Probiotic microsphere preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to a probiotic microsphere preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing a probiotic microsphere preparation inner core from a precursor solution through a water-in-oil emulsion calcium ion crosslinking method, and preparing the probiotic microsphere preparation with a core-shell structure from the probiotic microsphere preparation inner core through a # imgabs0 # L100 water-in-oil emulsion solvent volatilization method. According to the preparation method, a core-shell structure is constructed through water-in-oil emulsion calcium ion crosslinking and a solvent evaporation method, process parameters are clear, and repeatability is high. The obtained probiotic microsphere preparation can form physical-chemical double barriers in a gastric acid environment, and maintains the metabolic activity of thalli by virtue of the prebiotic effect of the fructo-oligosaccharide, so that the live bacterium delivery rate of 84.60% in a gastrointestinal tract simulation test is realized. The preparation prepared by the invention can be applied to treatment of inflammatory bowel diseases, can inhibit expression of proinflammatory factors and effectively promote repair of colonic mucosa, and is obviously superior to a traditional preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a probiotic microsphere preparation and a preparation method and application thereof. Background Art

[0002] Probiotics, as a class of active microorganisms beneficial to host health, play an important role in regulating intestinal flora balance, enhancing immune function, and improving metabolic disorders. However, during oral delivery, probiotics face the harsh environment of gastric acid and bile salts, which significantly reduces their bioactivity and makes it difficult for them to colonize and exert their efficacy in the intestine. Traditional probiotic preparations, such as freeze-dried powders or ordinary capsules, often lack effective protective mechanisms and have extremely low survival rates in the gastric acid environment of pH 1.5-3.0, severely limiting their effectiveness.

[0003] Microsphere encapsulation technology is an effective strategy for improving the stability and targeted delivery of probiotics. Encapsulating probiotics with natural or synthetic polymers can mitigate gastric acid erosion to a certain extent. However, existing microsphere preparations still have significant drawbacks: insufficient mechanical strength leads to rupture during gastrointestinal motility; some materials rapidly dissolve and release bacteria under low pH conditions; and the microsphere pore structure collapses during long-term storage, leading to probiotic inactivation. Furthermore, protective materials may affect the release kinetics of probiotics or pose biocompatibility issues. Summary of the Invention

[0004] The present invention aims to provide a probiotic microsphere preparation with simple process, high safety, excellent acid resistance and long-term stability, which can provide key technical support for the development of functional foods and microecological drugs.

[0005] To achieve the above object, the present invention provides a method for preparing a probiotic microsphere preparation, which comprises the following steps, calculated by weight:

[0006] Step 1: Prepare the inner core of the probiotic microsphere preparation:

[0007] Step 101: dissolving 2 to 4 parts of sodium alginate, 2 to 4 parts of fructooligosaccharides, 0.1 to 0.5 parts of glycerol, and 0.1 to 0.5 parts of calcium carbonate powder in 92 to 96 parts of deionized water, and stirring to obtain a quasi-precursor solution;

[0008] Step 102: adding probiotics to the quasi-precursor solution obtained in step 101 to a concentration of 108 to 109 cfu / ml to obtain a precursor solution;

[0009] Step 103: mixing the precursor solution obtained in step 102 with the oil phase, adding an emulsifier to obtain an emulsion;

[0010] Step 104: adding 1-2 ml of an acidic liquid to the emulsion obtained in step 103 to initiate a cross-linking reaction to obtain a probiotic microsphere preparation core;

[0011] Step 2: Construct the core-shell structure:

[0012] Step 201: Mix Eudragit 65-70 parts of L100, 5-10 parts of triethyl citrate, 20-30 parts of magnesium stearate, 333.75-341.25 parts of acetone, 511.75-523.25 parts of isopropyl alcohol, and 44.5-45.5 parts of water to obtain a coating solution;

[0013] Step 202: Disperse the probiotic microsphere preparation core obtained in step 104 in the coating solution, and stir at a speed of 800-1200 rpm for 30-60 minutes in a water bath at 37-40° C. to obtain microspheres;

[0014] Step 203: Wash the surface of the microspheres alternately with n-hexane and water to obtain a probiotic microsphere preparation.

[0015] In the above method, in step 103, the volume ratio of the oil phase to the precursor solution is 2 to 4:1; the emulsifier is Span 80, and the volume ratio of the emulsifier to the oil phase is 1:100 to 3:200.

[0016] In the above method, step 101 specifically includes:

[0017] Step 1011, dissolving sodium alginate in deionized water and stirring for 1 to 2 hours;

[0018] Step 1012: Add oligofructose and continue stirring for 1 to 2 hours;

[0019] Step 1013: add glycerol and stir for 10 to 30 minutes, and then add calcium carbonate powder and stir for 30 to 60 minutes;

[0020] Step 1014: Finally, add probiotics and stir for 10 to 20 minutes until uniform, to obtain a quasi-precursor solution.

[0021] In the above method, after step 104, the following steps are further included:

[0022] Step 105: Alternately washing with anhydrous ethanol and water to remove the surface oil phase of the probiotic microsphere preparation inner core obtained in step 104 to obtain a pure probiotic microsphere preparation inner core;

[0023] Step 106: The purified probiotic microsphere preparation core obtained in step 105 is sealed and stored at 4-8°C.

[0024] In the above method, the probiotic is at least one of Lactobacillus reuteri, Lactobacillus acidophilus, Saccharomyces boulardii, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium and Streptococcus thermophilus;

[0025] The oil phase is at least one of soybean oil, olive oil, mineral oil, liquid paraffin, silicone oil, castor oil and linseed oil.

[0026] In the above method, the acidic liquid is selected from at least one of glacial acetic acid, dilute hydrochloric acid, citric acid, lactic acid, succinic acid and formic acid.

[0027] In another aspect, the present invention provides a probiotic microsphere preparation obtained by the above-mentioned preparation method.

[0028] In another aspect, the present invention provides a use of the above-mentioned probiotic microsphere preparation in inflammatory bowel disease.

[0029] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0030] 1. The present invention achieves gastric acid protection and intestinal targeted release functions through core-shell structure design. After being treated in simulated gastric fluid for 2 hours, the probiotic survival rate of the core-shell microspheres AMS@Eud was as high as 92.81%, while the survival rate of free probiotics was only 0.04%; in further simulation of continuous gastrointestinal environment tests, the survival rate of AMS@Eud remained at 84.60%, proving that it can effectively resist gastric acid erosion and accurately release live bacteria in the intestine. This effect was verified by infrared spectroscopy and XPS analysis, confirming that Eudragit The L100 shell completely covers the inner core.

[0031] 2. The probiotic microsphere preparation of the present invention exhibits excellent long-term stability. After storage at 25°C for 35 days, the activity loss of AMS@EUD was 9.09%, significantly lower than the 38.88% of free probiotics; when stored at 4°C, the activity loss was reduced to 5.28%. L100, triethyl citrate and magnesium stearate, combined with acetone / isopropyl alcohol / water mixed solvent to form a dense protective layer.

[0032] 3. This invention has demonstrated its potential for clinical application through biosafety testing: After five days of co-culture with L929 cells, cell viability in the AMS@EUD group was not significantly different from that in the blank control group. Hemolysis rates were below 5% at all concentrations, meeting international safety standards for blood-contact materials. Furthermore, H&E staining revealed significantly reduced colonic inflammation and intact crypt structure in mice treated with AMS@EUD.

[0033] 4. This invention represents a breakthrough improvement over conventional technologies. The survival rate of uncoated microspheres in simulated gastric fluid is less than 5%, while the viability of free probiotics decreases by 38.88% after 35 days of storage. This invention demonstrates that the core-shell structure can simultaneously achieve efficient encapsulation of live bacteria (fluorescence staining reveals intact encapsulation) and synergistic physical and chemical protection, resolving the dual challenges of gastric acid inactivation and storage degradation in existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] In the following figures, AMS@Eud represents probiotic microspheres with a core-shell structure, which are prepared in Example 1; AMS represents the inner core of probiotic microspheres, which are prepared in Comparative Example 1; and Free probiotics represents free probiotics, which are obtained in Comparative Example 2.

[0036] Figure 1 This is the infrared spectrum of the probiotic microsphere preparation.

[0037] Figure 2 It is the X-ray photoelectron energy spectrum (XPS) of the probiotic microsphere preparation; Among them, A is the XPS full spectrum of Example 1 and Comparative Example 1; B is the O1s fine spectrum of Comparative Example 1; C is the O1s fine spectrum of Example 1; D is the Ca 2p fine spectrum of Comparative Example 1; E is the Ca 2p fine spectrum of Example 1.

[0038] Figure 3 : are scanning electron micrographs of the probiotic microsphere preparation; wherein, A is a scanning electron micrograph of comparative example 1; B is a carbon element analysis of the EDS of comparative example 1; C is a calcium element analysis of the EDS of comparative example 1; D is a scanning electron micrograph of example 1; E is a carbon element analysis of the EDS of example 1; and F is a calcium element analysis of the EDS of example 1.

[0039] Figure 4 This is a laser confocal microscopy image of the probiotic microsphere preparation. Figure 4 A in the figure is a bright-field image from a laser confocal microscope; Figure 4 B in the figure is a fluorescence image obtained by laser confocal microscopy; Figure 4 C in the figure is a superimposed image of a laser confocal microscope.

[0040] Figure 5These are the results of the simulated gastrointestinal tolerance test of the probiotic microsphere preparation; SGF stands for simulated gastric fluid; SIF stands for simulated intestinal fluid; and SGI stands for simulated gastrointestinal treatment.

[0041] Figure 6 1 is a graph showing the storage test results of the probiotic microsphere preparation; wherein, A is the result of Example 1 and Comparative Example 2 stored at 4°C for 35 days; B is the result of Example 1 and Comparative Example 2 stored at 25°C for 35 days.

[0042] Figure 7 Figures 1 and 2 are the results of the biocompatibility test of probiotic microspheres; Figure A is a laser confocal microscopy image of the biocompatibility test of Example 1, Comparative Example 1, and the blank group; Figure B is an optical density graph at 450 nm after co-culture of the extracts of Example 1, Comparative Example 1, and the blank group with L929; Figure C is a graph of cell viability after co-culture of the extracts of Example 1, Comparative Example 1, and the blank group with L929.

[0043] Figure 8 The figures are the hemolysis rate test results of the probiotic microspheres; A is the intuitive image result diagram of the hemolysis performance of Example 1, Comparative Example 1 and the positive control group; B is the quantitative analysis image result diagram of the hemolysis performance of Example 1, Comparative Example 1 and the positive control group.

[0044] Figure 9 Figures 1 and 2 show the results of in vivo delivery tests of probiotic microspheres. Figure A shows the weight changes of mice at different time periods after oral administration of Example 1, Comparative Example 2, and dextran sulfate sodium to mice for 7 days. Figure B shows the changes in the disease activity index of mice at different time periods after oral administration of Example 1, Comparative Example 2, and dextran sulfate sodium to mice for 7 days. Figure C shows the quantitative statistics of colon length of mice after oral administration of Example 1, Comparative Example 2, and dextran sulfate sodium to mice for 7 days. Figure D shows the visual representation of colon length of mice after oral administration of Example 1, Comparative Example 2, and dextran sulfate sodium to mice for 7 days.

[0045] Figure 10 H&E staining image of interpenetrating polymer network hydrogel containing collagen fibers.

[0046] Figure 11 The figures are the immunological staining test results of the interpenetrating polymer network hydrogel containing collagen fibers; wherein, A is the immunohistochemical staining result of TNF-α; and B is the statistical result of TNF-α expression level. DETAILED DESCRIPTION

[0047] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental and detection methods described in each example are conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified. The ratios in the following examples are ratios by mass unless otherwise specified.

[0048] Example 1

[0049] This embodiment provides a method for preparing a probiotic microsphere preparation, which comprises the following steps, calculated by weight:

[0050] Step 1: Prepare the inner core of the probiotic microsphere preparation:

[0051] Step 101: Dissolve 2 parts of sodium alginate, 2 parts of oligofructose, 0.1 parts of glycerol, and 0.1 parts of calcium carbonate powder in 95 parts of deionized water to obtain a quasi-precursor solution. The specific steps are as follows:

[0052] Step 1011, dissolving sodium alginate in deionized water and stirring for 1 hour;

[0053] Step 1012: Add oligofructose and continue stirring for 1 hour;

[0054] Step 1013: add glycerol and stir for 15 minutes, and then add calcium carbonate powder and stir for 40 minutes;

[0055] Step 1014: Finally, add probiotics and stir for 20 minutes until uniform to obtain a quasi-precursor solution.

[0056] Step 102: Add Lactobacillus reuteri to the quasi-precursor solution obtained in step 1014 and mix until the mixture is 10 8 cfu / ml, and the precursor solution was obtained;

[0057] Step 103: Mix the precursor solution obtained in step 102 with soybean oil, add Span 80, and obtain an emulsion, wherein the volume ratio of soybean oil to precursor solution is 2:1; the volume ratio of Span 80 to soybean oil is 1:100;

[0058] Step 104: adding 2 ml of glacial acetic acid to the emulsion obtained in step 103 to initiate a cross-linking reaction to obtain a probiotic microsphere preparation core;

[0059] Step 105: washing with anhydrous ethanol and water in sequence to remove the oil phase on the surface of the probiotic microsphere preparation inner core obtained in step 104 to obtain a pure probiotic microsphere preparation inner core;

[0060] Step 106: The purified probiotic microsphere preparation core obtained in step 105 is sealed and stored at 4-8°C.

[0061] Step 2: Construct the core-shell structure:

[0062] Step 201: Mix Eudragit 65.00 parts of L100, 5.00 parts of triethyl citrate, 20.00 parts of magnesium stearate, 333.75 parts of acetone, 511.75 parts of isopropyl alcohol, and 44.50 parts of water were added to obtain a coating solution;

[0063] Step 202: Disperse the purified probiotic microsphere preparation cores stored in step 106 in the coating solution, and stir at 80 rpm for 30 minutes in a 37° C. water bath to obtain microspheres;

[0064] Step 203: Wash the microsphere surface with n-hexane and water in sequence to obtain a probiotic microsphere preparation, called AMS@Eud.

[0065] Example 2

[0066] This embodiment provides a method for preparing a probiotic microsphere preparation, which comprises the following steps, calculated by weight:

[0067] Step 1: Prepare the inner core of the probiotic microsphere preparation:

[0068] Step 101: Dissolve 3 parts of sodium alginate, 3 parts of oligofructose, 0.3 parts of glycerol, and 0.3 parts of calcium carbonate powder in 93 parts of deionized water to obtain a quasi-precursor solution. The specific steps are as follows:

[0069] Step 1011, dissolving sodium alginate in deionized water and stirring for 2 hours;

[0070] Step 1012: Add oligofructose and continue stirring for 2 hours;

[0071] Step 1013: add glycerol and stir for 30 minutes, and then add calcium carbonate powder and stir for 60 minutes;

[0072] Step 1014: Finally, add probiotics and stir for 20 minutes until uniform to obtain a quasi-precursor solution.

[0073] Step 102: Add Lactobacillus reuteri to the quasi-precursor solution obtained in step 1014 and mix until the mixture is 10 9 cfu / ml, and the precursor solution was obtained;

[0074] Step 103: Mix the precursor solution obtained in step 102 with soybean oil, add Span 80, and obtain an emulsion, wherein the volume ratio of the oil phase to the precursor solution is 3:1; and the volume ratio of Span 80 to the oil phase is 1.3:100;

[0075] Step 104: adding 1.5 ml of glacial acetic acid to the emulsion obtained in step 103 to initiate a cross-linking reaction to obtain a probiotic microsphere preparation core;

[0076] Step 105: washing with anhydrous ethanol and water in sequence to remove the oil phase on the surface of the probiotic microsphere preparation inner core obtained in step 104 to obtain a pure probiotic microsphere preparation inner core;

[0077] Step 106: The purified probiotic microsphere preparation core obtained in step 105 is sealed and stored at 4-8°C.

[0078] Step 2: Construct the core-shell structure:

[0079] Step 201: Mix Eudragit 67.50 parts of L100, 7.50 parts of triethyl citrate, 25.00 parts of magnesium stearate, 337.50 parts of acetone, 517.50 parts of isopropyl alcohol, and 45.00 parts of water were added to obtain a coating solution;

[0080] Step 202: Disperse the purified probiotic microsphere preparation cores stored in step 106 in the coating solution, and stir at 1000 rpm for 45 minutes in a 38.5° C. water bath to obtain microspheres;

[0081] Step 203: Wash the surface of the microspheres with n-hexane and water in sequence to obtain a probiotic microsphere preparation.

[0082] Example 3

[0083] This embodiment provides a method for preparing a probiotic microsphere preparation, which comprises the following steps, calculated by weight:

[0084] Step 1: Prepare the inner core of the probiotic microsphere preparation:

[0085] Step 101: dissolve 4 parts of sodium alginate, 4 parts of oligofructose, 0.5 parts of glycerol, and 0.5 parts of calcium carbonate powder in 92 parts of deionized water to obtain a quasi-precursor solution. The specific steps are as follows:

[0086] Step 1011, dissolving sodium alginate in deionized water and stirring for 1.5 hours;

[0087] Step 1012: Add oligofructose and continue stirring for 1.5 hours;

[0088] Step 1013: add glycerol and stir for 10 minutes, and then add calcium carbonate powder and stir for 50 minutes;

[0089] Step 1014: Finally, add probiotics and stir for 10 minutes until uniform to obtain a quasi-precursor solution.

[0090] Step 102: Add Lactobacillus reuteri to the quasi-precursor solution obtained in step 1014 and mix until the mixture is 110 9 cfu / ml, and the precursor solution was obtained;

[0091] Step 103: Mix the precursor solution obtained in step 102 with soybean oil, add Span 80, and obtain an emulsion, wherein the volume ratio of the oil phase to the precursor solution is 4:1; and the volume ratio of Span 80 to the oil phase is 3:200;

[0092] Step 104: adding 2 ml of formic acid to the emulsion obtained in step 103 to initiate a cross-linking reaction to obtain a probiotic microsphere preparation core;

[0093] Step 105: washing with anhydrous ethanol and water in sequence to remove the oil phase on the surface of the probiotic microsphere preparation inner core obtained in step 104 to obtain a pure probiotic microsphere preparation inner core;

[0094] Step 106: The purified probiotic microsphere preparation core obtained in step 105 is sealed and stored at 4-8°C.

[0095] Step 2: Construct the core-shell structure:

[0096] Step 201: Mix Eudragit 70 parts of L100, 10 parts of triethyl citrate, 30 parts of magnesium stearate, 341.25 parts of acetone, 523.25 parts of isopropyl alcohol, and 45.50 parts of water were added to obtain a coating solution;

[0097] Step 202: Disperse the purified probiotic microsphere preparation cores stored in step 106 in the coating solution, and stir at 1200 rpm for 60 minutes in a 40° C. water bath to obtain microspheres;

[0098] Step 203: Wash the surface of the microspheres with n-hexane and water in sequence to obtain a probiotic microsphere preparation.

[0099] Example 4

[0100] First, this embodiment provides two comparative examples:

[0101] Comparative Example 1: The difference between this comparative example and Example 1 is that steps 3 and 4 are omitted, and only the probiotic-loaded calcium alginate hydrogel microsphere core, referred to as AMS, is finally prepared.

[0102] Comparative Example 2: This comparative example differs from Example 1 in that steps 1, 2, 3 and 4 are omitted, and only free probiotics are used, which are referred to as Free probiotics.

[0103] Secondly, this example provides infrared detection tests, X-ray photoelectron spectroscopy tests, scanning electron microscopy tests, and laser confocal microscopy tests of the gastric acid-resistant and long-lasting stable probiotic microsphere preparation.

[0104] 1. Infrared detection test of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0105] The potassium bromide tableting method was used for analysis. The AMS@Eud prepared in Example 1, the AMS prepared in Comparative Example 1 and the Eudragit After the L100 sample is dried, it is mixed with potassium bromide and ground evenly, then placed in a tableting mold and pressed into thin slices for spectral analysis at 4000 cm -1 ~400cm -1 The wave number segment was scanned 32 times and the average value was taken. The resolution was set to 4 cm. -1 , the infrared detection results of probiotic microsphere preparations are as follows Figure 1 As shown. Figure 1 It can be seen that the broad peak in the high-frequency region of AMS is caused by the stretching vibration of hydroxyl groups, while the peaks in the fingerprint region of 1500-1600 cm -1 and 1000-1200cm -1 The C-O-C vibrations of carboxylate groups and oligofructose, respectively, correspond to the broad peaks in the high-frequency region (3200-3600 cm -1 ) is by Eudragit The peak is shifted to a lower wave number and its shape is broadened. The fingerprint region is 1700 cm -1 Close composite peaks are derived from Eudragit Superposition of the ester C=O portion of L100 and the calcium alginate group. 1500-1600 cm -1 The peak attenuation is attributed to Eudragit The ion exchange or coordination interaction between the carboxylic acid groups of L100 and the calcium ions of calcium alginate, in general, the broadening of the hydroxyl peak in the high-frequency region, and the peak shift and intensity change in the fingerprint region further confirm the Eudragit L100 forms a shell protection structure on the surface of AMS.

[0106] 2. X-ray photoelectron spectroscopy test of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0107] X-ray photoelectron spectroscopy (XPS) was used for analysis. The hydrogel samples prepared in Example 1 and Comparative Example 1 were dried and degreased, then placed on a sample table and fixed with conductive adhesive to ensure that the sample surface was flat, clean, and free of contamination. After the instrument stabilized, a full spectrum scan was performed first, followed by a narrow spectrum scan of the characteristic peaks. Photoelectron energy distribution data was collected to analyze the elemental composition and chemical state of the samples. The XPS spectrum results of Example 1 and Comparative Example 1 are shown in Figure 2. Figure 2 As shown. Figure 2 As can be seen from A in the figure, the significant enhancement of the C1S signal intensity of AMS@Eud is attributed to Eudragit The carbon-rich polymer structure of L100. The shift of O 1S signal comes from the introduction of oxygen-containing functional groups (such as carbonyl and ether bonds), which is the key of Eudragit Inherent in L100. Figure 2 As can be seen from B to D in Figure 1, the main peak in the O1S spectrum of AMS@Eud is 532.51 eV, corresponding to Eudragit CO / C=O moieties in the L100 matrix. This shift in binding energy and peak intensity indicates that the Eudragit At the same time, the decrease in the intensity of the Ca 2P signal in AMS@Eud is caused by the shell coverage, which proves that Eudragit L100 shields the inner core of the probiotic microspheres. Overall, the enhanced C1S signal changes the O1S chemical environment and weakens the Ca 2p signal, which together demonstrates the successful formation of Eudragit on the AMS surface. L100 shell.

[0108] 3. Scanning electron microscopy test of gastric acid-resistant and long-lasting stable probiotic microsphere preparation

[0109] The working distance (WD) was set to 10.6 mm to observe the surface structure of the gastric acid-resistant and long-lasting stable probiotic microsphere preparations of Example 1 and Comparative Example 1. The scanning electron microscopy results are as follows: Figure 3 As shown. Figure 3 It can be seen that the surface morphology of AMS@Eud is smoother than the rough morphology of uncoated AMS, which is consistent with the formation of a uniform polymer shell. EDS elemental analysis supports these findings, and no calcium was detected on the surface of AMS@Eud, indicating that the Eudragit L100 completely covers the inner core of the probiotic microsphere preparation.

[0110] 4. Laser confocal microscopy test of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0111] The probiotics were stained with 1% rhodamine B dye for 10 minutes, and the excess dye was washed off. Steps 1 to 4 of Example 1 were repeated to obtain a probiotic microsphere preparation loaded with fluorescent probiotics. The microsphere preparation was placed on a laser confocal culture dish and observed under 546 nm laser irradiation. The results of the laser confocal microscope were as follows: Figure 4 As shown. Figure 4 It can be seen that obvious red fluorescence can be seen in the probiotic microsphere preparation, which clearly confirms that living probiotics are encapsulated in the core-shell structure.

[0112] Example 5

[0113] This example provides simulated gastrointestinal tolerance testing, storage testing, biocompatibility testing, hemolysis testing, in vivo delivery performance testing, H&E staining testing, and immunological staining testing of a gastric acid-resistant and long-lasting stable probiotic microsphere formulation.

[0114] Comparative Example 1 and Comparative Example 2 in this embodiment are derived from the comparative example in Example 4.

[0115] 1. Simulated gastrointestinal tolerance test of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0116] According to the principles specified in the Chinese Pharmacopoeia, simulated gastric fluid and simulated intestinal fluid were prepared. 1 g of pepsin was dissolved in 100 ml of deionized water, and the pH was adjusted to 1.2 with 1.0 M hydrochloric acid solution to prepare simulated gastric fluid (SGF). Similarly, 1 g of trypsin was dissolved in 100 ml of 0.05 M potassium phosphate buffer, and the pH was adjusted to 6.8 with 1.0 M NaOH solution to prepare simulated intestinal fluid (SIF). The AMS@Eud prepared in Example 1 and the free probiotics obtained in Comparative Example 2 were added to simulated gastric fluid and incubated for 2 hours and 4 hours at 37°C, respectively. In order to further test the tolerance of the prepared probiotic microsphere preparation under simulated continuous gastrointestinal tract, the AMS@Eud prepared in Example 1 and the free probiotics obtained in Comparative Example 2 were first incubated in simulated gastric fluid for 2 hours, and then resuspended in simulated intestinal fluid for 4 hours. The bacterial activity was detected by the plate count method. The results of the simulated gastrointestinal tract are as follows: Figure 5 shown.

[0117] Depend on Figure 5In simulated gastric fluid, free Lactobacillus reuteri was extremely sensitive to the environment, with a survival rate of only 0.04% after 2 hours, while AMS@Eud retained 92.81±2.45% of viable bacteria. In simulated intestinal fluid, the viability of the probiotics in AMS@Eud was higher than that of the free probiotics after 4 hours, but a t-test showed no significant difference (p<0.05), likely due to the neutral pH reducing direct bacterial stress. Under simulated continuous gastrointestinal conditions, the viability of the free probiotics plummeted to 0.06%, while the survival rate of AMS@Eud remained at 84.60%. These results demonstrate that the core-shell structure of AMS@Eud provides strong protection against gastric acid, enabling the probiotics to transit through the harsh gastrointestinal environment.

[0118] 2. Storage test of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0119] The AMS@Eud prepared in Example 1 and the free probiotics obtained in Comparative Example 2 were stored in sterile Falcon tubes at 4°C and 25°C, respectively. The viability of the encapsulated probiotics and free probiotics in the AMS@Eud were evaluated at 0, 1, 4, 7, 14, 21, 28, and 35 days. The storage performance of the probiotics is shown in Figure 2. Figure 6 As shown, the calculation formula for bacterial viability loss is as follows:

[0120] Bacterial viability loss = (N 实验组各时刻细菌数 -N 初始时刻细菌数 ) / (N 初始时刻细菌数 )×100%

[0121] Depend on Figure 6 It can be seen that compared with the free probiotics in Comparative Example 2, the AMS@Eud prepared in Example 1 significantly improved the stability of probiotics. At 4°C, the activity of the free probiotics decreased by 22.73% after storage for 35 days, while the AMS@Eud still maintained 94.72% activity (a loss of 5.28%). At 25°C, the activity loss of the free probiotics was 38.88%, significantly higher than the 9.09% loss in the AMS@Eud. The superior performance of AMS@Eud is attributed to the synergistic effect: Eudragit The physical protection of the L100 shell and the prebiotic-mediated metabolic support of oligofructose are particularly evident at high temperatures.

[0122] 3. Biocompatibility testing of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0123] L929 cells were selected and determined by the Cell Count Kit-8 (CCK-8) method, and 100 μL of cell suspension was prepared in a 96-well plate. The culture plate was placed in an incubator for pre-culture for 24 hours (37°C, 5% CO2), and 10 μL of the probiotic microsphere preparation extract obtained in Example 1 and the probiotic microsphere core extract obtained in Comparative Example 1 were added to the culture plate. The extract acquisition method was referred to the standard GB / T 16886.12-2017. At the same time, the control group (Control) was only added with sterile PBS solution, and the culture plate was placed in the incubator again. 10 μL of CCK-8 solution was added to each well at 1 day, 3 days, and 5 days of incubation, respectively. After adding CCK-8 solution, the mixture was incubated for 2 hours, and the optical density (OD) value at 450 nm was measured with an enzyme marker. The test results are as follows: Figure 7 and Figure 8 As shown, the calculation formula of cell viability is as follows, and the laser confocal microscope image of the biocompatibility test is as follows Figure 7 The statistical results of the biocompatibility test are shown in Figure 8 As shown:

[0124] Cell viability = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%

[0125] Depend on Figure 7 It can be seen that after the probiotic microspheres prepared in Example 1 of the present invention and the probiotic microsphere cores prepared in Comparative Example 1 were co-cultured with L929 cells, a large amount of green fluorescence signals could still be observed, indicating that the probiotic microspheres prepared in the present invention have good biocompatibility and safety. Figure 8 It can be seen that there was no significant difference in cell viability and proliferation after 1, 3 and 5 days of co-incubation between AMS and AMS@Eud and the control group (p>0.05), confirming that neither preparation induced cytotoxic effects.

[0126] 4. Hemolytic performance test of gastric acid-resistant and long-lasting stable probiotic microsphere preparation

[0127] The AMS@Eud prepared in Example 1 and the AMS extract (0.5 ml) prepared in Comparative Example 1 were mixed with red blood cell suspensions (0.5 ml) of different concentrations (25, 50, 100, 200 mg / ml), incubated for 1 hour, and centrifuged. The extract acquisition method can refer to the standard GB / T 16886.12-2017. The positive control group (Positive) used 0.5 ml of 0.1% TRITON, and the blank control group (Control) used 0.5 ml of PBS buffer. The absorbance (Ab) of the supernatant at 540 nm was measured using an ultraviolet spectrophotometer. The hemolysis rate is calculated by the following formula, and the intuitive image of the hemolysis rate test is as follows: Figure 8 As shown in A, the quantitative analysis of the hemolysis rate test is as follows Figure 8 As shown in B:

[0128] Hemolysis rate = (Ab 实验组 -Ab 空白组 ) / (Ab 阳性组 -Ab 空白组 )×100%

[0129] Depend on Figure 8 As shown in A, the extracts of AMS@Eud prepared in Example 1 and AMS prepared in Comparative Example 1 do not significantly damage the structure and function of the red blood cell membrane when in contact with red blood cells. Red blood cells can maintain their normal morphology and integrity and maintain normal physiological functions. Figure 8 As shown in Figure B, the hemolysis rates of the extracts of AMS@Eud prepared in Example 1 and AMS prepared in Comparative Example 1 were both less than 5%, meeting international safety standards for blood-contact biomaterials. These results demonstrate the biocompatibility and hemocompatibility of AMS@Eud prepared in Example 1 and AMSd prepared in Comparative Example 1, ensuring their safety for in vivo use.

[0130] 5. In vivo delivery performance test of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0131] C57BL / 6J mice were randomly divided into 4 groups, with 6 mice in each group: (1) control group: normal saline (0.2 ml) was administered orally; (2) DSS group: dextran sulfate sodium (DSS) (3% w / v) in drinking water + normal saline (0.2 ml) was administered orally; (3) free Lactobacillus reuteri group: DSS (3% w / v) was added to drinking water + 2×10 9CFU / ml free probiotic suspension (0.2ml); (4) Microsphere group: DSS (3% w / v) + probiotic microsphere suspension in drinking water was gavage (0.2ml). Body weight and disease activity index (DAI) were measured every day to assess the severity of the disease. The mice were killed on the 7th day, the colon was collected, and the length was measured. The disease activity index is a comprehensive score based on the percentage of weight loss of the patient (sick animal) (no change in weight is 0, 1-5 is 1 point, 5-10 is 2 points, 10-15 is 3 points, and greater than 15 is 4 points), stool consistency (normal is 0, loose stool is 2 points, diarrhea is 4 points) and stool bleeding (normal is 0, occult blood is 2 points, and overt bleeding is 4 points). The total score of the three results is divided by 3 to obtain the DAI value. The calculation formula of weight change and disease activity index is as follows, and the weight change curve is as follows. Figure 9 As shown in A, the disease activity index changes as Figure 9 As shown in B, the quantitative statistical results of colon length are as follows Figure 9 As shown in C, the colon pictures of each group are as follows Figure 9 As shown in D.

[0132] Weight change = (W 第n天 -W 第一天 ) / W 第一天 ×100%

[0133] Disease Activity Index = (Score 体重 +Score 大便粘稠 +Score 大便出血 ) / 3

[0134] Depend on Figure 9 As shown in Figure A, the dynamic changes in body weight over 7 days showed that the DSS-treated mice had the most significant weight loss (8.38%), while oral administration of free probiotics reduced the decline to 6.00%. Notably, mice treated with the probiotic microsphere formulation showed the slowest weight loss, emphasizing its protective effect against DSS-induced morbidity. Figure 9 As shown in Figure B, the disease activity index (DAI) score also reflects this trend. AMS@Eud reduced DAI to 1.70±0.30 (significantly lower than the DSS group (3.33±0.40)), reflecting the improvement of clinical symptoms. Figure 9 As shown in Figures C and D, AMS@Eud-treated mice retained a significantly longer colon (6.39±0.35 cm) compared with the DSS group (5.04±0.61 cm), indicating that epithelial damage was alleviated and DSS-mediated intestinal toxicity was reduced.

[0135] 6. H&E staining test of gastric acid-resistant and long-lasting stable probiotic microsphere preparation

[0136] The DSS inflammatory bowel disease model established by in vivo delivery performance testing was selected, and mice were euthanized at the predetermined time for histological observation. H&E staining: The tissue sections were dewaxed and hydrated, then stained with hematoxylin to highlight the blue color of the cell nuclei. Then, acid alcohol was used for differentiation to remove excess dye; then, eosin was used to stain the cytoplasm to make it appear red; finally, the sections were dehydrated, transparentized, and sealed for microscopic observation. The results are as follows: Figure 10 shown.

[0137] The gastric acid-resistant and long-lasting stable probiotic microsphere preparation of the present invention has shown excellent effects in promoting the recovery of inflammatory bowel disease. Figure 10 As can be seen from the H&E images, more inflammatory cells appeared in the DSS group. This is because DSS destroyed the epithelial monolayer barrier of the large intestine during gavage, resulting in damage to intestinal epithelial cells and increased intestinal permeability. The colon of the control group mice maintained an intact structure, characterized by a well-organized epithelial layer, preserved goblet cells and a clear crypt structure. In contrast, DSS-induced mice showed severe colon damage: complete loss of goblet cells, crypt collapse, extensive inflammatory cell infiltration, and mucosal ulcers. Mice given the free probiotics obtained in Example 2 showed partial recovery of colon structure and reduced inflammation, but local ulcers persisted. It is worth noting that the AMS@Eud treatment prepared in Example 1 resulted in substantial structural recovery, with minimal ulcers, preserved crypt morphology and a significant reduction in inflammatory cell infiltration.

[0138] 7. Immunological staining test of gastric acid-resistant and long-lasting stable probiotic microsphere preparations

[0139] The DSS inflammatory bowel disease model established by in vivo delivery performance testing was selected, and mice were euthanized at a predetermined time for immunological observation. TNF-α immunohistochemical staining: First, tissue sections were prepared and fixed on glass slides, followed by dewaxing and hydration; heat-induced antigen retrieval was used to expose the antigen, and then endogenous peroxidase was inactivated to reduce background staining; protein blocking was then performed to prevent nonspecific binding; specific primary antibodies were added for incubation, and enzyme-labeled secondary antibodies were added after washing and incubation; after washing again, color development was performed with a colorant, and the positive area was colored; finally, nuclear staining was performed to enhance tissue structure contrast, and the slides were sealed to preserve the staining results for easy observation under a microscope. The results are shown in Figure 2. Figure 11 shown.

[0140] Depend on Figure 11The results indicate that TNF-α expression levels in infected wounds treated with the gastric acid-resistant, long-lasting, and stable probiotic microsphere preparation were lower, consistent with the H&E staining results. This suggests that the gastric acid-resistant, long-lasting, and stable probiotic microsphere preparation has excellent anti-inflammatory properties against DSS-induced inflammatory bowel disease in mice and promotes colon repair. During the recovery process of inflammatory bowel disease, the gastric acid-resistant, long-lasting, and stable probiotic microsphere preparation can effectively reduce the inflammatory response, promote intestinal repair, and alleviate structural damage.

[0141] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A method for preparing a probiotic microsphere preparation, characterized in that: Calculated by mass, comprising the following steps: Step 1: Prepare the inner core of the probiotic microsphere preparation: Step 101: dissolving 2 to 4 parts of sodium alginate, 2 to 4 parts of fructooligosaccharides, 0.1 to 0.5 parts of glycerol, and 0.1 to 0.5 parts of calcium carbonate powder in 92 to 96 parts of deionized water, and stirring to obtain a quasi-precursor solution; Step 102: Add probiotics to the quasi-precursor solution obtained in step 101 and prepare to 10 8 ~10 9 cfu / ml, and the precursor solution was obtained; Step 103: mixing the precursor solution obtained in step 102 with the oil phase, adding an emulsifier to obtain an emulsion; Step 104: adding 1-2 ml of an acidic liquid to the emulsion obtained in step 103 to initiate a cross-linking reaction to obtain a probiotic microsphere preparation core; Step 2: Construct the core-shell structure: Step 201: Mixing 65-70 parts of L100, 5-10 parts of triethyl citrate, 20-30 parts of magnesium stearate, 333.75-341.25 parts of acetone, 511.75-523.25 parts of isopropyl alcohol, and 44.5-45.5 parts of water to obtain a coating solution; Step 202: Disperse the probiotic microsphere preparation core obtained in step 104 in the coating solution, and stir at a speed of 800-1200 rpm for 30-60 minutes in a water bath at 37-40° C. to obtain microspheres; Step 203: Wash the surface of the microspheres alternately with n-hexane and water to obtain a probiotic microsphere preparation.

2. The method according to claim 1, characterized in that In step 103 , the volume ratio of the oil phase to the precursor solution is 2 to 4:1; the emulsifier is Span 80, and the volume ratio of the emulsifier to the oil phase is 1:100 to 3:

200.

3. The method according to claim 1, characterized in that The step 101 specifically includes: Step 1011, dissolving sodium alginate in deionized water and stirring for 1 to 2 hours; Step 1012: Add oligofructose and continue stirring for 1 to 2 hours; Step 1013: add glycerol and stir for 10 to 30 minutes, and then add calcium carbonate powder and stir for 30 to 60 minutes; Step 1014: Finally, add probiotics and stir for 10 to 20 minutes until uniform, to obtain a quasi-precursor solution.

4. The method according to claim 1, wherein After step 104, the following steps are further included: Step 105: Alternately washing with anhydrous ethanol and water to remove the surface oil phase of the probiotic microsphere preparation inner core obtained in step 104 to obtain a pure probiotic microsphere preparation inner core; Step 106: The purified probiotic microsphere preparation core obtained in step 105 is sealed and stored at 4-8°C.

5. The method according to claim 1, wherein The probiotic is at least one of Lactobacillus reuteri, Lactobacillus acidophilus, Saccharomyces boulardii, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium and Streptococcus thermophilus; The oil phase is at least one of soybean oil, olive oil, mineral oil, liquid paraffin, silicone oil, castor oil and linseed oil.

6. The method according to claim 1, characterized in that The acidic liquid is selected from at least one of glacial acetic acid, dilute hydrochloric acid, citric acid, lactic acid, succinic acid and formic acid.

7. A probiotic microsphere preparation, characterized in that: The probiotic microsphere preparation is prepared by the preparation method of any one of claims 1 to 9.

8. Use of the probiotic microsphere preparation according to claim 7 in inflammatory bowel disease.

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