Preparation method and application of lysozyme-modified adhesive butyrate microcapsules

Abstract

The application provides a lysozyme-modified adhesive butyrate microcapsule preparation method and application thereof, and belongs to the technical field of medicine manufacturing. The microcapsule has a core-shell structure, the outer layer material is a thiolated pectin microsphere, and the inside is wrapped with sodium butyrate-loaded egg white lysozyme nanoparticles. The application proves through in-vitro and in-vivo experiments that the microcapsule has good gastrointestinal stability and colon targeting, can prolong the intestinal retention time, improve the effective concentration of butyric acid in the colon, shield the bad smell, significantly improve the intestinal homeostasis related to colitis, and improve the drug taking acceptance. The application provides a green, safe and efficient technical solution for the preparation of exogenous butyrate drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological drugs, and particularly relates to a preparation method of lysozyme-modified adhesive butyrate microcapsules and application thereof. BACKGROUND

[0002] Ulcerative colitis (UC) is a common intestinal disease, which not only causes physiological and psychological burden to patients, but also brings heavy economic pressure to the public health system. Studies have shown that the level of butyric acid in the feces of UC patients is significantly reduced, and sodium butyrate (SB) plays an important role in maintaining intestinal homeostasis and regulating adaptive and innate immunity.

[0003] However, there are multiple challenges in the precise delivery of oral sodium butyrate to the colon (the main distribution area of intestinal symbiotic bacteria): sodium butyrate has a pungent odor, which seriously affects the sensory acceptance of its oral preparation; as a sodium salt, it is easily absorbed in the upper digestive tract, and the proportion entering the colon is limited; at the same time, it has a short retention time in the intestine, and the drug efficacy is limited. SUMMARY

[0004] The purpose of the present application is to provide a lysozyme-modified butyrate microcapsule, which realizes efficient encapsulation and protection of butyrate through a double-layer structure of thiolated pectin and egg white lysozyme. Not only can it target the delivery of butyrate to the colon, but also can prolong the retention time of butyrate in the colon through thiol-mediated intestinal adhesion, significantly improve the effective concentration of butyric acid in the UC state, and bring more obvious health benefits to the intervention of UC and other diseases.

[0005] The present application provides a butyrate microcapsule, comprising thiolated pectin and egg white lysozyme nanoparticles loaded with butyrate embedded in the thiolated pectin;

[0006] The mass ratio of the thiolated pectin and the egg white lysozyme nanoparticles loaded with butyrate is 0.5-2:1;

[0007] The mass ratio of butyrate and egg white lysozyme in the egg white lysozyme nanoparticles loaded with butyrate is 1:1-5.

[0008] Preferably, the thiol density of the thiolated pectin is 317.19-586.72 µmol / g.

[0009] Preferably, the mass ratio of the thiolated pectin and the egg white lysozyme nanoparticles loaded with butyrate is 1:1.

[0010] Preferably, the mass ratio of butyrate and egg white lysozyme in the egg white lysozyme nanoparticles loaded with butyrate is 1:3.

[0011] The application provides a preparation method of the butyrate microcapsule, and comprises the following steps:

[0012] The butyrate is encapsulated in egg white lysozyme nanoparticles to obtain egg white lysozyme nanoparticles loaded with butyrate;

[0013] The egg white lysozyme nanoparticles loaded with butyrate and a thiolated pectin solution are mixed to prepare an aqueous phase, and an emulsion microsphere is prepared by using a reverse emulsification method;

[0014] The emulsion microsphere is crosslinked under anaerobic conditions, impurities are removed, and the butyrate microcapsule is obtained.

[0015] Preferably, the method for encapsulating butyrate in egg white lysozyme nanoparticles comprises the following steps: mixing a butyrate solution and an egg white lysozyme solution, and ultrasonic treatment after heating treatment to obtain egg white lysozyme nanoparticles encapsulating sodium butyrate;

[0016] The temperature of the heating treatment is 80-90 DEG C, the time of the heating treatment is 30-60 min, and the pH value of the mixed system during the heating treatment is 2-4.

[0017] Preferably, the method for preparing the emulsion microsphere by using the reverse emulsification method comprises the following steps: mixing an emulsifier and a continuous phase to obtain an oil phase, and mixing and emulsifying the oil phase and an aqueous phase to obtain the emulsion microsphere;

[0018] The rotation speed of the emulsification is 5000-15000 rpm, and the time of the emulsification is 2-4 min.

[0019] The volume ratio of the oil phase to the aqueous phase is 20:1, and the volume ratio of the emulsifier to the continuous phase is 1:20-30.

[0020] Preferably, the time of the crosslinking is 6-12 h, and the rotation speed of the crosslinking is 250-350 rpm.

[0021] The application provides application of the butyrate microcapsule or the butyrate microcapsule prepared by the preparation method in preparation of a medicine for preventing and / or treating colitis.

[0022] The application provides a medicine for preventing and / or treating colitis, which comprises the butyrate microcapsule or the butyrate microcapsule prepared by the preparation method and a pharmaceutically acceptable adjuvant.

[0023] The butyrate microcapsule provided by the present application comprises thiolated pectin and butyrate-loaded egg white lysozyme nanoparticles embedded in the thiolated pectin; the mass ratio of the thiolated pectin and the butyrate-loaded egg white lysozyme nanoparticles is 0.5-2:1; and the mass ratio of butyrate and egg white lysozyme in the butyrate-loaded egg white lysozyme nanoparticles is 1:1-5. The butyrate microcapsule is encapsulated by a double-layer structure experiment, and the outer layer of thiolated pectin can improve the intestinal adhesion and significantly prolong the retention of the microcapsule. The thiolated pectin can also form a disulfide bond with mucin in the colonic mucus layer, thereby effectively slowing down the mechanical removal of the intestine and prolonging the local retention time of butyrate in the colon. Meanwhile, the thiolated pectin in the microcapsule system can resist the degradation of gastrointestinal digestive enzymes, and at the same time has the property of prebiotics and can be selectively utilized by colonic microorganisms; and the egg white lysozyme nanoparticles can intelligently respond to the colonic digestive enzyme environment. The two can synergistically achieve the stable protection of butyrate in the upper digestive tract and the precise controlled release in the colon. The particle size, encapsulation rate, drug loading rate and in-vitro simulated gastrointestinal release performance of the butyrate microcapsule are determined, the particle size of the butyrate microcapsule is 1.0-25.64 µm, the butyrate encapsulation rate is 26%-93.1%, and the drug loading rate is 5.5%-12.5%. The release rate of butyrate in artificial gastric juice is less than 10%, and the release rate in artificial intestinal juice is less than 30%, which shows excellent gastrointestinal stability and colon targeting. At the same time, the microcapsule system constructed based on thiolated pectin and egg white lysozyme can effectively mask the unpleasant odor of butyrate. The results of gastrointestinal fluorescence imaging of mice show that the butyrate microcapsule has good intestinal adhesion and can achieve enrichment and positioning in the colon, and can effectively prevent and treat colitis. It can be seen that the butyrate microcapsule can effectively reduce the colon shortening and inflammatory response by increasing the effective concentration of butyrate in the colon of UC mice, and promote the reconstruction of the mucus layer thickness, and improve the intestinal homeostasis of UC patients.

[0024] The preparation method of the butyrate microcapsule provided by the present application selects natural pectin and egg white lysozyme as main raw materials, which are safe, edible and non-toxic, suitable for oral preparations, meet the safety standards of functional foods and nutritional products, and have the characteristics of green safety and mild preparation. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The correlation analysis results of the thiol density of the microcapsule obtained under different crosslinking and curing times and the rheological properties of the mucus, wherein A is the thiol content result, B is the viscosity result, and C is the comparison of storage modulus (G') and loss modulus (G'') (MU represents pig small intestinal mucus); note: different letters represent significant differences between groups P <0.05;

[0026] Figure 2Figure 1 is a morphology determination result of the microcapsule, wherein A is a particle size distribution result of the SB-LYS nanoparticle, B is a particle size distribution result of the SB-LYS-PSH microcapsule, C is a scanning electron microscope image, and D is a transmission electron microscope image;

[0027] Figure 3 Figure 2 is an FTIR image of the microcapsule;

[0028] Figure 4 Figure 3 is a butyrate release curve of the microcapsule at different time points in vitro simulated digestion of the stomach and small intestine;

[0029] Figure 5 Figure 4 is a gastrointestinal tract imaging image (A) and a fluorescence intensity column chart (B) of the microcapsule at different time points in mice; wherein indicates a significant difference within the group P <0.05; indicates a significant difference within the group P <0.01;

[0030] Figure 6 Figure 5 is a principal component analysis image of an electronic nose sensor of the microcapsule;

[0031] Figure 7 Figure 6 is an effect of the microcapsule on the short-chain fatty acid level of the UC mice; wherein A is an acetic acid determination result, B is a propionic acid determination result, C is a butyric acid determination result, and D is a total short-chain fatty acid determination result; note: different letters indicate a significant difference between groups P <0.05;

[0032] Figure 8 Figure 7 is an effect of the microcapsule on the colon length of the UC mice; A is a colon length statistical result of the UC mice in each group, and B is a colon length morphological image of the UC mice in each group; note: different letters indicate a significant difference between groups P <0.05;

[0033] Figure 9 Figure 8 is an effect of the microcapsule on the inflammation condition of the UC mice; wherein A is a staining result, and B is a pathology score result; note: different letters indicate a significant difference between groups P <0.05;

[0034] Figure 10 Figure 9 is an effect of the microcapsule on the mucus layer thickness of the UC mice, wherein A is a staining result, and B is the thickness of the acid mucin layer and the neutral mucin layer; note: different letters indicate a significant difference between groups P <0.05. DETAILED DESCRIPTION

[0035] The application provides a butyrate microcapsule, which comprises thiolated pectin and lysozyme nanoparticles loaded with butyrate embedded in the thiolated pectin.

[0036] The mass ratio of the thiolated pectin and the butyrate-loaded egg white lysozyme nanoparticles is 0.5-2:1.

[0037] The mass ratio of the butyrate and the egg white lysozyme in the butyrate-loaded egg white lysozyme nanoparticles is 1:1-5.

[0038] In the present application, the butyrate microcapsules have a core-shell structure, the thiolated pectin serves as an outer shell, increases adhesion to the colonic mucus layer, resists gastrointestinal digestive enzymes, has prebiotic properties, thereby promoting the residence time of the active ingredient butyrate in the colon, precisely targets the colon for release, and also provides raw materials for microorganisms in the colon. The butyrate-loaded egg white lysozyme nanoparticles are preferably egg white lysozyme nanoparticles encapsulating butyrate inside. The egg white lysozyme nanoparticles can intelligently respond to the colonic digestive enzyme environment, and in cooperation with the thiolated pectin, achieve stable protection of butyrate in the upper gastrointestinal tract and precise controlled release in the colon. The butyrate as a pharmaceutical active ingredient can be sodium butyrate, magnesium butyrate or potassium butyrate, etc.

[0039] In the present application, the mass ratio of the thiolated pectin and the butyrate-loaded egg white lysozyme nanoparticles is preferably 0.5-2:1, and can be 1:1 or 1.5:1.

[0040] In the present application, the mass ratio of the butyrate and the egg white lysozyme in the butyrate-loaded egg white lysozyme nanoparticles is 1:1-5, and can be 1:2, 1:3 and 1:4. The mass ratio of butyrate and egg white lysozyme affects the particle size distribution of the nanoparticles and the drug loading rate and encapsulation rate of the microcapsules. The results of the present application show that when the ratio is 1:5, 1:4 and 1:3, the PDI results of the obtained nanoparticles show that the system particle size distribution is relatively uniform; when the ratio is 1:1, the PDI significantly increases, suggesting that the nanoparticles are excessively aggregated, resulting in a slightly uneven system distribution. In terms of encapsulation rate and drug loading rate, the encapsulation rates of 1:5, 1:4 and 1:3 are 97.1±2.7%, 96.1±1.6% and 95.3±2.0% respectively, which are significantly higher than 32.0±3.3% under the condition of 1:1; the drug loading rate gradually increases from 1:5 to 1:3 (16.3±1.8%, 19.4±2.6%, 24.1±0.6%), and reaches 24.2±0.9% under the condition of 1:1, which is not significantly different from 1:3. It is speculated that the reason may be that the hydrophobic inner cavity of lysozyme has limited binding sites, and the encapsulation capacity has an upper limit, and excessive butyrate no longer increases the encapsulation efficiency.

[0041] In the present application, the thiol density of the thiolated pectin is preferably 317.19-586.72 µmol / g. The preparation method of the thiolated pectin preferably comprises the following steps: dissolving pectin, mixing and reacting with cysteine under the action of EDC and NHS, removing impurities by dialysis to obtain thiolated pectin. The dialysis time is preferably 48 h, and the dialysis is sequentially performed in the following procedures: 5.0 mM HCl solution for 12 h, 5.0 mM HCl solution containing 1.0% NaCl for 12 h, 5.0 mM HCl solution for 12 h, and 1.0 mM HCl solution for 12 h.

[0042] The present application provides a preparation method of the butyrate microcapsule, comprising the following steps:

[0043] The butyrate is encapsulated in egg white lysozyme nanoparticles to obtain egg white lysozyme nanoparticles loaded with butyrate;

[0044] The egg white lysozyme nanoparticles loaded with butyrate and a thiolated pectin solution are mixed to prepare an aqueous phase, and an emulsion microsphere is prepared by reverse phase emulsification;

[0045] The emulsion microsphere is crosslinked under anaerobic conditions, and impurities are removed to obtain a butyrate microcapsule.

[0046] The present application encapsulates butyrate in egg white lysozyme nanoparticles to obtain egg white lysozyme nanoparticles loaded with butyrate.

[0047] In the present application, the method for encapsulating butyrate in egg white lysozyme nanoparticles preferably comprises the following steps: mixing a butyrate solution and an egg white lysozyme solution, heating treatment, and then ultrasonic treatment to obtain egg white lysozyme nanoparticles encapsulating sodium butyrate. The concentration of the butyrate solution is preferably 0.5-2 mg / mL, and can be 1-1.5 mg / mL. The concentration of the egg white lysozyme solution is preferably 0.5-2 mg / mL, and can be 1-1.5 mg / mL. The heating treatment temperature is preferably 80-90℃, and can be 82-88℃, or can be 85℃. The heating treatment time is preferably 30-60 min, and can be 35-55 min, or can be 40-50 min, or can be 45 min. The pH value of the mixed system during the heating treatment is preferably 2-4, or can be 3. The heating treatment causes partial conformation unfolding of the egg white lysozyme and initiates a nanogelation reaction. The ultrasonic treatment power is preferably 100-200 W, and the working time is 5-10 s, the interval is 10 s, and the total time is 5-10 min. The ultrasonic treatment utilizes cavitation effect to promote uniform dispersion of molecules and reduce particle size, so that egg white lysozyme nanoparticles encapsulating butyrate with uniform particle size are finally obtained.

[0048] In the present application, the method for preparing emulsion microspheres by reverse phase emulsification method comprises mixing emulsifier and continuous phase to obtain oil phase, mixing and emulsifying the oil phase and water phase to obtain emulsion microspheres. The emulsifier is preferably sorbitan fatty acid ester (Span series), such as Span 80. The continuous phase is preferably mineral oil (such as liquid paraffin) or vegetable oil (such as corn oil). The rotation speed of emulsification is preferably 5000-15000 rpm, which can be 8000-12000 rpm, and can be 10000 rpm. The emulsification time is preferably 2-4 min, which can be 3 min. The volume ratio of the oil phase and the water phase is preferably 20:1. The volume ratio of the emulsifier and the continuous phase is preferably 1:20-30. The crosslinking time is preferably 6-12 h, which can be 6-10 h, or can be 6-8 h. The crosslinking rotation speed is preferably 250-350 rpm, which can be 300 rpm.

[0049] The butyrate microcapsules prepared by the present application have a butyrate release rate of less than 10% in artificial gastric juice (2 hours) and a release rate of less than 30% in artificial intestinal juice (6 hours).

[0050] The present application provides the use of the butyrate microcapsules or the butyrate microcapsules prepared by the preparation method in the preparation of a medicine for preventing and / or treating colitis.

[0051] In the embodiments of the present application, the butyrate microcapsules can effectively avoid release in the upper digestive tract after oral administration, achieve colon positioning delivery, significantly slow down intestinal clearance, prolong the residence time of butyrate in the colon tissue, significantly improve the effective concentration of butyrate in UC state, and further improve the intestinal homeostasis of colitis mice.

[0052] The present application provides a medicine for preventing and / or treating colitis, which comprises the butyrate microcapsules or the butyrate microcapsules prepared by the preparation method and a pharmaceutically acceptable excipient.

[0053] In the present application, the dosage form of the medicine preferably comprises oral preparations, such as tablets, powders, suspensions, capsules, granules, etc. The excipient is conventionally selected according to the dosage form of the medicine. The preparation method of the medicine is not particularly limited in the present application, and the medicine dosage form preparation known in the art can be used.

[0054] The preparation method and application of the lysozyme-modified adhesive butyrate microcapsules provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0055] Example 1

[0056] Preparation method of thiolated pectin polysaccharide

[0057] A 1.25% (w / v) commercial pectin (PEC) solution was prepared and the pH of the solution was adjusted to 4.0, EDC and NHS powders were added to a final concentration of 50 mM, followed by stirring for 15 min. After the stirring time was reached, cysteine powder was added to the mixed solution at a mass ratio of 0.6, 0.9, 1.5 to commercial pectin, after which the pH of the solution was adjusted to 5.0 and the reaction was continued for 3 h at a stirring rate of 300 r / min. When the reaction was completed, the pH of the solution was adjusted to 6.0 and the reaction was continued for 30 min. It is worth noting that the entire synthesis process was carried out in a nitrogen atmosphere and in the dark. After the reaction was completed, in order to remove excess cysteine, EDC and NHS, a dialysis bag with a molecular weight cut-off of 3000 Da was used and the resulting mixed solution was sequentially dialyzed for 48 h in the dark: 5.0 mM HCl for 12 h, 5.0 mM HCl containing 1.0% NaCl for 12 h, 5.0 mM HCl for 12 h, 1.0 mM HCl for 12 h. Until no cysteine was detected in the dialysate, indicating the successful synthesis of thiolated pectin.

[0058] Example 2

[0059] A method for preparing an adhesive butyrate microcapsule with colon targeting and intestinal adhesion

[0060] An egg white lysozyme aqueous solution (concentration 2 mg / mL) was mixed with a sodium butyrate aqueous solution (concentration 0.4 mg / mL) in equal volume ratio, controlling the mass ratio of sodium butyrate to egg white lysozyme to be 1:5; an egg white lysozyme aqueous solution (concentration 1.5 mg / mL) was mixed with a sodium butyrate aqueous solution (concentration 0.5 mg / mL) in equal volume ratio, controlling the mass ratio of sodium butyrate to egg white lysozyme to be 1:3; an egg white lysozyme aqueous solution (concentration 2 mg / mL) was mixed with a sodium butyrate aqueous solution (concentration 2 mg / mL) in equal volume ratio, controlling the mass ratio of sodium butyrate to egg white lysozyme to be 1:1. Stir at 300 rpm under the condition of pH 3 until the mixed system is uniform. Subsequently, the mixed solution was heated at 90°C for 40 min to initiate the nanogelation reaction. After cooling, the solution was subjected to probe-type ultrasonic treatment (power 150 W, working 8 s, intermittent 10 s, total time 8 min), using the cavitation effect to promote uniform dispersion of molecules and reduce particle size, finally obtaining egg white lysozyme nanoparticles with uniform particle size and encapsulated butyrate.

[0061] The thiolated pectin prepared in Example 1 was dissolved in deionized water to prepare a 2% (w / v) aqueous solution. Using the reverse phase emulsification method, the emulsifier Span 80 was first added to liquid paraffin at a volume ratio of 1:25 (Span 80: liquid paraffin, volume ratio), stirred until completely dissolved, and a uniform oil phase was formed. The sodium butyrate-encapsulated egg white lysozyme nanoparticles were dispersed in the above thiolated pectin aqueous solution at a mass ratio of 1:2, 1:1, and 2:1. The two phases were mixed at a volume ratio of 20:1, and then emulsified using a high-speed homogenizer at 10,000 rpm for 3 min to form emulsion microspheres. After emulsification, oxygen was introduced into the emulsion at room temperature, and the mixture was stirred at 300 rpm for 8 h to allow the thiolated pectin to crosslink and solidify. After the reaction was completed, the liquid paraffin was removed by centrifugation at 8,000 rpm for 10 min, and the residual oil phase was washed with n-hexane. Finally, the resulting product was dried at 35°C to obtain adhesive butyrate microcapsules.

[0062] The particle size distribution and average particle size were measured by dynamic light scattering (DLS) on a laser particle size analyzer Zetasizer Nano-ZS90. The field emission scanning electron microscope (FE-SEM) was used to observe the nanoparticles after freeze-drying. In order to better observe the sample morphology, the conductivity of the nanoparticle powder was enhanced by gold spraying treatment. Transmission electron microscopy (TEM) was used to observe the structure of the nanoparticles. 10 μL of nanoparticle dispersion was dropped onto a 400-mesh carbon support film mesh, and the excess dispersion was absorbed with filter paper. The sample was then observed under an acceleration voltage of 80 kV.

[0063] The thiol content was determined as follows: 5 mg / mL microcapsule aqueous solution and 0.5 M phosphate buffer (pH 8.0) were prepared, and a 0.03% (w / v) DTNB solution was prepared using the buffer. 0.2 mL of the microcapsule solution was mixed with 1.8 mL of the buffer, 2 mL of the DTNB solution was added, and the mixture was mixed and reacted in the dark for 15 min. The supernatant was measured for absorbance at a wavelength of 412 nm, and the thiol content was calculated based on the cysteine standard curve.

[0064] In addition, the encapsulation efficiency (EE) and loading efficiency (LE) of sodium butyrate in the obtained microcapsules were calculated according to the following formulas:

[0065] EE (%) = encapsulated sodium butyrate mass / total sodium butyrate added mass x 100% Formula I;

[0066] LE (%) = encapsulated sodium butyrate mass / total microcapsule mass x 100% Formula II.

[0067] The test results include: SB-LYS nanoparticles as the research object, under the conditions of butyrate and egg white lysozyme mass ratio of 0.2-1, preparation and characterization, the results are shown in Table 1.

[0068] Table 1 Comparison of average particle size, polydispersity index (PDI) and butyrate encapsulation rate and drug loading rate of SB-LYS nanoparticles prepared under different mass ratios

[0069]

[0070] Note: Lowercase letters indicate significant differences between sample groups p <0.05).

[0071] The particle size of all prepared particles is concentrated in the nanometer range (167.0±7.4-354.1±8.7 nm). With the increase of butyrate ratio (1:5, 1:4, 1:3, 1:1), the average particle size shows a gradual increasing trend (167.0±7.4, 180.1±5.2, 208.1±4.0 and 354.1±8.7 nm, respectively). This phenomenon shows that high proportion of butyrate combines with egg white lysozyme more widely through electrostatic interaction and hydrogen bond, prompting part of the protein structure to unfold and intermolecular crosslinking occurs, eventually forming larger aggregates. The polydispersity index (PDI) further verifies this trend, PDI reflects the uniformity of particle size distribution, and the smaller the value, the more concentrated the distribution. When the ratio is 1:5, 1:4, 1:3, the PDI of the obtained nanoparticles is 0.364±0.020, 0.379±0.037, 0.355±0.037, respectively, showing that the particle size distribution of the system is relatively uniform; when the ratio is 1:1, the PDI significantly increases to 0.502±0.013, indicating that the excessive aggregation of nanoparticles leads to uneven distribution of the system. In terms of encapsulation rate and drug loading rate, the encapsulation rates of 1:5, 1:4, 1:3 are 97.1±2.7%, 96.1±1.6% and 95.3±2.0%, respectively, which are significantly higher than that of 1:1, which is 32.0±3.3%. Drug loading rate gradually increases from 1:5 to 1:3 (16.3±1.8%, 19.4±2.6%, 24.1±0.6%), and reaches 24.2±0.9% under the condition of 1:1, which has no significant difference with 1:3. The reason is that the hydrophobic lumen of lysozyme has limited binding sites, and the encapsulation capacity has an upper limit, and excessive butyrate does not increase the encapsulation efficiency. In summary, when the mass ratio of butyrate to egg white lysozyme is 1:3, it has better particle size uniformity and encapsulation performance, which is suitable for the construction of subsequent microcapsule system.

[0072] Table 2 Average particle size of microcapsules prepared under different shear forces and the effect of different mass ratios of SB-LYS nanoparticles to thiolated pectin on butyrate encapsulation rate and drug loading rate in microcapsules

[0073]

[0074] Note: Lowercase letters indicate significance between samples within the same group, and uppercase letters indicate significance between sample groups. p <0.05).

[0075] In the reverse emulsification process for preparing microcapsules, different stirring speeds (5000–15000 rpm) effectively controlled the droplet size (Table 2). At a stirring speed of 5000 rpm, the resulting microcapsules had a larger particle size (22 ± 3.64 µm); at 10000 rpm, the particle size decreased to 5.5 ± 1.32 µm; and at 15000 rpm, the particle size further decreased to 1.5 ± 0.50 µm. Regarding encapsulation performance, the large-diameter microcapsules (22 ± 3.64 µm) exhibited a higher encapsulation efficiency, superior to the small- and medium-diameter microcapsules (5.5 ± 1.32 µm and 1.5 ± 0.50 µm). This is mainly attributed to their larger internal cavity, which provides a more stable containment space for the SB-LYS nanoparticles; while the small-diameter microcapsules, due to their limited internal space, had limited encapsulation capabilities. As the proportion of SB-LYS nanoparticles in the system increased (i.e., the mass ratio of SB-LYS to thiolated pectin increased from 1:2 to 1:1, and then to 2:1), the overall encapsulation efficiency showed a decreasing trend. This indicates that within a limited encapsulation space, once the amount of nanoparticles exceeds a certain threshold, the encapsulation efficiency cannot be further improved. Instead, the excessively dense distribution and the tendency of the system to become oversaturated negatively impact the encapsulation effect. Further analysis of the drug loading rate revealed that when the mass ratio of SB-LYS to thiolated pectin increased from 1:2 to 1:1, the drug loading rate significantly improved; however, when the ratio continued to increase to 2:1, the improvement in drug loading rate was not significant. This indicates that the system was already close to the saturation loading limit of nanoparticles at this point, and further increasing the proportion of nanoparticles had limited effect on enhancing drug loading capacity.

[0076] After reverse emulsification, the thiol groups between thiolized pectin molecules can undergo covalent cross-linking under oxygen conditions, thereby achieving microcapsule curing. Different curing times (6–12 h) have a significant impact on the thiol density of the microcapsules and their interaction with the mucus.

[0077] Figure 1 The results show the thiol density of the microcapsules and their interaction with the mucus after curing for 6 h, 9 h, and 12 h. Figure 1 As shown in Figure A, the thiol density of the microcapsules gradually decreased with the extension of curing time. The thiol densities corresponding to 6 h, 9 h, and 12 h of curing were 586.72±7.93 µmol / g, 466.7±14.1 µmol / g, and 317.19±13.15 µmol / g, respectively. Figure 1As shown in Fig. 3B, the viscosity of the solution of the microcapsules obtained by the three curing times decreased with the increase of the shear rate after mixing with natural mucus, showing a typical shear-thinning characteristic. Among them, the viscosity of the 6 h curing group was significantly higher than that of the 9 h and 12 h groups. Figure 1 Fig. 3C further shows that in the dynamic rheological test, the storage modulus (G') of all samples was greater than the loss modulus (G''), showing an elastic dominant characteristic. As an indicator of the strength of the gel network, the higher the value of G', the more stable the structure. The results showed that the modulus of the 6 h curing group was the highest. Comprehensive analysis shows that the changes of G' and G'' are positively correlated with the density of sulfhydryl groups, that is, the higher the content of sulfhydryl groups, the stronger the interaction between the microcapsules and the mucus.

[0078] In the subsequent performance verification, the SB-LYS nanoparticles prepared under the condition of a butyrate to egg white lysozyme mass ratio of 1:3 were selected, and the SB-LYS-PSH microcapsules were constructed under the process conditions of an SB-LYS nanoparticle to thiolated pectin mass ratio of 0.2-5:1, an inverse emulsification shear force of 5000 rpm, and a curing time of 6 h. Under the conditions, the SB-LYS nanoparticles Figure 2 Fig. 4A) and the SB-LYS-PSH microcapsules Figure 2 Fig. 4B) both showed a single peak shape, indicating that the system had good uniformity. Among them, the average particle size of the SB-LYS nanoparticles was 208.1±4.0 nm, the average particle size of the SB-LYS-PSH microcapsules was 22±3.64 µm, and the density of sulfhydryl groups was 586.72±7.93 µmol / g. The scanning electron microscope (SEM, Figure 2 Fig. 4C) and the transmission electron microscope (TEM, Figure 2 Fig. 4D) observation results further confirmed that the SB-LYS nanoparticles showed a typical core-shell structure, directly revealing the hydrophobic interaction between the SB alkane chain and the hydrophobic residues of lysozyme, indicating that the SB was effectively encapsulated and formed stable nanoparticles. After loading the SB-LYS nanoparticles into the microcapsules, the SB-LYS-PSH microcapsules showed a rough surface morphology, and nanoparticles were distributed on the surface and inside of the microcapsules.

[0079] Example 3

[0080] Characterization of FTIR structure

[0081] The structure characteristics of sodium butyrate, egg white lysozyme and thiolated pectin were further verified by FTIR spectrum. The specific steps were as follows: about 1 mg of dried sample was mixed with 200 mg of potassium bromide, ground and pressed into a tablet, and then scanned by a Nicolet iN10 Fourier transform infrared spectrometer. The scanning range was set to 4000-400 cm -1 .

[0082] The test results are as follows Figure 3 As shown, the most distinctive characteristic peak of pure sodium butyrate is located at 937 cm⁻¹. -1 1254 cm -1 and 1561 cm -1 , representing the unsaturated CH bending vibration, CO stretching vibration, C=O stretching vibration, and CH stretching vibration, respectively. In the FTIR spectrum of egg white lysozyme, at 1542 cm⁻¹... -1 1657 cm -1 and 3302 cm -1 The characteristic spectra at 937 cm⁻¹ represent the vibrations of amide II (-CN and -NH), amide I (-C=O), and -OH, respectively. In the spectrum of SB-LYS nanoparticles, the characteristic spectrum at 937 cm⁻¹ represents the vibrations of amide II (-CN and -NH), amide I (-C=O), and -OH, respectively. -1 1254 cm -1 and 1561 cm -1 There was no obvious peak for sodium butyrate in the wavenumber region; only the peak for egg white lysozyme at 1542 cm⁻¹ was retained. -1 1657cm -1 and 3302 cm -1 The characteristic peak at [value missing] indicates that sodium butyrate was successfully encapsulated into egg white lysozyme nanoparticles. Furthermore, the main characteristic peak of thiolated pectin PSH is at 954 cm⁻¹. -1 1013cm -1 1043cm -1 1106 cm -1 and 1538 cm -1 When SB-LYS nanoparticles were encapsulated in PSH microcapsules, characteristic peaks of egg white lysozyme and thiolated pectin were observed, indicating that SB-LYS nanoparticles were distributed on both the surface and inside the microcapsules. This is consistent with the SEM results. Figure 2 Corresponding to C).

[0083] Example 4

[0084] In vitro simulated gastric and small intestinal digestive microcapsules

[0085] In the in vitro simulation of gastric digestion process, first in 10 mL SGF solution according to 2000 U / mL added pepsin until it is completely dissolved, followed by the addition of 10 mg sodium butyrate sample and 100 mg SB-LYS-PSH microcapsule sample, and the solution pH value is adjusted to 3. In a 37°C water bath shaker digestion, at different time points 0 min, 30 min, 60 min, 90 min, 120 min each take out 200 μL digestion solution, after enzyme inactivation through 0.22 μm water film, then use HPLC to determine the SB content, the specific steps are: using C18 chromatographic column for chromatographic analysis, the selected mobile phase is chromatographically pure acetonitrile and 0.1% phosphoric acid solution mixture, wherein the volume ratio of acetonitrile and phosphoric acid solution is 20:80. The flow rate of the system is set to 1.0 mL / min, the working wavelength of the ultraviolet detector is set to 206 nm, and the working temperature of the chromatographic column is maintained at 30°C, the injection amount is 20 μL.

[0086] In the in vitro simulation of small intestinal digestion process, first in 9 mL SIF solution add 10 mM bile and according to 100 U / mL added trypsin until completely dissolved, then mix the remaining 9 mL SGF solution with 9 mL SIF solution, and adjust the solution pH to 7. In a 37°C water bath shaker digestion, at different time points 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, 360 min each take out 200 μL digestion solution, after enzyme inactivation through 0.22 μm water film, use HPLC to determine the SB content, the method is the same as above.

[0087] The test results are shown in Figure 4 . Figure 4The release profiles of SB-LYS nanoparticles and SB-LYS-PSH microcapsules under in vitro simulated gastrointestinal digestion conditions were demonstrated, in which unencapsulated SB was used as a control, which had good stability in both simulated gastric and intestinal environments. The results showed that in the SGF stage, the SB-LYS nanoparticles had a faster release in the first 30 min, with a release rate of 18.3 ± 3.6%, and showed a stable release rule in 30-120 min. At the end of 120 min SGF digestion, the final SB release rate was 46.0 ± 4.8%. After reaching the SIF stage, the SB in the SB-LYS nanoparticles was still continuously released, and the cumulative release rate reached 86.9 ± 4.4% after 180 min of small intestine digestion, and then remained basically constant until the end of 360 min of small intestine digestion. In contrast, after encapsulating SB-LYS nanoparticles into PSH microcapsules, the SB release rate of SB-LYS-PSH microcapsules was 9.7 ± 2.6% at the end of SGF digestion, and the cumulative SB release rate was only 26.6 ± 8.0% after 360 min of SIF digestion. The results showed that encapsulating SB into egg white lysozyme nanoparticles could delay the release of SB, but due to the easy degradation of egg white lysozyme by enzymes present in the gastrointestinal tract, the protection ability of SB was limited. However, after encapsulating SB-LYS nanoparticles into thiolated pectin microcapsules, the stability of thiolated pectin in the gastrointestinal environment significantly improved the ability of the composite carrier to resist gastric and intestinal digestion, effectively avoiding the release of SB in the gastrointestinal tract and achieving colon targeting.

[0088] Example 5

[0089] Intestinal adhesion verification experiment of microcapsules

[0090] Cy5 dye was encapsulated into microcapsules by replacing butyrate according to the method of Example 2, and C57BL / 6J mice (4-6 weeks, about 23 g) were purchased from Hangzhou Medical College and divided into 3 groups: Cy5 group, Cy5-LYS group and Cy5-LYS-PSH group. According to the Cy5 of 0.16 mg / kg, the mice were gavaged, the dose was 400 μL, and the mice were dissected at different time points of 1 h, 2 h, 6 h, 12 h and 24 h. The whole gastrointestinal tract was taken out and the fluorescence intensity was observed using PerkinElmer IVIS Lumina III small animal live optical imaging system, the excitation wavelength of Cy5 was 650 nm, and the emission wavelength was 670 nm. Living Image software was used for image acquisition and fluorescence intensity analysis.

[0091] The experimental results are shown in Figure 5 . Figure 5 The gastrointestinal tract of mice was imaged in Figure 5 A) and the corresponding fluorescence intensityFigure 5 As a result, free Cy5 molecules have started to move from the small intestine to the colon after 1 h of administration, and part of the Cy5 release from Cy5-LYS is due to the degradation of egg white lysozyme by pepsin and the like, while there is almost no release of Cy5 molecules from the Cy5-LYS-PSH microcapsules. After 2 h of administration, the free Cy5 molecules have been transported to the colon, and Cy5-LYS can be observed to have obvious fluorescence in the small intestine, and the Cy5-LYS-PSH microcapsules still have only a few fluorescent molecules released. After 6 h, the fluorescence intensity of the free Cy5 molecules in the intestinal tract gradually decreases, and after 24 h, it has basically disappeared. Cy5-LYS is basically transported to the colon after 6 h and gradually metabolized, and after 24 h, no fluorescence is basically observed. While the Cy5-LYS-PSH microcapsules still exist in the small intestine after 6 h and 12 h, they reach the colon after 24 h and a high fluorescence intensity is observed in the colon, indicating that the Cy5 molecules in the microcapsules are released in large quantities in the colon. This result fully proves that the microcapsules can effectively avoid the premature release of fluorescent molecules in the upper digestive tract, target them to the colon and gradually release them, while the adhesion of thiolated pectin effectively slows down the clearance of Cy5 by the intestine, increasing the retention time of Cy5 in the colon tissue.

[0092] Example 6

[0093] Odor masking verification

[0094] Odor masking verification of the microcapsules for sodium butyrate can be measured by a Heracles II electronic nose system. The instrument includes an automatic sampler, MXT-5 (non-polar) and MXT-1701 (weakly polar) capillary columns, and two flame ionization detectors (FIDs). After calibration, 20 μL of n-alkane (C6-C16) standard was first analyzed using the same chromatographic conditions as the sample for peak calibration and retention index (RI) calculation. Next, 200 mg of sodium butyrate powder, LYS-PSH and SB-LYS-PSH microcapsules were added to 20 mL sample bottles, respectively, and the headspace gas in the sample bottle was saturated by incubating at 45°C for 30 min. 800 μL of headspace gas was captured for detection at a rate of 125 μL / s for 45 s, and data analysis was performed using the AroChemBase database.

[0095] The results of the test include: when the molecules are absorbed on the coating of the microbalance quartz crystal surface of the electronic nose sensor, the frequency of oscillation changes in proportion to the mass absorbed, and the resulting change in current is measured. The data of the sensor array is usually presented using multivariate statistical techniques, such as Principle component analysis (PCA), which transforms the original sensor signals into variables that are linear combinations of the signals. Figure 6The masking effect of SB-LYS-PSH microcapsules on the special odor of sodium butyrate is shown. The variance contribution rate of principal component 1 (PCA1) is 99.9997%, and the variance contribution rate of principal component 2 (PCA2) is 0.0007%. The cumulative variance contribution rate of the first two principal components is 100%, which is much higher than the general statistical limit of 85%. This indicates that the two principal components contain all the original information of the samples, and the differences between different samples are significant and have good discrimination. Further analysis and comparison show that SB is located on the positive half-axis of PCA1, and the complex carrier egg white lysozyme-mercapto pectin (LYS-PSH) and SB-LYS-PSH are located on the negative half-axis of PCA1. LYS-PSH and SB-LYS-PSH are far away from SB, indicating that the odor system of SB encapsulated into microcapsules has changed significantly compared with pure SB. The odor of SB-LYS-PSH microcapsules encapsulating SB is most similar to that of the egg white lysozyme-mercapto pectin complex carrier.

[0096] Example 7

[0097] Verification of microcapsules in improving the effective concentration of butyric acid in the colon of UC mice and relieving intestinal homeostasis of UC mice

[0098] All animal experiments were approved and conducted in accordance with the relevant regulations of China Institute of Metrology and the legal requirements of China animal research (Approval No. ZJCLA-IACUC-20010431). The specific steps are as follows: C57BL / 6J mice (4-6 weeks, 23 g or so, 60) were purchased from Hangzhou Medical College and raised in a specific pathogen-free environment (22±1℃). The mice were fed with standard purified feed and allowed to drink water freely, and the adaptation period was one week. After the adaptation period, the mice were randomly divided into 6 groups (10 in each group, 5 in each cage), corresponding to the control group (Control), model group (DSS), SB group, LYS-PSH group and SB-LYS-PSH group. During the 10-day experimental period, the mice in the Control group could drink water freely, and the mice in the other groups were allowed to drink 1.5% DSS solution freely on day 0. The body weight, diarrhea symptoms and blood in stool of the mice were monitored and scored every day. According to the modeling of the mice, 1.5% DSS induced obvious disease characteristics on the 5th day, so the mice were changed to drink water freely on the 5th day, and different reagents were administered at a dose of 400 µL / day. The blank group and the model group were administered with normal saline. After the collection of feces on the 10th day, all the mice were anesthetized, and blood samples were collected by orbital blood collection. The colon, cecal contents and colon contents were collected immediately after sacrifice, frozen in liquid nitrogen and stored at -80℃ for use.

[0099] Determination of short-chain fatty acid level

[0100] Approximately 100 mg of cecal content was weighed into 500 μΐ of deionized water, vortexed and filtered using a 0.45 μιη water membrane. The short chain fatty acid levels were determined using gas chromatography. The conditions for the determination were: detector and injector temperature both set to 240 °C, N2 as carrier gas at a flow rate of 19 mL / min. The temperature program was 100 °C for 0.5 min, followed by an increase of 4 °C / min to 180 °C. The injection volume was 1 μΐ, and the total test time was 25 min. The short chain fatty acid content was calculated from a standard curve in the range of 0-50 mM.

[0101] Observation of colon tissue sections

[0102] HE staining: The sections were placed in xylene I for 8 min, xylene II for 8 min, xylene III for 8 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then water for 2 min. Subsequently, the sections were stained with hematoxylin for 5 min, differentiated with hydrochloric acid for 2 s, blued with ammonia water for 15-30 s, and washed with water. Next, the sections were dehydrated with 95% ethanol, stained with eosin for 5-8 s, absolute ethanol I for 30 s, absolute ethanol II for 2.5 min, absolute ethanol III for 2.5 min, xylene I for 2.5 min, xylene II for 2.5 min, and finally mounted with neutral balsam. PAS staining: The sections were placed in xylene I for 20 min, xylene II for 20 min, absolute ethanol I for 10 min, absolute ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then distilled water. Subsequently, the sections were oxidized with 1% aqueous high iodine acid for 10 min, washed with water, placed in Schiff’s reagent and stained in the dark for 15-30 min, washed with water, and the nuclei were counterstained with hematoxylin for 1-2 min, washed with water, differentiated with 1% hydrochloric acid in ethanol for a few seconds, washed with water, blued with ammonia water for 10-30 s, and washed with water. Next, the sections were dehydrated and cleared in the following order: 95% ethanol I for 5 min; 95% ethanol II for 5 min; absolute ethanol I for 5 min; absolute ethanol II for 5 min; xylene I for 5 min; xylene II for 5 min; and finally mounted with neutral balsam. AB staining: The sections were placed in Aliein blue staining solution (pH 2.5) for 2-5 min, washed with water, stained with nuclear fast red for 5 min, washed with water, cleared with xylene for 5 min, and finally mounted with neutral balsam.

[0103] The results of the tests include:

[0104] The present application is based on the fact that the content of butyric acid in the colon under UC state decreases significantly, and a colon-targeted delivery microcapsule with adhesion for exogenous butyric acid supplement is designed. Therefore, the evaluation of the improvement of short-chain fatty acid levels, especially butyric acid content, in the intestinal tract of UC mice is an important basis for verifying that SB-LYS-PSH microcapsules can be used as an exogenous butyric acid supplement carrier. Figure 7 , the concentrations of acetic acid, propionic acid, butyric acid and total short-chain fatty acids in the cecum of DSS group mice were much lower than those of the control group. In contrast, only the intervention of SB-LYS-PSH microcapsules significantly increased the concentration of butyric acid in the cecum of UC mice. This result shows that the butyrate released by SB-LYS-PSH microcapsules after reaching the colon can increase the effective concentration of butyric acid. Surprisingly, the concentrations of acetic acid, propionic acid and total SCFAs also increased accordingly, which also confirms the positive role of butyric acid supplementation in regulating intestinal homeostasis, and when the intestinal homeostasis is restored, the ability of intestinal flora to metabolize short-chain fatty acids also improves.

[0105] As shown in Figure 8 , after anatomical analysis of the colon of mice, it was found that the colon length of mice after DSS treatment was significantly shortened compared with the control group. The SB group and LYS-PSH group did not have a significant improvement effect on the shortening of the colon length of UC mice. However, the SB-LYS-PSH microcapsule group significantly improved the shortening of the colon, and there was no significant difference in colon length between the control group.

[0106] As shown in Figure 9 A, by observing the H&E staining of the colon tissue, the tissue structure of the control group was complete, the gland structure was clear, and there was no inflammatory cell infiltration phenomenon. On the contrary, the colon tissue structure of the DSS treatment group was significantly damaged, the glands lost normal morphology, and was accompanied by a large number of inflammatory cell infiltration. In addition, the SB group and LYS-PSH complex carrier group did not effectively improve the intestinal damage caused by DSS. However, in the SB-LYS-PSH microcapsule treatment group, obvious tissue structure recovery was observed, the intestinal wall thickness was normal, and inflammatory cell infiltration was significantly reduced, indicating that the adhesive butyrate microcapsule has a significant intervention effect on UC. The colon histopathology score Figure 9 B) further confirms the superiority of SB-LYS-PSH microcapsules in inhibiting intestinal inflammation, and the score is close to the control group and much lower than other treatment groups.

[0107] As shown in Figure 10As shown, the colonic mucus layer is mainly composed of MUC2 secreted by goblet cells, which is an important barrier to isolate intestinal contents from epithelial cells. However, UC will cause goblet cells to be depleted, MUC2 secretion to be reduced, and thus the mucus layer to be thin, so that intestinal bacteria can directly contact epithelial cells to cause inflammation. In order to further determine the change of the mucus layer, the present application adopts PAS staining (periodic acid-schiff staining) for neutral mucin mucus layer observation and AB staining (alcian blue staining) for acid mucin mucus layer observation Figure 10 in the middle A) and the thickness of the mucus layer is calculated Figure 10 in the middle B). It can be known from Figure 10 the middle A that the control group mice have rich colonic goblet cells, clear mucus layer structure, and large mucus layer thickness. The DSS group has large-area loss of goblet cells, and the mucus layer structure cannot be observed. In comparison, the intervention of the SB group and the LYS-PSH group does not observe obvious mucus layer structure, however, the SB-LYS-PSH microcapsule after encapsulating butyrate obviously shows the restored mucus layer structure and thickness.

[0108] In summary, the SB-LYS-PSH microcapsule can be used as an efficient delivery carrier of exogenous butyric acid, which significantly improves the concentration of butyric acid in the colonic tissue of the UC mice. The enrichment of butyric acid not only can play an important role in regulating intestinal immunity, effectively alleviate the colonic shortening and inflammatory reaction of the UC mice, but also can stimulate the secretion of mucin by goblet cells, thereby restoring the thickness of the mucus layer, and embodies the significant improvement effect on the intestinal homeostasis of the UC mice.

[0109] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A butyrate microcapsule, characterized by, The butyrate microcapsule comprises the following steps: The mass ratio of the thiolated pectin and the butyrate-loaded egg white lysozyme nanoparticles is 0.5-2:

1. The mass ratio of the butyrate and the egg white lysozyme in the butyrate-loaded egg white lysozyme nanoparticles is 1:1-5. The butyrate microcapsule is prepared by the following steps: The butyrate is encapsulated in the egg white lysozyme nanoparticles to obtain the butyrate-loaded egg white lysozyme nanoparticles; the method for encapsulating the butyrate in the egg white lysozyme nanoparticles is to mix a butyrate solution and an egg white lysozyme solution, and then to obtain the butyrate-loaded egg white lysozyme nanoparticles by ultrasonic treatment after heating treatment; the heating treatment is performed at a temperature of 80-90℃. The butyrate-loaded egg white lysozyme nanoparticles and a thiolated pectin solution are mixed to prepare an aqueous phase, and an emulsion microsphere is prepared by reverse phase emulsification; The emulsion microsphere is crosslinked in an oxygen condition, and impurities are removed to obtain the butyrate microcapsule.

2. The butyrate microcapsules according to claim 1, characterized in that, The thiol density of the thiolated pectin is 317.19-586.72 µmol / g.

3. The butyrate microcapsules according to claim 1, wherein The mass ratio of the thiolated pectin and the butyrate-loaded egg white lysozyme nanoparticles is 1:

1.

4. The butyrate salt microcapsules according to any one of claims 1 to 3, characterized in that, The mass ratio of the butyrate and the egg white lysozyme in the butyrate-loaded egg white lysozyme nanoparticles is 1:

3.

5. The process for the production of the butyric acid salt microcapsule according to any one of claims 1 to 4, characterized by, The butyrate microcapsule comprises the following steps: The butyrate is encapsulated in the egg white lysozyme nanoparticles to obtain the butyrate-loaded egg white lysozyme nanoparticles; the method for encapsulating the butyrate in the egg white lysozyme nanoparticles is to mix a butyrate solution and an egg white lysozyme solution, and then to obtain the butyrate-loaded egg white lysozyme nanoparticles by ultrasonic treatment after heating treatment; the heating treatment is performed at a temperature of 80-90℃. The butyrate-loaded egg white lysozyme nanoparticles and a thiolated pectin solution are mixed to prepare an aqueous phase, and an emulsion microsphere is prepared by reverse phase emulsification; The emulsion microsphere is crosslinked in an oxygen condition, and impurities are removed to obtain the butyrate microcapsule.

6. The preparation method according to claim 5, characterized in that, The heating treatment is performed for 30-60 min; and the pH value of the mixed system during the heating treatment is 2-4.

7. The preparation method according to claim 5, characterized in that, The emulsion microsphere is prepared by reverse phase emulsification, wherein an emulsifier is mixed with a continuous phase to obtain an oil phase, and the oil phase and an aqueous phase are mixed to emulsify to obtain the emulsion microsphere. The rotation speed during the emulsification is 5000-15000 rpm; and the emulsification is performed for 2-4 min. The volume ratio of the oil phase and the aqueous phase is 20:1; and the volume ratio of the emulsifier and the continuous phase is 1:20-30.

8. The preparation method according to claim 5, characterized in that, The crosslinking is performed for 6-12 h; and the rotation speed during the crosslinking is 250-350 rpm.

9. The butyrate microcapsule of any one of claims 1-4 or prepared by the method of any one of claims 5-8 is used in the preparation of a drug for preventing and / or treating colitis.

10. A medicament for preventing and / or treating colitis, characterized by, The butyrate microcapsule of any one of claims 1-4 or prepared by the method of any one of claims 5-8 is used in the preparation of a drug for preventing and / or treating colitis.

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