Microspheres entrapped with probiotics and ginsenoside as well as preparation method and application of microspheres

Abstract

The invention discloses a microsphere entrapped with probiotics and ginsenoside as well as a preparation method and application of the microsphere, and aims to realize efficient treatment of flora imbalance inflammatory bowel disease (IBD). The microspheres adopt a bi-component synergistic entrapment technology, can stably bear probiotics and ginsenoside, effectively avoid inactivation or degradation of the probiotics and ginsenoside in gastric acid and cholate environments, and ensure directional release and efficient utilization of the probiotics and ginsenoside in intestinal tracts. The microspheres not only provide a protection effect as a delivery carrier, but also construct a'microreactor 'system, and provide a suitable microenvironment for survival of probiotics and conversion of ginsenoside. Probiotics secrete a specific enzyme to promote prototype ginsenoside to be converted into rare ginsenoside with higher activity and stronger solubility while regulating intestinal flora balance and relieving intestinal inflammation, so that the bioavailability and efficacy of the rare ginsenoside are remarkably improved. According to the microsphere system, the technical bottlenecks of the existing treatment means in the aspects of activity maintenance, directional release and conversion utilization are broken through by realizing the synergistic effect of the probiotics and the ginsenoside, and a brand-new strategy and a technical platform are provided for treatment of flora imbalance related diseases such as IBD.

Description

Technical Field

[0001] This invention relates to the field of biomaterial preparation technology, and in particular to a microsphere loaded with probiotics and ginsenosides, its preparation method and application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic inflammatory bowel diseases, primarily including ulcerative colitis and Crohn's disease. Its pathogenesis is complex, involving genetic, immune, environmental factors, and gut microbiota imbalance. Clinical manifestations include abdominal pain, diarrhea, weight loss, and bloody stools; severe cases may lead to intestinal stricture, perforation, or cancer. Existing treatments (such as immunosuppressants and biologics) have unstable efficacy, significant side effects, and are difficult to control or cure long-term. Current research indicates that gut microbiota imbalance is considered a key pathogenic factor for IBD. In IBD patients, the gut microbiota shows a decrease in beneficial bacteria and an increase in harmful bacteria, leading to abnormal immune responses and exacerbating inflammation. Therefore, restoring gut microbiota balance has become an important research direction in IBD treatment.

[0003] Probiotics, as beneficial live microorganisms, can regulate the balance of intestinal flora, modulate immune responses, treat inflammatory bowel disease, alleviate microbe-associated diarrhea, and even have potential effects on the prevention and treatment of colon cancer. However, the stable delivery and maintenance of high activity of probiotics in the intestine remain key issues for their effectiveness. While traditional powder formulations and capsule delivery methods can protect probiotics to some extent, their survival rate and activity are significantly reduced under the influence of highly acidic gastric juices and bile salts, limiting their therapeutic effects. Furthermore, single-component probiotics also suffer from long onset times and limited therapeutic efficacy. Therefore, effectively protecting probiotics and ensuring their smooth release and colonization in the intestine has become a crucial challenge in developing highly active probiotic delivery systems.

[0004] Ginseng, a traditional Chinese medicine with a long history, possesses a wide range of pharmacological effects, including antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective functions. The main active components of ginseng are ginsenosides, primarily including native ginsenosides (R1, Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, Rd, Rg1, etc.) and rare ginsenosides (Rg3, Rh2, Rh3, Rk1, Rk2, aPPT, and aPPD, etc.). These saponins exhibit significant anti-inflammatory, antioxidant, hypoglycemic, and antitumor biological activities, and show particular potential in regulating intestinal microecology and treating inflammatory bowel diseases. However, the large molecular structure and low water solubility of native ginsenosides limit their bioavailability, resulting in low absorption efficiency in vivo and hindering optimal therapeutic effects. The conversion process of ginsenosides—that is, transforming native ginsenosides into rare ginsenosides with smaller molecular weights, higher solubility, and greater activity—is crucial for improving their bioactivity and bioavailability. Studies have shown that the rich gut microbiota can convert ginsenosides through enzymatic hydrolysis, but the efficiency of this process is affected by the type and quantity of bacteria, and the conversion of ginsenosides usually requires specific enzyme systems and gut environment. Furthermore, existing oral formulations cannot ensure their targeted delivery, conversion, and absorption in the gut.

[0005] In recent years, microspheres have attracted widespread attention as an advanced drug delivery carrier. By adjusting their size, surface properties, and the selection of encapsulated components, microspheres can provide effective drug release control. Microsphere delivery systems can not only effectively protect the encapsulated active ingredients (such as probiotics and ginsenosides) from degradation in the acidic environment of the stomach, but also precisely release them in the intestine and improve drug bioavailability. Microspheres can enhance the efficacy of drugs through targeted delivery, while delaying drug release to achieve sustained-release and timed-release effects, making them particularly suitable for intestinal active drug delivery. Summary of the Invention

[0006] This invention relates to microspheres encapsulating probiotics and ginsenosides, their preparation method, and applications, particularly suitable for treating inflammatory bowel disease with gut microbiota imbalance. The microspheres of this invention can simultaneously encapsulate probiotics and ginsenosides, achieving stable, targeted delivery and active release of both in the intestine. As a "microreactor," the microspheres not only protect probiotics from damage by the gastrointestinal environment but also promote the conversion of ginsenosides by probiotics by providing a suitable reaction environment. Therefore, this microreaction system can regulate the intestinal microecological environment through probiotics and improve the conversion and utilization efficiency of ginsenosides through their enzymatic action. The synergistic effect of both significantly improves the therapeutic effect of inflammatory bowel disease with gut microbiota imbalance.

[0007] A microsphere encapsulating probiotics and ginsenosides, wherein the wall material of the microsphere is alginate or a composite system of alginate and one or more polymeric materials, to achieve synergistic protection, targeted release, and intestinal-targeted therapeutic effects of probiotics and ginsenosides. The wall material may be combined with EDTA-Ca... 2+ Ba 2+ Ionic crosslinking agents further enhance the structural stability and functional diversity of microspheres, with the interior of the microspheres containing probiotic liquid and prototype ginsenosides.

[0008] Preferably, the alginate has a molecular weight of 50-200 kDa and a concentration range of 8%-12%; the polymeric material includes chitosan, gelatin, ginseng polysaccharide, resistant starch, pectin, guar gum, hydroxypropyl methylcellulose (HPMC) and its derivatives. Preferably, the wall material of the microspheres is alginate-EDTA-Ca 2+ .

[0009] Preferably, the concentration of the probiotics is 1×10⁻⁶. 6 -1×10 9 CFU / mL.

[0010] Preferably, the concentration of the original ginsenoside is 10-20 g / L.

[0011] Preferably, the microspheres have a particle size of 100-200 micrometers.

[0012] Preferably, the probiotics are Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium, or Lactobacillus fermentum.

[0013] A method for preparing microspheres loaded with probiotics and ginsenosides, comprising the following steps: Step 1: First, weigh 20-22 wt.% CaCl2 and 36-38 wt.% EDTA, and adjust the pH to 7.5 to prepare CaCl2. 2+ -EDTA solution, then mixed with an equal volume of 40 wt. % alginate to obtain sodium alginate (alginate-EDTA-Ca 2+ ) solution; Step 2: In the alginate-EDTA-Ca 2+ Aqueous solution was obtained by adding probiotic bacterial solution washed with PBS and proto-ginsenosides to the solution. Step 3: Weigh 0.5 ml of glacial acetic acid and dissolve it in 500 ml of microdroplet solution to generate oil and obtain an oil phase solution; Step 4: Start the microfluidic device, place the aqueous solution in the second channel and the oil solution in the first channel, connect and adjust the device to begin preparing microspheres; wherein, the flow rates of the aqueous solution and the oil solution are 15 μL / min and 20 μL / min, respectively; Step 5: After preparing probiotic microspheres of uniform size, collect the solidified microspheres using centrifuge tubes to obtain alginate hydrogel microspheres containing probiotics. Step 6: After collection, wash with PBS to obtain dispersed microspheres of the probiotic and ginsenoside microreaction system for treating dysbiosis-related inflammatory bowel disease.

[0014] Step 7: Take 10g of 20wt% alginate hydrogel solution and resuspend the prepared probiotic and ginsenoside microspheres for treating dysbiosis-induced inflammatory bowel disease in the hydrogel.

[0015] Application of microspheres containing probiotics and ginsenosides in the treatment of inflammatory bowel disease with dysbiosis.

[0016] The beneficial effects of this invention are: This invention utilizes a microfluidic device to prepare sodium alginate microspheres encapsulating probiotics and ginsenosides for oral delivery. This effectively protects the probiotics and ensures successful delivery and colonization of the intestines. The polymeric microspheres can undergo cross-linking reactions in low-pH gastric juice and bile salts, allowing the Ca2+ in the sodium alginate solution to... 2+ The microspheres chelate with EDTA to form a stable gel structure, thus tightly encapsulating probiotics and preventing their inactivation by gastric acid and bile salts, thereby improving the survival rate and encapsulation efficiency of probiotics. These microspheres not only protect probiotics from gastric acid but also ensure stable release of probiotics in the intestines, allowing them to function in regulating the gut microbiota.

[0017] In the intestinal environment, sodium alginate microspheres release ginsenosides and probiotics through dissolution. The original ginsenosides are converted into rarer ginsenosides (such as Rg3 and Rh2) by enzymes secreted by the probiotics. These rarer ginsenosides have significant anti-inflammatory, antioxidant, and immunomodulatory effects, helping to eliminate harmful bacteria in the intestines and reduce intestinal inflammation. Simultaneously, the probiotics in the microspheres colonize the intestines, regulating the gut microbiota and enhancing the intestinal barrier function, thereby further promoting intestinal health.

[0018] Through this microsphere encapsulation system, the present invention can not only inhibit the growth of harmful bacteria in the intestine, but also effectively achieve the colonization of probiotics in the intestine, synergistically enhance the anti-inflammatory effect of ginsenosides, restore and maintain the balance of intestinal flora, thereby effectively improving and treating inflammatory bowel disease caused by flora imbalance, and has significant therapeutic effects. Attached Figure Description

[0019] Figure 1 Data graph of the Microsphere prepared in Example 1 of this invention alleviating the inflammatory response caused by DSS; Figure 2 H&E tissue staining and histological scoring data of Example 1 of the present invention; Figure 3 The TUNEL staining results of Example 1 of the present invention are shown in the figure. Detailed Implementation

[0020] A microsphere encapsulating probiotics and ginsenosides, wherein the wall material of the microsphere is alginate or a composite system of alginate and one or more polymeric materials, to achieve synergistic protection, targeted release, and intestinal-targeted therapeutic effects of probiotics and ginsenosides. The wall material may be combined with EDTA-Ca... 2+ Ba 2+ Ionic crosslinking agents further enhance the structural stability and functional diversity of microspheres, with the interior of the microspheres containing probiotic liquid and prototype ginsenosides.

[0021] Preferably, the alginate has a molecular weight of 50-200 kDa and a concentration range of 8%-12%; the polymeric material includes chitosan, gelatin, ginseng polysaccharide, resistant starch, pectin, guar gum, hydroxypropyl methylcellulose (HPMC) and its derivatives. Preferably, the wall material of the microspheres is alginate-EDTA-Ca 2+ .

[0022] Preferably, the concentration of the probiotics is 1×10⁻⁶. 6 -1×10 9 CFU / mL.

[0023] Preferably, the concentration of the original ginsenoside is 10-20 g / L.

[0024] Preferably, the microspheres have a particle size of 100-200 micrometers.

[0025] Preferably, the probiotics are Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium, or Lactobacillus fermentum.

[0026] A method for preparing microspheres loaded with probiotics and ginsenosides, comprising the following steps: Step 1: First, weigh 20-22 wt.% CaCl2 and 36-38 wt.% EDTA, and adjust the pH to 7.5 to prepare CaCl2. 2+ -EDTA solution, then mixed with an equal volume of 40 wt.% alginate, to obtain alginate-EDTA-Ca 2+ liquid; Step 2: In the alginate-EDTA-Ca 2+ Aqueous solution was obtained by adding probiotic bacterial solution washed with PBS and proto-ginsenosides to the solution. Step 3: Weigh 0.5 ml of glacial acetic acid and dissolve it in 500 ml of microdroplet solution to generate oil and obtain an oil phase solution; Step 4: Start the microfluidic device, place the aqueous solution in the second channel and the oil solution in the first channel, connect and adjust the device to begin preparing microspheres; wherein, the flow rates of the aqueous solution and the oil solution are 15 μL / min and 20 μL / min, respectively; Step 5: After preparing probiotic microspheres of uniform size, collect the solidified microspheres using centrifuge tubes to obtain alginate hydrogel microspheres containing probiotics. Step 6: After collection, wash with PBS to obtain dispersed microspheres of the probiotic and ginsenoside microreaction system for treating dysbiosis-related inflammatory bowel disease.

[0027] Step 7: Take 10g of 20wt% alginate hydrogel solution and resuspend the prepared probiotic and ginsenoside microspheres for treating dysbiosis-induced inflammatory bowel disease in the hydrogel.

[0028] Application of microspheres containing probiotics and ginsenosides in the treatment of inflammatory bowel disease with dysbiosis.

[0029] The technical solutions adopted in this invention will be further explained and described below with reference to specific embodiments.

[0030] Example 1: A method for preparing microspheres loaded with probiotics and ginsenosides, the specific steps are as follows: Step 1: Weigh 8g CaCl2 powder, 3g sodium alginate powder and 5g EDTA and dissolve them in 50ml ultrapure water. Stir thoroughly and mix evenly to obtain the aqueous phase of sodium alginate microspheres without bacterial solution. Step 2: Add PBS-washed, anti-inflammatory *Lactobacillus plantarum* and prototype ginsenoside Rg1 to the aqueous phase of sodium alginate microspheres obtained in Step 1 (without bacterial culture) to prepare a sodium alginate solution containing ginsenoside and *Lactobacillus plantarum*, wherein the concentration of *Lactobacillus plantarum* is 1 × 10⁻⁶. 7 -1×10 9 CFU / mL, the concentration of the original ginsenosides is 10-20g / L; Step 3: Weigh 0.5 ml of glacial acetic acid and dissolve it in 500 ml of micro-droplet oil. Shake well to obtain an oil phase solution.

[0031] Step 4: Add 5 mL of oil phase solution and 1 mL of prepared aqueous phase solution to the 5 mL oil phase reservoir (channel 1) and the 1.5 mL aqueous phase reservoir (channel 2) of the microfluidic device, respectively. Prepare microspheres by using the flow rates of the aqueous phase solution and the oil phase solution at 15 μL / min and 20 μL / min, respectively. The aqueous phase solution is the sodium alginate solution containing ginsenosides and Lactobacillus plantarum obtained in Step 2. Step 5: Collect the uniformly sized microspheres prepared in a 5ml centrifuge tube, and take out the oil generated from the bottom droplets of the 5ml centrifuge tube; Step 6: Add twice the volume of demulsifier (Drop-Surf) to the microspheres and shake to demulsify; centrifuge at 2500 rpm for 1 min and remove the bottom demulsifier; repeat this step once.

[0032] Step 7: Finally, solidified sodium alginate microspheres are obtained and dispersed in PBS buffer; the resulting sodium alginate microspheres are probiotic microspheres used to treat inflammatory bowel disease with dysbiosis.

[0033] Example 2: The steps in this example are the same as in Example 1, except that: Step 2: Add PBS-washed Lactobacillus rhamnosus bacterial solution and prototype ginsenoside Rg1 to the aqueous phase of sodium alginate microspheres obtained in Step 1 (without bacterial solution) to prepare a sodium alginate solution containing ginsenoside and Lactobacillus rhamnosus, wherein the concentration of Lactobacillus rhamnosus is 1×10⁻⁶. 6 -1×10 9 CFU / g(mL), the concentration of the original ginsenoside is 10-20g / L; Lactobacillus rhamnoides is better able to regulate the intestinal flora than Lactobacillus plantarum, forming a biological barrier on the intestinal mucosa and enhancing the host's intestinal mucosal barrier capacity. It can also attach to the host's intestinal epithelial cells and achieve a balanced state by regulating the structure and function of the microecological community in the host's intestine, thereby improving the function of the host's digestive system. Furthermore, it has outstanding performance in terms of resistance to gastric acid and bile, and can enter the human intestine alive, while Lactobacillus plantarum dies before entering the intestine due to the action of gastric acid and bile without being encapsulated.

[0034] Example 3: The steps in this example are the same as in Example 1, except that: Step 3: Unlike Example 1, this example uses a 1% succinic acid solution as the oil phase solution, which is dissolved and shaken evenly in 500ml of microdroplet-generated oil.

[0035] Succinic acid was washed away using the same demulsification method to obtain probiotic microspheres. Succinic acid mainly plays the same role as acetic acid, that is, it releases calcium ions through acid decomposition and induces the cross-linking and formation of sodium alginate microspheres. At the same time, succinic acid also has good biocompatibility and has little impact on the activity of probiotics.

[0036] Example 4: The steps in this example are the same as in Example 1, except that: Step 1: Unlike Example 1, in this example, 8g of CaCl2 powder, 1.5g of sodium alginate, 1.5g of chitosan and 5g of EDTA are weighed and dissolved in 50ml of ultrapure water, and thoroughly stirred to obtain a chitosan (or inulin) solution.

[0037] In this embodiment, a natural polymer (chitosan) was used to replace part of the sodium alginate. Chitosan, as a natural biopolymer, possesses excellent biodegradability and biocompatibility. Compared to sodium alginate crosslinking alone, the composite crosslinking system with added chitosan may exhibit higher mechanical strength and better structural stability. Especially when used in vivo, the addition of chitosan helps improve the stability of microspheres or capsules in the intestinal environment.

[0038] Example 5: Preparation method of microspheres using sodium alginate-chitosan composite system as wall material To further enhance the gastric acid tolerance, structural stability, and protective effect of the microspheres on probiotics, this embodiment improves the wall material based on Example 1 by combining sodium alginate with chitosan to construct functional microspheres with a multi-layered protective structure.

[0039] Step 1: Weigh 3g of sodium alginate and 1.5g of low molecular weight chitosan (degree of deacetylation ≥85%) into 50mL of ultrapure water, add 2g of CaCl2 and 4g of disodium ethylenediaminetetraacetate (EDTA-Na2), stir at room temperature for 2 hours to fully dissolve and form a homogeneous mixed solution to obtain the aqueous phase system of the composite wall material.

[0040] Step 2: Add *Lactiplantibacillus plantarum* (treated with PBS) and prototype ginsenoside Rg1 to the aqueous phase of the composite wall material obtained in Step 1, stir until homogeneous, and the final bacterial concentration is 1×10⁻⁶. 7 ~ 1×10 9 CFU / mL, ginsenoside concentration is 10 ~ 20 g / L.

[0041] Step 3: Prepare the oil phase using the same method as in Example 1, that is, add 0.5 mL of glacial acetic acid to 500 mL of microdroplet-generated oil, shake to mix, and obtain the oil phase.

[0042] Step 4: Add the aqueous solution of the composite wall material obtained in Step 2 and the oil phase obtained in Step 3 to channels 2 and 1 of the microfluidic device, respectively. Set the flow rate to 15 μL / min for the aqueous phase and 20 μL / min for the oil phase to form microspheres.

[0043] Step 5: Collect the formed uniform microspheres using a 5mL centrifuge tube, discard the bottom oil phase, and set aside.

[0044] Step 6: Add an equal volume of demulsifier Drop-Surf, shake to mix, and centrifuge at 2500 rpm for 1 min to remove the lower demulsified phase; repeat this step once to completely remove any oil phase residue.

[0045] Step 7: Resuspend the obtained composite wall material microspheres in PBS buffer to obtain a probiotic / ginsenoside microsphere preparation with sodium alginate-chitosan double-layer coating, and the particle size is controlled in the range of 50~200 μm.

[0046] Example 6: The steps in this example are the same as in Example 1, except that: Step 2: Unlike Example 1, in this example, Lactobacillus plantarum, Bacillus lactis, and Lactobacillus rhamnosus bacterial suspensions washed with PBS were added to the sodium alginate solution, and 10 g / L of the original ginsenosides Rg1, Rb1, and Rg3 (in a 1:1:1 ratio) were added at the same time to obtain an aqueous solution.

[0047] This embodiment combines multiple ginsenosides and different probiotics. The combined use of various probiotic strains can complement each other in terms of enzyme systems and metabolic functions. This complementarity can improve the intestinal environment through the complementary use of multiple metabolites (such as short-chain fatty acids), inhibit the growth of harmful bacteria, and enhance the intestinal immune barrier. Furthermore, various probiotics, by secreting different enzymes such as β-glucosidase, lactase, and protease, collectively promote the more efficient conversion of ginsenosides, especially into rare saponins such as Rg3, Rh2, and CK, thereby improving the bioavailability and biological functionality of ginsenosides. Rare saponins generally possess stronger biological activities, including enhanced anti-inflammatory, antioxidant, and immunomodulatory functions. Therefore, the combined application of multiple probiotics and ginsenosides helps to synergistically enhance the therapeutic effect on enterocolitis caused by dysbiosis.

[0048] Efficacy verification: 1) The role of the microspheres prepared in Example 1 in alleviating the DSS-induced inflammatory response in colitis. Studies have shown that tumor necrosis factor-α (TNF-α) can synergistically act with various inflammatory factors to induce the production of inflammatory mediators in the body, thereby participating in the development and progression of inflammatory diseases such as ulcerative colitis. Therefore, detecting TNF-α levels can be used to assess the degree of inflammatory response and disease status. TNF-α can promote cell apoptosis and induce the secretion of interleukin-1β (IL-1β) and interleukin-6 (IL-6), thereby amplifying the inflammatory response. IL-1β, as a key member of the IL-6 family, has significant pro-inflammatory activity, can induce the expression of various inflammatory factors, and promote lymphocyte infiltration by upregulating adhesion molecules, thereby activating the immune response. In addition, glutathione peroxidase (GSH-PX) is one of the important indicators for evaluating intestinal oxidative stress and antioxidant defense capacity in IBD, reflecting the body's ability to cope with free radical attacks and is closely related to the degree of inflammatory damage.

[0049] In this embodiment, the following is adopted: Figure 1 The microspheres shown were used to treat a DSS-induced colitis mouse model, and the levels of inflammation-related factors were measured. The results showed that, compared with the control group, the levels of malondialdehyde (MDA), TNF-α, and IL-1β in the colonic tissue of the DSS-induced group were significantly increased, indicating significant oxidative stress and inflammatory response. After treatment with microspheres, these indicators significantly decreased, and were significantly lower than those in the DSS group. Furthermore, the level of GSH-PX in the DSS-induced group was significantly decreased, while the level of GSH-PX in the microsphere-treated tissue was significantly increased. These results indicate that the prepared microspheres have good anti-inflammatory effects and can effectively reduce the levels of inflammatory factors in the DSS-induced acute colitis model, thereby regulating the immune response and alleviating colitis symptoms.

[0050] 2) H&E staining and histological scoring Figure 2Microscopic images of H&E-stained colon tissue sections from mice in different treatment groups. As shown in the images, the colon tissue structure of mice in the Control group was normal, with regularly arranged mucosal epithelial cells, no necrosis, deformation, or inflammatory cell infiltration, abundant goblet cells, and clear and intact crypt structures. In contrast, the colon tissue of mice in the DSS-treated group showed significant pathological changes, including severe epithelial cell damage, a significant reduction in the number of goblet cells, extensive inflammatory cell infiltration of the mucosa and submucosa, damaged and deformed crypt structures, disordered arrangement, and loss of villous structures in some areas. After treatment with the microspheres prepared in Example 1 of this invention, the pathological improvement of mouse colon tissue was significant. Tissue sections showed that the mucosal surface structure was nearly intact, the number of goblet cells was significantly increased, inflammatory cell infiltration was significantly reduced, and the crypt structure was basically restored, similar to the Control group. This result is also consistent with the corresponding histological scoring data, indicating that the microspheres have good tissue repair and anti-inflammatory effects.

[0051] Figure 3 Further TUNEL staining analysis results were presented to assess colonic tissue cell apoptosis. The results showed that a large number of apoptotic cells were present in the colonic tissue of mice treated with DSS, mainly concentrated in the apical region of the intestinal villi, consistent with the distribution characteristics of inflammatory lesions. After treatment with Microspheres, the number of TUNEL-positive cells was significantly reduced, suggesting that the microspheres have the effect of inhibiting DSS-induced epithelial cell apoptosis and protecting intestinal tissue structure.

[0052] In summary, the results of this embodiment demonstrate that the probiotic microspheres described in this invention exhibit good effects in alleviating DSS-induced acute colitis, repairing intestinal mucosal damage, and inhibiting tissue apoptosis.

[0053] It should be noted that the above embodiments are merely one of the preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. For those skilled in the art, various equivalent substitutions or modifications made to the technical solutions without departing from the essential concept of the present invention should be included within the scope of protection of the present invention.

[0054] It should be noted that the above embodiments are merely one of the preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. For those skilled in the art, various equivalent substitutions or modifications made to the technical solutions without departing from the essential concept of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microsphere encapsulating probiotics and ginsenosides, characterized in that: The wall material of the microspheres is alginate or a composite system of alginate and one or more polymeric materials to achieve synergistic protection, targeted release, and intestinal-targeted therapeutic effects of probiotics and ginsenosides. The wall material can be combined with EDTA-Ca 2+ 、Ba 2+ Ionic crosslinking agents further enhance the structural stability and functional diversity of microspheres, with the interior of the microspheres containing probiotic liquid and prototype ginsenosides.

2. The microspheres encapsulating probiotics and ginsenosides according to claim 1, characterized in that: The alginate has a molecular weight of 50-200 kDa and a concentration range of 8%-12%; the polymeric materials include chitosan, gelatin, ginseng polysaccharide, resistant starch, pectin, guar gum, hydroxypropyl methylcellulose (HPMC) and its derivatives.

3. The microspheres encapsulating probiotics and ginsenosides according to claim 2, characterized in that: The wall material of the microspheres is alginate-EDTA-Ca 2+ .

4. The microspheres encapsulating probiotics and ginsenosides according to claim 3, characterized in that: The concentration of the probiotics is 1×10⁻⁶. 6 -1×10 9 CFU / mL.

5. The microspheres encapsulating probiotics and ginsenosides according to claim 4, characterized in that: The concentration of the original ginsenosides is 10-20 g / L.

6. The microspheres encapsulating probiotics and ginsenosides according to claim 5, characterized in that: The microspheres have a particle size of 100-200 micrometers.

7. The microspheres encapsulating probiotics and ginsenosides according to claim 6, characterized in that: The probiotics are Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium, and Lactobacillus fermentum.

8. A method for preparing microspheres loaded with probiotics and ginsenosides, used to prepare the microspheres loaded with probiotics and ginsenosides as described in claim 7, characterized in that: The specific steps are as follows: Step 1: First, weigh 20-22 wt.% CaCl2 and 36-38 wt.% EDTA, and adjust the pH to 7.5 to prepare CaCl2. 2+ -EDTA solution, then mixed with an equal volume of 40 wt.% alginate, to obtain alginate-EDTA-Ca 2+ liquid; Step 2: In the alginate-EDTA-Ca 2+ Aqueous solution was obtained by adding probiotic bacterial solution washed with PBS and proto-ginsenosides to the solution. Step 3: Weigh 0.5 ml of glacial acetic acid and dissolve it in 500 ml of microdroplet solution to generate oil and obtain an oil phase solution; Step 4: Start the microfluidic device, place the aqueous solution in the second channel and the oil solution in the first channel, connect and adjust the device to begin preparing microspheres; wherein, the flow rates of the aqueous solution and the oil solution are 15 μL / min and 20 μL / min, respectively; Step 5: After preparing probiotic microspheres of uniform size, collect the solidified microspheres using centrifuge tubes to obtain alginate hydrogel microspheres containing probiotics. Step 6: After collection, wash with PBS to obtain dispersed microspheres of the probiotic and ginsenoside microreaction system for treating dysbiosis-induced inflammatory bowel disease; Step 7: Take 10g of 20wt% alginate hydrogel solution and resuspend the prepared probiotic and ginsenoside microspheres for treating dysbiosis-induced inflammatory bowel disease in the hydrogel.

9. The use of microspheres containing probiotics and ginsenosides as described in any one of claims 1 to 7 in the treatment of inflammatory bowel disease with dysbiosis.

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