spraying, and after static curing, separation and washing, primary gel microspheres are obtained. S4. Forming a composite adhesive layer: The primary gel microspheres obtained in S3 are sequentially soaked and coated with cysteine-modified chitosan solution and fucoidan solution. After each coating, they are separated and washed to obtain composite adhesive wet microspheres. After drying, the probiotic sustained-release microspheres for the gastrointestinal tract are obtained.

[0007] During preparation, storage, and oral administration into the gastrointestinal tract, probiotics are susceptible to decreased activity due to multiple adverse factors, including gastric acid, bile salts, digestive enzymes, and dehydration stress. While existing single or conventional composite wall materials such as alginate and pectin can improve encapsulation efficiency and gastrointestinal tolerance to some extent, they often suffer from insufficient gastric protection, rapid intestinal release, and microspheres swelling in simulated gastric juice but rapidly eroding or disintegrating in simulated intestinal juice. This leads to premature leakage of probiotics, making it difficult to simultaneously meet the requirements of gastric protection, intestinal sustained release, and local retention. On the other hand, traditional probiotic microspheres focus more on the physical encapsulation of the bacteria themselves, which can hinder the colonization and efficacy of probiotics in areas with damaged intestinal mucosal barriers and inflammatory microenvironments, thus limiting their actual gastrointestinal conditioning effects.

[0008] Preferably, the probiotic freeze-dried powder in S1 includes Lactobacillus rhamnosus freeze-dried powder and / or Dubois nystagmus freeze-dried powder; the weight ratio of succinate-7-O-β-D-glucuronide to γ-polyglutamic acid is 1:(3-12); the weight ratio of the probiotic freeze-dried powder to succinate-7-O-β-D-glucuronide is (20-100):1.

[0009] Dubois nystatin can be used to regulate the local intestinal immune tolerance and metabolic microenvironment, mitigating the adverse effects of the inflammatory microenvironment on probiotic colonization and efficacy. Lactobacillus rhamnosus enhances intestinal mucosal adhesion, increases tight junction protein expression, and improves intestinal epithelial barrier function. When combined, Dubois nystatin improves the local intestinal metabolism and immune microenvironment, while Lactobacillus rhamnosus enhances intestinal mucosal barrier stability and local bacterial colonization, thus creating a synergistic effect of immunomodulation and barrier repair in the gastrointestinal tract.

[0010] The sustained-release microspheres used in this invention have a core material composed of probiotics, γ-polyglutamic acid, and jujubenin-7-O-β-D-glucuronide. γ-polyglutamic acid forms a hydrophilic protective microenvironment around the bacteria, reducing damage caused by freeze-drying, storage, and gastric juice environments, and improving the survival rate of probiotics in acidic environments. Jujubenin-7-O-β-D-glucuronide, as a functional component co-loaded with the probiotics, participates in intestinal mucosal barrier protection and local inflammation regulation after microsphere release, thereby improving the microenvironment for subsequent colonization and efficacy of the probiotics. γ-polyglutamic acid can improve the survival of probiotics in acidic media and simulated gastric juice, while jujubenin-7-O-glucuronide can inhibit TNF-α-induced tight junction damage and reduce intestinal mucosal barrier damage.

[0011] The inventors discovered that sodium alginate, as the main spheroidizing framework material, can rapidly form an encapsulation network; after low-methoxyl pectin and sodium alginate jointly participate in calcium ion cross-linking, they can improve the structural integrity and density of the gel layer, reduce the rapid infiltration of gastric juice, and reduce the premature release of probiotics in the stomach; at the same time, this type of acidic polysaccharide composite gel has good targeted release potential and a long colonic retention time in the colonic environment, thus making it more conducive for the encapsulated probiotics to be released and exert their effects in the lower digestive tract.

[0012] Preferably, the weight ratio of sodium alginate to low-methoxyl pectin in S2 is 1:(0.5-1.2); the volume ratio of the core material mixture to the composite wall material solution is 1:(2-8); and the mass concentration of sodium alginate in the composite wall material solution is 0.5-5%.

[0013] Preferably, the electrostatic spraying conditions in S3 are as follows: nozzle inner diameter 0.1-0.8 mm, spraying voltage 5-20 kV, spraying distance 5-15 cm, and propulsion speed 10-50 mL / h; the curing liquid is an aqueous solution containing calcium ions with a mass concentration of 0.5-5%; the amount of curing liquid used is 5-20 times the volume of the embedding liquid; and the standing curing time is 10-60 min.

[0014] In addition to the composite sustained-release layer, this invention employs cysteine-modified chitosan and fucoidan to prepare a composite adhesion layer. Cysteine-modified chitosan is a thiolized chitosan; the thiol groups on its molecular chain can exchange thiol / disulfide bonds or form disulfide bonds with cysteine-rich regions in intestinal mucus glycoproteins, thereby significantly enhancing the adhesion of microspheres to the intestinal mucus layer and prolonging local residence time. Fucoidan, a negatively charged sulfated polysaccharide, can form a relatively stable polyelectrolyte composite layer with the positively charged cysteine-modified chitosan, further improving the density and structural stability of the outer interface, reducing bacterial loss during gastrointestinal transit, and providing better protection and sustained release for probiotics under simulated digestion conditions. This solves the problems of insufficient gastric protection, excessively rapid intestinal release, and short local residence time in existing probiotic preparations.

[0015] Preferably, the mass concentration of cysteine-modified chitosan in the cysteine-modified chitosan solution in S4 is 0.1-1%; the mass concentration of fucoidan in the fucoidan solution is 0.05-0.5%; the pH of the cysteine-modified chitosan solution is 4.5-6.0; the treatment time of the primary gel microspheres in the cysteine-modified chitosan solution is 5-30 min, and the treatment time in the fucoidan solution is 5-20 min.

[0016] Preferably, the cysteine-modified chitosan is prepared by a method comprising the following steps: dissolving chitosan in an acidic aqueous solution, adding L-cysteine ​​or its hydrochloride, performing an amidation reaction in the presence of a condensing agent, and then dialysis and drying the mixture to obtain cysteine-modified chitosan.

[0017] Preferably, the condensing agent comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its hydrochloride, and N-hydroxysuccinimide; the weight ratio of chitosan to L-cysteine ​​hydrochloride is 1:(0.2-2); the amidation reaction is carried out under light-protected conditions for 2-8 hours.

[0018] Preferably, the sterile buffer solution in S1 is a phosphate buffer solution with a concentration of 0.005-0.05 mol / L and a pH of 6.0-7.5; the mixing operation in S1 is carried out at 15-30℃ and a stirring speed of 100-400 rpm; the preparation temperature of the composite wall material solution in S2 is 30-40℃; and the drying in S4 is freeze drying or low-temperature vacuum drying.

[0019] Preferably, the composite adhesive wet microspheres in S4 are further coated before drying to increase the number of cysteine-modified chitosan layers and fucoidan layers.

[0020] The probiotic sustained-release microspheres for the gastrointestinal tract prepared by the method described above have a multi-layer structure, comprising, from the inside out: a core layer containing probiotics, jujubein-7-O-β-D-glucuronide and γ-polyglutamic acid; an inner wall layer composed of calcium alginate-low methoxy pectin gel; and an outer adhesion layer formed by alternating assembly of cysteine-modified chitosan and fucoidan.

[0021] The beneficial effects of this invention are: 1. This invention provides a probiotic sustained-release microsphere for the gastrointestinal tract and its preparation method. By using *Duboscirella nystatin* and *Lactobacillus rhamnosus* as the core material of a compound probiotic, and further introducing γ-polyglutamic acid and juglone-7-O-β-D-glucuronide into the core material, the survival stability of the probiotics during microsphere preparation and freeze-drying can be significantly improved. γ-polyglutamic acid helps to form a hydrophilic protective microenvironment around the bacteria, reducing dehydration stress and freeze-drying damage, while juglone-7-O-β-D-glucuronide helps to improve the internal microenvironment of the core material and enhance system stability. After the two bacteria are combined, *Duboscirella nystatin* is more effective in regulating the local intestinal metabolism and immune microenvironment, while *Lactobacillus rhamnosus* is more effective in enhancing intestinal mucosal adhesion and barrier stability, thus forming a certain degree of functional complementarity.

[0022] 2. This invention uses sodium alginate and low-methoxyl pectin to prepare the inner wall layer of a composite gel, and cysteine-modified chitosan and fucoidan as the outer composite adhesive layer, which can simultaneously help protect the gastric segment, provide sustained release in the intestinal segment, and prolong the local residence time in the intestine. After calcium ion cross-linking, sodium alginate and low-methoxyl pectin can form a relatively dense gel network, thereby reducing the rapid inward penetration of gastric juice and reducing the premature leakage of probiotics in the stomach; the outer composite structure formed by cysteine-modified chitosan and fucoidan further improves the stability and density of the microsphere surface and enhances its adhesion to the intestinal mucus layer.

[0023] 3. This invention achieves significant advantages in maintaining viable bacteria, protecting the stomach, slow-release in the intestine, and overall stability of probiotic sustained-release microspheres through synergistic optimization of the core material composition, the proportion of the gel inner wall layer, and the outer composite coating material. It can effectively solve the problems of decreased activity, insufficient gastric protection, and excessively rapid intestinal release in existing probiotic preparations after freeze-drying. Detailed Implementation

[0024] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0025] The raw materials described in this application are partially described; all other raw materials not described are commercially available. Chitosan, CAS No.: 9012-76-4, degree of deacetylation: 90%, viscosity: 50-200 mPa·s, provided by Suzhou Jiaye Biotechnology Co., Ltd.

[0026] Dubois nystocia was purchased from the China Center for Type Culture Collection, strain number: CCTCC NO: M20252889.

[0027] Lactobacillus rhamnosus was purchased from the China General Microbiological Culture Collection Center, strain number: CGMCC No. 29103.

[0028] γ-Polyglutamic acid was purchased from Kunshan Shengan Biotechnology Co., Ltd., model number: D1UL.

[0029] Glucuronine-7-O-β-D-glucuronide was purchased from Chengdu Medtronic Technology Co., Ltd., product number: RM2267.

[0030] Low-methoxyl pectin was purchased from Hubei Bocare New Materials Co., Ltd., model: bocare-1. Example 1

[0031] A method for preparing probiotic sustained-release microspheres for the gastrointestinal tract includes the following steps: S1. Take 0.1 parts by weight of jujube extract-7-O-β-D-glucuronide, add 20 parts by weight of sterile phosphate buffer solution with a concentration of 0.01 mol / L and a pH of 6.7, and stir at 25°C and 300 rpm for 30 min; add 0.60 parts by weight of γ-polyglutamic acid, and continue stirring for 15 min to obtain the functional core material base solution; add 5.0 parts by weight of Lactobacillus rhamnosus lyophilized powder and 3.0 parts by weight of Dubois nystagmus lyophilized powder to the functional core material base solution, and stir at 20°C and 120 rpm for 10 min to obtain the core material mixture; S2. Take 2 parts by weight of sodium alginate and 1.2 parts by weight of low methoxyl pectin, add them to 80 parts by weight of sterile water, and stir for 40 minutes at 35°C and 350 rpm to obtain a composite wall material solution; add all the core material mixture obtained in S1 to the composite wall material solution, continue stirring at 25°C and 200 rpm for 10 minutes, and let stand for 15 minutes to remove bubbles to obtain an embedding solution; S3. The embedding solution obtained in S2 is loaded into an electrostatic spraying device and added dropwise to a 2.5 wt% calcium chloride aqueous solution through a 0.25 mm inner diameter nozzle under the conditions of a spraying voltage of 10 kV, a spraying distance of 8 cm, and a propulsion speed of 25 mL / h. The solution is allowed to stand and solidify for 20 min. The microspheres are collected by filtration and washed with sterile water to obtain primary gel microspheres. The amount of calcium chloride aqueous solution used is 10 times the total volume of the embedding solution. S4. Add 0.35 parts by weight of cysteine-modified chitosan to 100 parts by weight of 0.5 wt% acetate-sodium acetate buffer solution, adjust the pH to 5.2, and stir at room temperature until completely dissolved to obtain a cysteine-modified chitosan coating solution; separately take 0.15 parts by weight of fucoidan, add 100 parts by weight of sterile water and stir to dissolve to obtain a fucoidan solution; add the primary gel microspheres obtained in S3 to the cysteine-modified chitosan coating solution, and gently stir at 25℃ and 80 rpm for 10 min to form a first cationic adhesion layer on the surface of the microspheres; after filtration, wash with sterile water, and then transfer to the fucoidan solution, continue to process at 25℃ and 80 rpm for 8 min, filter, and wash with sterile water again to obtain composite adhesive wet microspheres, freeze-dry, and obtain probiotic sustained-release microspheres for gastrointestinal use.

[0032] The preparation method of the cysteine-modified chitosan powder is as follows: Take 2 parts by weight of chitosan and add it to 180 parts by weight of 1.5 wt% acetic acid aqueous solution. Stir at 25°C and 400 rpm for 40 minutes to obtain a chitosan solution. Add 1.5 parts by weight of L-cysteine ​​hydrochloride to the chitosan solution and adjust the pH to 5.0. Then add 1.2 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.8 parts by weight of N-hydroxysuccinimide. React at room temperature in the dark for 4 hours. After the reaction is completed, dialyze. Freeze dry to obtain cysteine-modified chitosan powder. Example 2

[0033] It is basically the same as Example 1, except that: no Lactobacillus rhamnosus lyophilized powder was added in S1, only 8.0 parts by weight of Dubois nystagmus lyophilized powder was added. Example 3

[0034] It is basically the same as Example 1, except that: no lyophilized Dubois nystomatitis powder was added in S1, only 8.0 parts by weight of Lactobacillus rhamnosus lyophilized powder was added. Example 4

[0035] It is basically the same as Example 1, except that the ratio of sodium alginate and low-methoxyl pectin in S1 is 1:1. Comparative Example 1

[0036] It is basically the same as Example 1, except that the cysteine-modified chitosan in S4 is replaced with chitosan. Comparative Example 2

[0037] It is basically the same as Example 1, except that: in S1, jujube-7-O-β-D-glucuronide was not added, but replaced with an equal amount of sterile phosphate. Comparative Example 3

[0038] It is basically the same as Example 1, except that: γ-polyglutamic acid is not added in S1, but is replaced by an equal amount of sterile phosphate. Comparative Example 4

[0039] It is basically the same as Example 1, except that the same amount of Lactobacillus rhamnosus lyophilized powder and Dubois nystagmus lyophilized powder are directly mixed and do not have a microsphere structure. Test Example 1

[0040] Methods for testing encapsulation efficiency and viability retention: 1.00 g of wet microspheres from the examples and comparative examples were added to 10 mL of 0.10 mol / L sodium citrate solution and vortexed for 5 min to allow the microspheres to unencapsulate and release bacteria. After serial dilution, plate counting was performed using MRS solid medium to determine the number of viable bacteria after encapsulation. The plate counts were performed under anaerobic conditions at 37℃ for 48 h, and the resulting number of viable bacteria was the total number of viable bacteria. For Comparative Example 4, since no microsphere structure was formed, encapsulation efficiency was not measured; only the number of viable bacteria before and after freeze-drying and the viability retention rate after freeze-drying were determined.

[0041] Samples were taken before and after freeze-drying, and the cells were unpacked and counted according to the above method. The viable cell retention rate after freeze-drying was calculated.

[0042] The calculation formula is as follows: Encapsulation rate (%) = Actual number of viable bacteria in the encapsulated microspheres / Total number of viable bacteria in the feed × 100%; Viable cell retention rate after freeze-drying (%) = (Number of viable cells after freeze-drying / Number of viable cells before freeze-drying) × 100%; Each group was repeated in triplicate, and the results are expressed as mean ± standard deviation. The results are shown in Table 1.

[0043] Table 1 Results of encapsulation efficiency and viability retention tests

[0044] Test Example 2 Test methods: (1) Simulated gastric juice (SGF) Artificial gastric juice formula: Each liter contains 3.2g of pepsin and 2.0g of NaCl, adjusted to pH 1.2 with hydrochloric acid, and sterilized through a 0.22μm filter membrane.

[0045] (2) Simulated intestinal fluid (SIF) Artificial intestinal fluid formula: Each liter contains 10.0g of trypsin and 6.8g of potassium dihydrogen phosphate, with pH adjusted to 6.8 using NaOH, and sterilized by passing through a 0.22μm filter membrane.

[0046] (3) Gastrointestinal tolerance test Take 1.00 g of each group of lyophilized microspheres, and for Comparative Example 4, take a mixture of free bacteria with the same total viable count. Add the mixture to 20 mL of simulated gastric fluid and shake at 37 °C and 100 rpm for 2 h. After treatment, immediately adjust the pH of the system to 6.8-7.0 with sterile NaHCO3 solution to stop the gastric fluid from continuing to act. Then centrifuge, take a sample, add 10 mL of 0.10 mol / L sodium citrate solution to uncapsulate for 5 min and count. For Comparative Example 4, count directly and calculate the survival rate of the simulated gastric fluid after 2 h.

[0047] Another sample treated with gastric juice was centrifuged and transferred to 20 mL of simulated intestinal juice. The sample was then shaken at 37°C and 100 rpm for 4 hours. The samples were counted again, and the overall survival rate after 2 hours in gastric juice and 4 hours in intestinal juice was calculated.

[0048] (4) Sustained-release test Take 1.00g of each group of lyophilized microspheres and treat them in 20mL of simulated gastric fluid at 37℃ and 100rpm for 2h. Measure the number of viable bacteria released in the gastric fluid stage. Then transfer the remaining sample to 20mL of simulated intestinal fluid for further treatment. Take samples and count them at 1h, 2h, 4h, and 8h. Count the bacteria after decapsulation with 0.10mol / L sodium citrate and calculate the cumulative release rate.

[0049] The calculation formula is as follows: Survival rate (%) = (Number of viable bacteria after treatment / Number of viable bacteria before treatment) × 100%; Cumulative release rate (%) = (Cumulative number of viable bacteria released in the medium / Initial total number of viable bacteria in the microspheres) × 100%; Each group was repeated in triplicate, and the results are expressed as mean ± standard deviation. The results are shown in Table 2.

[0050] Table 2 Results of simulated gastrointestinal tolerance and sustained-release performance

[0051] The above results show that the probiotic sustained-release microspheres prepared by this invention for the gastrointestinal tract exhibit good effects in terms of encapsulation rate, viable bacterial retention rate after freeze-drying, survival rate after 2 hours in simulated gastric juice, overall survival rate after 4 hours in intestinal juice following 2 hours in gastric juice, and low release during the gastric juice stage and continuous release during the intestinal juice stage. Examples 2 and 3 used either a single *Duboscirella nystatus* or a single *Lactobacillus rhamnosus*, respectively, and their test results were slightly lower than those of Example 1, indicating that the microsphere structure of this invention has good protective effects against both single and dual bacteria. Example 1, however, is superior to Examples 2 and 3, indicating that the dual-bacterial combination can further improve the overall system's stability and gastrointestinal tolerance. This is because *Duboscirella nystatus* is more inclined to improve the local intestinal metabolism and immune microenvironment, while *Lactobacillus rhamnosus* is more inclined to enhance adhesion and barrier stability. The combination of the two is more conducive to forming a functionally complementary synergy within the microspheres, thus exhibiting better effects under freeze-drying protection and gastrointestinal stress conditions. Example 4 only changed the ratio of sodium alginate to low-methoxyl pectin from 2:1.2 in Example 1 to 1:1. After this adjustment, the encapsulation efficiency, viable bacteria retention rate after freeze-drying, and protective effect in simulated gastric juice were all lower than in Example 1. At the same time, the cumulative release rate in the gastric juice stage slightly increased. This indicates that although a 1:1 ratio can still form a usable composite gel layer, its density and cross-linking stability are not as good as the ratio used in Example 1. This shows that changes in the ratio of sodium alginate to low-methoxyl pectin affect the structural density and media permeability of the calcium cross-linking network, thereby affecting the gastric protective ability and intestinal release process of probiotics. The ratio in Example 1 is more conducive to balancing low gastric release and gradual intestinal release.

[0052] In Comparative Example 1, replacing cysteine-modified chitosan with ordinary chitosan significantly reduced the encapsulation efficiency, viable bacteria retention rate after freeze-drying, and survival rate in simulated gastric juice, while significantly increasing the cumulative release rate during the simulated gastric juice stage. This indicates that cysteine-modified chitosan is not a common coating material in this invention, but rather enhances the composite density between the outer layer and fucoidan through its thiolized structure, thereby improving the structural stability of the microspheres in the gastric juice environment. Replacing it with ordinary chitosan weakens the stability of the outer composite structure, making it easier for gastric juice to penetrate inwards. This reduces the protective effect of the microspheres on probiotics, resulting in higher premature release and lower viable bacteria retention rate.

[0053] In Comparative Example 2, after removing guarin-7-O-β-D-glucuronide, the encapsulation efficiency did not change significantly compared to Example 1. However, the viable bacteria retention rate, simulated gastric juice survival rate, and overall survival rate all decreased after freeze-drying. This indicates that although guarin-7-O-β-D-glucuronide is not the core wall material component that determines microsphere formation and main acid resistance protection, as a functional core material component, it can improve the internal environment and system stability of the core material, thereby playing an auxiliary role in promoting the survival of probiotics under freeze-drying and gastrointestinal stress conditions. In Comparative Example 3, the removal of γ-polyglutamic acid significantly reduced both the encapsulation rate and the viable cell retention rate after freeze-drying. The survival rate after 2 hours in simulated gastric fluid and the overall survival rate after 4 hours in intestinal fluid were also significantly lower. This is because γ-polyglutamic acid has strong hydrophilicity and water-retention capacity, forming a protective microenvironment around the bacteria, reducing dehydration stress during freeze-drying and acidic damage in the gastric fluid environment. When it is removed, the buffering and protective effect inside the core material weakens, making the bacteria more susceptible to inactivation under freeze-drying and acidic conditions, thus exhibiting a significantly reduced viable cell retention rate and gastrointestinal tolerance. Comparative Example 4, an unencapsulated mixture of free bacteria, showed the lowest viable cell retention rate after freeze-drying. Its survival rate after 2 hours in simulated gastric fluid and the overall survival rate were also far lower than those of the examples and other comparative examples, indicating that direct mixing of probiotics alone cannot resist adverse environments such as freeze-drying, gastric fluid, and intestinal fluid.

Claims

1. A method for preparing probiotic sustained-release microspheres for the gastrointestinal tract, characterized in that, Includes the following steps: S1. Preparation of core material mixture: Mix probiotic freeze-dried powder, jujube extract-7-O-β-D-glucuronide and γ-polyglutamic acid evenly in sterile buffer to obtain core material mixture; S2. Preparation of embedding solution: Sodium alginate and low-methoxyl pectin are dissolved in sterile water to obtain a composite wall material solution. Then, the core material mixture obtained in S1 is added to the composite wall material solution, mixed evenly, and allowed to stand to remove bubbles to obtain the embedding solution. S3. Preparation of primary gel microspheres: The embedding solution obtained in S2 is added to the curing solution by electrostatic spraying, and after static curing, separation and washing, primary gel microspheres are obtained. S4. Forming a composite adhesive layer: The primary gel microspheres obtained in S3 are sequentially soaked and coated with cysteine-modified chitosan solution and fucoidan solution. After each coating, they are separated and washed to obtain composite adhesive wet microspheres. After drying, the probiotic sustained-release microspheres for the gastrointestinal tract are obtained.

2. The preparation method according to claim 1, characterized in that, The probiotic freeze-dried powder in S1 includes Lactobacillus rhamnosus freeze-dried powder and / or Dubois nystagmus freeze-dried powder; the weight ratio of succinin-7-O-β-D-glucuronide to γ-polyglutamic acid is 1:(3-12); the weight ratio of the probiotic freeze-dried powder to succinin-7-O-β-D-glucuronide is (20-100):

1.

3. The preparation method according to claim 1, characterized in that, The weight ratio of sodium alginate to low-methoxyl pectin in S2 is 1:(0.5-1.2); the volume ratio of the core material mixture to the composite wall material solution is 1:(2-8); and the mass concentration of sodium alginate in the composite wall material solution is 0.5-5%.

4. The preparation method according to claim 1, characterized in that, The electrostatic spraying conditions in S3 are as follows: nozzle inner diameter 0.1-0.8 mm, spraying voltage 5-20 kV, spraying distance 5-15 cm, and propulsion speed 10-50 mL / h; the curing solution is an aqueous solution containing calcium ions with a mass concentration of 0.5-5%; the amount of curing solution used is 5-20 times the volume of the embedding solution; and the standing curing time is 10-60 min.

5. The preparation method according to claim 1, characterized in that, The mass concentration of cysteine-modified chitosan in the cysteine-modified chitosan solution in S4 is 0.1-1%; the mass concentration of fucoidan in the fucoidan solution is 0.05-0.5%; the pH of the cysteine-modified chitosan solution is 4.5-6.0; the treatment time of the primary gel microspheres in the cysteine-modified chitosan solution is 5-30 min, and the treatment time in the fucoidan solution is 5-20 min.

6. The preparation method according to claim 1, characterized in that, The cysteine-modified chitosan is prepared by a method comprising the following steps: dissolving chitosan in an acidic aqueous solution, adding L-cysteine ​​or its hydrochloride, performing an amidation reaction in the presence of a condensing agent, and then dialysis and drying the mixture to obtain cysteine-modified chitosan.

7. The preparation method according to claim 6, characterized in that, The condensing agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its hydrochloride, and N-hydroxysuccinimide; the weight ratio of chitosan to L-cysteine ​​hydrochloride is 1:(0.2-2); the amidation reaction is carried out under light-protected conditions for 2-8 hours.

8. The preparation method according to claim 1, characterized in that, The sterile buffer solution in S1 is a phosphate buffer solution with a concentration of 0.005-0.05 mol / L and a pH of 6.0-7.5; the mixing operation in S1 is carried out at 15-30℃ and a stirring speed of 100-400 rpm; the preparation temperature of the composite wall material solution in S2 is 30-40℃; the drying in S4 is freeze drying or low-temperature vacuum drying.

9. The preparation method according to claim 1, characterized in that, The composite adhesive wet microspheres in S4 are further coated before drying to increase the number of cysteine-modified chitosan and fucoidan layers.

10. The probiotic sustained-release microspheres for the gastrointestinal tract prepared by the method according to any one of claims 1-9, characterized in that, The microspheres have a multi-layered structure, comprising, from the inside out: a core layer containing probiotics, jujube-7-O-β-D-glucuronide and γ-polyglutamic acid; an inner wall layer composed of calcium alginate-low methoxy pectin gel; and an outer adhesion layer formed by alternating assembly of cysteine-modified chitosan and fucoidan.

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