Preparation method of synbiotics-loaded pH-responsive emulsion beads and application of synbiotics-loaded pH-responsive emulsion beads in targeted improvement of colitis

Abstract

The invention belongs to the technical field of microorganisms, and discloses a preparation method of synbiotics-loaded pH-responsive emulsion beads and application of the synbiotics-loaded pH-responsive emulsion beads in targeted improvement of colitis. Taking a mixed solution of activated and cultured probiotic bacterial sludge and fructo-oligosaccharide as a W1 phase; mixing the W1 phase with the oil phase, shearing and homogenizing to obtain a W1 / O emulsion; dispersing into a W2-phase aqueous solution dissolved with polysaccharide-protein mixed nanoparticles, and shearing and homogenizing to obtain a W1 / O / W2 dual emulsion; and cross-linking the double emulsion with polylysine, adding a sodium alginate solution, and then dropwise adding into a calcium chloride solution for curing to obtain the emulsion condensate beads. The synbiotics are loaded in the emulsion gel beads, so that the emulsion gel beads have excellent biocompatibility, enzyme triggering property and pH responsiveness. Experiments show that the emulsion gel beads improve the UC model rat colon shortening, repair the intestinal mucosa barrier, reduce the content of inflammatory factors IL-1beta, IL-6 and TNF-alpha, increase the content of an anti-inflammatory factor IL-10, increase the expression of tight junction proteins ZO-1, Claudin-1 and Occludin and regulate the intestinal flora balance of rats through synergistic interaction.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a preparation method of pH-responsive emulsion beads loaded with synbiotics and its application in targeted improvement of colitis. Background Art

[0002] Ulcerative colitis, as one of the two main forms of inflammatory bowel disease, is a non-specific chronic inflammatory disease of the colon. Patients usually suffer from a series of symptoms such as chronic abdominal pain, diarrhea, intestinal obstruction, fever, bloody stools, fatigue, and weight loss. In terms of the gastrointestinal tract, the typical characteristics of patients are the imbalance of inflammatory homeostasis and the damage of the intestinal epithelial barrier, and its incidence shows an increasing trend globally. Research shows that the onset of ulcerative colitis may be related to genetics, immune regulation, environment, and intestinal microorganisms. However, the exact pathogenesis of ulcerative colitis is still unclear. Existing treatment methods, such as antibiotics, corticosteroids, and immunomodulators, although having achieved certain success, are not suitable for long-term treatment due to the certain side effects of drug treatment.

[0003] In recent years, the relationship between the onset of ulcerative colitis and the intestinal flora has become a research hotspot. Research has proved that there are significant differences in the intestinal flora between people and animals with ulcerative colitis and healthy people. Patients with ulcerative colitis mostly show varying degrees of intestinal flora dysbiosis, and often involve changes in the number of multiple flora. Experiments show that using methods such as probiotic drug therapy and intestinal flora transplantation to improve the intestinal microecology of the body can have a good adjuvant treatment effect on people and animals with chronic ulcerative colitis. However, after being processed, stored, and transported to the harsh strong acid and strong base environment of the gastrointestinal tract, the number and activity of probiotics will both decline, resulting in poor treatment effects. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a preparation method of pH-responsive emulsion beads loaded with synbiotics.

[0005] The above technical object of the present invention is achieved through the following technical solutions: A preparation method of pH-responsive emulsion beads loaded with synbiotics, comprising the following steps:

[0006] S1: Under anaerobic conditions and at a temperature of 30-40°C, single colonies of probiotic strains are subjected to continuous two 12-24-hour activation cultures in a culture medium, and then centrifuged at a speed of 1000-5000 rpm for 2-15 minutes at a temperature of 0-10°C to obtain probiotic mud;

[0007] S2: The probiotic mud (1.0×10 8 ~1.0×10 12CFU / mL) and fructooligosaccharide with a mass concentration of 2-5% are dispersed in sterile distilled water and vortexed to form W 1 aqueous phase; polyglyceryl ricinoleate is used as a surfactant and fully stirred and dissolved in corn oil to obtain an oil phase; the oil phase and W 1 aqueous phase are mixed and stirred at a rotation speed of 8000-12000 rpm to prepare W 1 / O emulsion;

[0008] S3: Zein powder is fully dissolved in an ethanol aqueous solution of 70-80% under stirring conditions at 20-30 °C to obtain a zein alcohol solution; apple pectin powder is dissolved in deionized water at 20-30 °C and stirred for 2-3 h to obtain an apple pectin aqueous solution; the zein alcohol solution and the apple pectin aqueous solution are mixed, and the mass ratio of zein to apple pectin is 2:1, and then stirred at a rotation speed of 7000 rpm for 5-8 minutes; finally, rotary evaporation is carried out at 45 °C to obtain a zein-apple pectin nanoparticle mixed solution; after adjusting the pH of the solution to 4.0 with 1M HCl, W 2 phase aqueous solution;

[0009] S4: The W 1 / O emulsion is dispersed in the W 2 phase aqueous solution, and the volume ratio of the W 2 phase aqueous solution to the W 1 / O emulsion is 4:6; homogenize at a rotation speed of 5000 rpm for 1-5 minutes to obtain a W 1 / O / W 2 double emulsion;

[0010] S5: Take 5 mL of W 1 / O / W 2 double emulsion, crosslink with polylysine with a mass concentration of 0.1% under stirring at a rotation speed of 150 rpm, then add 2 mL of sodium alginate with a mass concentration of 2% for mixing, and inject the mixed emulsion into a CaCl 2 solution to prepare emulsion beads.

[0011] Furthermore, the probiotic strain in step S1 is one of Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium thermophilum.

[0012] Furthermore, the mass concentration of polyglyceryl ricinoleate in corn oil in step S2 is 0.5-4%.

[0013] Furthermore, the mass concentration of the zein-apple pectin mixed nanoparticles in the W 2 phase aqueous solution in step S3 is 1-5%.

[0014] To achieve the above object, the present invention also provides the use of the emulsion beads prepared by the above method for preparing emulsion beads in the targeted improvement of colitis.

[0015] In summary, the present invention has the following beneficial effects: In this application, probiotic sludge is first cultured and an aqueous phase is prepared, and then it is mixed with an oil phase to prepare a W 1 / O emulsion, and then zein and apple pectin are used to prepare a W 1 phase aqueous solution, and finally a W 2 loaded with synbiotics is obtained. 1 / O / W 2 double emulsion; then, by adding polylysine to fill the porous network of calcium alginate, a dense three-dimensional gel structure is further formed as a barrier, thereby preparing emulsion beads loaded with synbiotics, achieving the targeted release of synbiotics, and ensuring the quantity and activity of probiotics reaching the colon site. It can alleviate the colon shortening symptoms of rats in the repair of ulcerative colitis, reduce the inflammatory response of colon tissues, reduce the contents of inflammatory factors IL-1β, IL-6 and TNF-α in serum, increase the content of anti-inflammatory factor IL-10 at the same time, increase the expression of tight junction proteins ZO-1, Claudin-1 and Occludin in colon tissues, regulate the intestinal flora structure, and improve the symptoms of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flowchart of a method for preparing a pH-responsive emulsion bead loaded with synbiotics according to an embodiment of the present invention;

[0017] Figure 2 is a schematic diagram of the number of viable probiotics in vitro digestion simulation of microgel balls prepared from zein-apple pectin mixed nanoparticles with different concentrations according to an embodiment of the present invention (different lowercase letters above the column chart represent significant differences (P<0.05));

[0018] Figure 3 is a schematic diagram of in vitro digestion simulation of probiotic microgel balls prepared from zein-apple pectin mixed nanoparticles with different concentrations according to an embodiment of the present invention;

[0019] Figure 4 is a schematic diagram of the colon morphology of rats in each group according to an embodiment of the present invention;

[0020] Figure 5 is a schematic diagram of the colon length of rats in each group according to an embodiment of the present invention;

[0021] Figure 6 is a schematic diagram of the colon tissue pathology of rats in each group according to an embodiment of the present invention;

[0022] Figure 7Schematic diagram of the contents of IL-1β, IL-6, TNF-α, and IL-10 in the sera of rats in each group of the embodiments of the present invention (*P<0.05, **P<0.01, ***P<0.001);

[0023] Figure 8 Schematic diagram of the comparison of the optical densities of tight junction proteins ZO-1, Claudin-1, and Occludin in the colon tissues of rats in each group of the embodiments of the present invention;

[0024] Figure 9 Schematic diagram of the relative mRNA expression levels of tight junction proteins ZO-1, Claudin-1, and Occludin in the colon tissues of rats in each group of the embodiments of the present invention;

[0025] Figure 10 Schematic diagrams of the rarefaction curves, abundance curves, Venn diagrams, principal component analysis (PCA), principal coordinate analysis (PcoA), Simpson index, and Chao1 index of the fecal microbiota of rats in each group of the embodiments of the present invention;

[0026] Figure 11 Schematic diagram of the relative abundances of the main microbial flora components of the fecal microbiota of rats in each group of the embodiments of the present invention at the phylum level;

[0027] Figure 12 Schematic diagram of the relative abundances of the main microbial flora components of the fecal microbiota of rats in each group of the embodiments of the present invention at the genus level. Detailed implementation manners

[0028] As Figure 1 shown, the embodiments of the present application disclose the preparation of a pH-responsive emulsion bead loaded with synbiotics, including the following steps:

[0029] S1: Under anaerobic conditions and at a temperature of 30-40°C, single colonies of probiotic strains are subjected to continuous activation culture in a medium for 12-24 hours twice, and then centrifuged at a speed of 1000-5000 rpm for 2-15 minutes at a temperature of 0-10°C to obtain probiotic bacterial mud. The probiotic strain is one of Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium thermophilum.

[0030] S2: The probiotic bacterial mud (1.0×10 8 -1.0×10 12 CFU / mL) and fructooligosaccharide with a mass concentration of 2-5% are dispersed in sterile distilled water and vortexed to form W 1An aqueous phase, where fructooligosaccharide is used as a prebiotic; polyglyceryl ricinoleate is used as a surfactant and is fully stirred and dissolved in corn oil to obtain an oil phase, where the mass concentration of polyglyceryl ricinoleate in corn oil is 0.5 - 4%; then the oil phase and the W 1 aqueous phase are mixed and stirred at a rotation speed of 8000 - 12000 rpm to prepare a W 1 / O emulsion.

[0031] S3: Zein powder is fully dissolved in a 70 - 80% ethanol aqueous solution under stirring conditions at 20 - 30 °C to obtain a zein alcohol solution; apple pectin powder is dissolved in deionized water at 20 - 30 °C and stirred for 2 - 3 h to obtain an apple pectin aqueous solution; the zein alcohol solution and the apple pectin aqueous solution are mixed, where the mass ratio of zein to apple pectin is 2:1, and then stirred with a high - speed homogenizer at a rotation speed of 7000 rpm for 5 - 8 minutes to mix the solution evenly; finally, rotary evaporation is carried out at 45 °C to obtain a zein - apple pectin nanoparticle mixed solution, where the mass concentration of zein - apple pectin mixed nanoparticles is 1 - 5%. After adjusting the pH of the solution to 4.0 with 1M HCl, a W 2 phase aqueous solution is obtained.

[0032] S4: The W 1 / O emulsion is dispersed in the W 2 phase aqueous solution, and the volume ratio of the W 2 phase aqueous solution to the W 1 / O emulsion is 4:6; homogenization is carried out with a T25 Ultra Turrax homogenizer at a rotation speed of 5000 rpm for 1 - 5 minutes to obtain a W 1 / O / W 2 double emulsion.

[0033] A higher W 1 / O volume ratio helps to increase the internal droplet space, thus encapsulating more probiotics. Zein - apple pectin mixed nanoparticles are used as the outer aqueous phase stabilizer for the formation of W 1 / O / W 2 double emulsion through the Pickering mechanism, which can ensure a sufficiently dense mixed nanoparticle as a solid and ordered physical barrier to prevent direct contact between digestive tract components (such as digestive enzymes and H + ) and probiotics; encapsulating probiotic strains in the internal aqueous phase using the double emulsion technology helps to protect probiotics from damage under harsh conditions.

[0034] S5: Take 5 mL of W 1 / O / W 2Double emulsion was crosslinked with polylysine at a mass concentration of 0.1% under stirring at 150 rpm, and then 2 mL of sodium alginate with a mass concentration of 2% was added for mixing. The mixed emulsion was injected into a CaCl 2 solution to prepare emulsion beads. Sodium alginate reacts with CaCl 2 to form calcium alginate. The added polylysine can fill the porous network of calcium alginate to further form a dense three-dimensional gel structure as a barrier.

[0035] The emulsion beads prepared by the above method can, on the one hand, provide external protection for probiotics, thereby protecting the strain activity and improving the bioavailability; on the other hand, by adding polylysine, sodium alginate, and CaCl 2 , a pH-sensitive polylysine-sodium alginate-Ca system is formed to encapsulate probiotics, thereby establishing a pH-sensitive and enzyme-triggered colon-targeted delivery system for the colon-targeted release of probiotics. Specifically, in the acidic environment of the stomach, the "box"-shaped gel shell formed by PLL-calcium alginate shows excellent tolerance to H + and proteases. At the same time, the adsorption of pectin on the surface of zein also effectively reduces the aggregation of W 1 / O / W 2 emulsion induced by protease and ion concentration through steric hindrance. In the neutral environment of the small intestine, due to the deprotonation of the carboxyl group (COO - ) of calcium alginate and the penetration of the reaction between OH - and Ca 2+ , the electrostatic repulsion between anionic groups is enhanced, prompting partial disassembly of the cross-linked network structure during digestion. This process is accompanied by partial decomposition of the gel shell, releasing the internal W 1 / O / W 2 emulsion. When the pH is lower than the pKa value (3.5) of pectin, the positively charged zein and the negatively charged pectin form a stable emulsifying system through electrostatic adsorption. After the coating disintegrates, the protein particles will aggregate at the isoelectric point. In the alkaline environment of the intestine, the carboxyl group of zein is deprotonated, and the surface then carries more negative charges. Under the electrostatic action, it repels the negatively charged apple pectin, resulting in the desorption of pectin from zein, promoting the flocculation of zein to form larger aggregates, with a significant increase in particle size and loss of emulsifying stability. The desorption of pectin from the surface of zein is ultimately attributed to electrostatic repulsion, which is beneficial to the step-by-step decomposition of W 1 / O / W 2 emulsion. Thus, the targeted release of synbiotics is achieved, ensuring the quantity and activity of probiotics reaching the colon site.

[0036] The embodiments of the present invention also provide the application of the emulsion beads prepared by the above preparation method in the targeted improvement of colitis. It has been experimentally proven that the emulsion beads prepared by the above preparation scheme can alleviate the colon shortening symptoms of UC rats, reduce the contents of inflammatory factors IL-1β, IL-6 and TNF-α in the serum, increase the content of anti-inflammatory factor IL-10 at the same time, increase the expression of tight junction proteins ZO-1, Claudin-1 and Occludin in the colon tissue, regulate the intestinal flora structure, and achieve the effect of repairing ulcerative colitis.

[0037] The present invention will be further described in detail through specific experiments below.

[0038] Materials and Reagents

[0039] 1. Main reagents: Lactobacillus reuteri was purchased from the China Center for Industrial Culture Collection Management. Polyglycerol ricinoleate, zein, fructooligosaccharide, polylysine, and sodium alginate were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Dextran sulfate sodium was purchased from Dalian Meilun Biotechnology Co., Ltd. Pepsin and pancreatin were purchased from Sigma-Aldrich Company, USA.

[0040] 2. Main instruments: Vacuum freeze dryer (Beijing Songyuan Huaxing Biotechnology Co., Ltd.), high-speed homogenizer (IKA (Guangzhou) Instrument and Equipment Co., Ltd.), ultra-micro nucleic acid and protein analyzer (Thermo Fisher Scientific), biological safety cabinet (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.).

[0041] 3. The results are reported as the mean ± standard deviation of three repeated measurements. SPSS 25.0 software was used to test the statistical significance of the mean differences by one-way analysis of variance (<0.05). GraphPad Prism 9.5 and Origin 2025 software were used for drawing.

[0042] Example 1 Preparation of Emulsion Beads Loaded with Synbiotics

[0043] S1: Under anaerobic conditions and at a temperature of 25°C, single colonies of Lactobacillus reuteri were continuously cultured in a medium for 24 hours twice, and then centrifuged at 1000 rpm for 10 minutes at 4°C to obtain Lactobacillus reuteri bacterial sludge.

[0044] S2: The Lactobacillus reuteri bacterial sludge (1.0×10 10 CFU / mL) and fructooligosaccharide with a mass concentration of 2% were dispersed in sterile distilled water and vortexed to form an aqueous phase W 1 where fructooligosaccharide was used as a prebiotic; polyglycerol ricinoleate with a mass concentration of 3% was used as a surfactant and fully stirred and dissolved in corn oil to obtain an oil phase; then the oil phase was mixed with W at a speed of 10000 rpm 1Mix and stir in the aqueous phase to obtain W 1 / O emulsion.

[0045] S3: Dissolve zein powder in 70% aqueous ethanol solution under stirring at 25 °C to obtain zein alcohol solution; dissolve apple pectin powder in deionized water at 25 °C and stir for 3 h to obtain apple pectin aqueous solution; mix the zein alcohol solution and the apple pectin aqueous solution, where the mass ratio of zein to apple pectin is 2:1, then stir with a high-speed homogenizer at 7000 rpm for 6 minutes to mix the solution evenly; finally, rotary evaporate at 45 °C to obtain a zein-apple pectin nanoparticle mixed solution, where the mass concentration of zein-apple pectin mixed nanoparticles is 3%; adjust the pH of the solution to 4.0 with 1M HCl to obtain W 2 phase aqueous solution.

[0046] S4: Disperse the W 1 / O emulsion in the W 2 phase aqueous solution, and the volume ratio of the W 2 phase aqueous solution to the W 1 / O emulsion is 4:6; homogenize with a T25 Ultra Turrax homogenizer at 5000 rpm for 2 minutes to obtain the W 1 / O / W 2 double emulsion.

[0047] S5: Take 5 mL of W 1 / O / W 2 double emulsion, crosslink it with polylysine with a mass concentration of 0.1% under stirring at 150 rpm, then add 2 mL of sodium alginate with a mass concentration of 2% for mixing, and inject the mixed emulsion into a CaCl 2 solution with a mass concentration of 1.5% to prepare emulsion beads.

[0048] Example 2 In vitro digestion simulation experiment of synbiotic-loaded emulsion beads

[0049] I. Protective effect of synbiotic-loaded emulsion beads on probiotics

[0050] 1. Determination method

[0051] Refer to the INFOGEST model to prepare simulated gastric fluid (SGF, 2 mg / mL NaCl, 3.3 mg / mL pepsin, 33 μg / mL CaCl 2 , adjust the pH to 2.0 with 1M HCl) and simulated intestinal fluid (SIF, 35 mg / mL bile salt and 150 mg / mL trypsin dissolved in PBS solution, 160 mg / mL NaCl, 1.32 mg CaCl2 , adjust the pH to 7.0 with 1M NaOH). Prepare the emulsion beads according to the preparation method of Example 1. In step S3, adjust the mass concentration of the zein - apple pectin mixed nanoparticles in the W 2 phase aqueous solution to 1%, 2%, 3%, 4% and 5% to obtain emulsion beads loaded with synbiotics prepared from five different concentrations of zein - apple pectin mixed nanoparticles (denoted as C1, C2, C3, C4 and C5 respectively).

[0052] Stomach: Dissolve 1.0 g of emulsion beads and pepsin in 9.0 mL of SGF solution (pH 2.0), place it in a constant temperature shaker at 37 °C, and shake at 100 rpm for 2 h. Small intestine: After 2 h, quickly add 7.5 mL of SIF to the in vitro digestive juice. Adjust the pH of the simulated solution to 7.0. Colon: Finally, adjust the pH value of the solution to 6.8. Take out 0.1 g of emulsion beads every 1 h during the digestion process and dissolve it in 9.9 mL of 5% sodium citrate solution by mass fraction. The released Lactobacillus reuteri was determined by the plate colony counting method. All stages of digestion were carried out in the dark.

[0053] 2. Protective effect of synbiotic - loaded emulsion beads on probiotics during digestion

[0054] It can be seen from Figure 2 that the viable cell count of Lactobacillus reuteri in each group before the experiment was greater than 8.00 log CFU / g. After 2 h of digestion, the free Lactobacillus reuteri showed specific sensitivity to gastric acid, and almost no visible probiotic growth was observed on the plate. After the gastric digestion was completed, the viable cell count of the emulsion beads prepared from different concentrations of zein - apple pectin mixed nanoparticles remained above 7.00 log CFU / g. After the small intestine digestion stage was completed, the survival rates of C1, C2, C3, C4 and C5 were 63.84%, 79.16%, 79.86%, 78.47% and 70.45% respectively. After the digestion was completed, the viable cell counts of C2, C3 and C4 remained above the internationally recommended amount (>6.00 log CFU / g), which were 6.61 ± 0.03, 6.46 ± 0.13, 6.47 ± 0.02 log CFU / g respectively.

[0055] II. Microscopic morphology of synbiotic - loaded emulsion beads

[0056] 1. Measurement method

[0057] Record the morphological characteristics of the emulsion beads at 0, 2, 4 and 6 h of simulated digestion through macroscopic imaging and optical microscopy techniques.

[0058] 2. Morphological changes of emulsion beads during digestion

[0059] It can be seen fromFigure 3 It can be seen that after 2 h of digestion (the end of gastric digestion), the emulsion beads maintained a complete spherical structure and were all suspended in the simulated gastric fluid. This indicates that under the stimulation of gastric acid and protease, calcium alginate was not degraded, and the three-dimensional network gel coating on the surface of the microspheres effectively maintained the stability of the delivery system. After 4 h (the end of small intestine digestion), some oil droplets could be observed floating on the upper layer of the simulated digestive fluid. The emulsion beads swelled and ruptured under neutral pH conditions, resulting in the decomposition of the oil escaping from the internal emulsion by bile salts and pancreatic enzymes, forming a turbid system. At 6 h (the end of colon digestion), obvious swelling was visible, and the structure of the emulsion beads was damaged. After the digestion in the colon stage ended, most of the emulsion beads remained in the digestive system. After adding probiotics, each bead contained multiple double emulsion droplets and the light transmittance became worse, indicating that Lactobacillus reuteri was successfully encapsulated, forming a dense structure. After gastric digestion, in addition to becoming transparent due to water absorption and swelling, the edges of the emulsion beads were relatively clear, and there was no obvious change in the internal emulsion. Under the action of enzymes, bile salts, and pH in the simulated intestinal environment, the microspheres swelled, the structure was gradually damaged, the internal emulsion escaped and disintegrated, and the probiotics were slowly released in a targeted manner.

[0060] Establishment of a rat model of ulcerative colitis in Example 3

[0061] Thirty male SD rats (6 - 8 weeks old) with a body weight of 180 - 220 g were selected. After 7 days of adaptive feeding, they were randomly divided into 1 blank control group (control group), 1 model control group (model group), and 3 experimental groups, with 6 rats in each group. The model control group and the experimental groups were subjected to model establishment. A dextran sulfate sodium (DSS) solution with a mass concentration of 4.5% was prepared and allowed to be freely consumed by the rats for 7 days to induce acute ulcerative colitis and construct a rat model of ulcerative colitis. The rats in the blank control group were freely given sterile water. After the model establishment treatment, the rats in the blank control group and the model control group were intragastrically administered PBS. In one of the experimental groups, the prepared emulsion beads loaded with synbiotics were intragastrically administered, designated as the bacteria bead group, with a dosage of 10 9 CFU / rat / day; one group was intragastrically administered a mixture of Lactobacillus reuteri and fructooligosaccharides, designated as the free group, with a dosage of 1 mL / rat / day; one group was intragastrically administered the empty shell of the emulsion beads without the mixture of Lactobacillus reuteri and fructooligosaccharides, designated as the empty bead group, with a dosage of 1.0 g / rat / day. Intragastric administration was performed once a day for 7 consecutive days. During this period, the rats were allowed to freely eat and drink sterile water.

[0062] Protective effect of emulsion beads loaded with synbiotics on DSS-induced ulcerative colitis rats in Example 4

[0063] I. Changes in the rat colon

[0064] 1. Measurement method

[0065] Perform anatomical operations in a biosafety cabinet. Take the section from 1 cm above the anus to the end of the cecum of the rats. After quickly rinsing with pre-cooled sterile saline, measure and record the colon length of each group. After thoroughly cleaning the colon, take some segment tissues and soak them in 4% paraformaldehyde solution for storage at 4°C for histological staining analysis. The remaining samples are stored at -80°C for subsequent experiments. Observe the histopathological changes through an optical microscope. The scoring includes epithelial damage, crypt damage, goblet cell depletion, and the degree of inflammatory cell infiltration.

[0066] 2. Effects of synbiotic-loaded emulsion beads on the colon of rats with DSS-induced ulcerative colitis

[0067] The results of colon length show that Figures 4 - 5 the experimental group is beneficial to alleviating the outcome of colon shortening caused by colitis. After the intervention of the beads, the colon shortening is significantly improved. It can be seen from Figure 6 that the successful establishment of the ulcerative colitis rat model is shown by pathological sections. The bead group intragastrically administered with the emulsion beads can maintain the crypt integrity, reduce inflammatory infiltration, and restore the mucosal layer to a thickness close to normal.

[0068] II. Changes in inflammatory factors in the serum of rats

[0069] 1. Measurement method

[0070] Collect orbital venous plexus blood samples from SD rats after anesthesia. Let them stand at room temperature and then centrifuge at 2000 rpm for 10 min. Aspirate the upper serum and store it in a -80°C refrigerator. Strictly detect the levels of inflammation-related cytokines in the serum, including IL-1β, IL-6, TNF-α, and IL-10, according to the enzyme-linked immunosorbent assay (ELISA) kit (Quanzhou Ruixin Biotechnology Co., Ltd.).

[0071] 2. Effects of synbiotic-loaded emulsion beads on serum inflammatory factors in rats with DSS-induced ulcerative colitis

[0072] It can be seen from Figure 7 that compared with the control group, the levels of IL-1β, IL-6, and TNF-α in the model group are significantly increased (P < 0.0001). After intragastric administration of the bead group, the above pro-inflammatory cytokines are significantly reduced, while there is no significant difference between the free group and the empty bead group (P > 0.05). On the contrary, compared with the control group, the level of IL-10 in the model group is significantly decreased (P < 0.001), indicating that the pro-inflammatory response is inhibited. The bead treatment significantly increases the level of IL-10 (P < 0.05), restoring it to a level close to that of the control group. In summary, the emulsion bead treatment can effectively regulate the expression of inflammatory factors in DSS-induced colitis. By reducing pro-inflammatory cytokines and enhancing the level of anti-inflammatory cytokines, the inflammatory response can be alleviated and mucosal healing can be promoted.

[0073] III. Changes in tight junction proteins in rat colon tissues

[0074] 1. Measurement method

[0075] Total RNA of colon tissues was extracted using Trizol reagent. It was reverse transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (+gDNA wiper). According to the instructions of 2x qPCR Mix (SYBR Green) (Heyuan LiJi, China), the reaction system was prepared, and the mRNA levels of genes were detected using a real-time fluorescence quantitative PCR instrument to measure the expression levels of ZO-1, Claudin-1, and Occludin mRNA.

[0076] 2. Effect of synbiotic-loaded emulsion beads on the repair of colon mucosa in DSS-induced ulcerative colitis rats

[0077] It can be seen from Figures 8 - 9 that the emulsion beads can increase the expression of tight junction proteins ZO-1, Claudin-1, and Occludin and restore the damaged intestinal mucosa.

[0078] IV. Changes in rat intestinal flora

[0079] 1. Measurement method

[0080] After the rats were fasted for 12 h, 2 - 3 feces of the rats were collected in a sterile cryopreservation tube under a sterile environment and stored in a -80°C refrigerator. The detection was completed by a professional biotechnology company. The intestinal flora analysis process includes DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics analysis. Total DNA was extracted according to the instructions of the reagent supplier. Amplification was performed using TransStart Fastpfu DNA polymerase (338F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’)). The PCR products were purified using the agcourt AMPure XP nucleic acid purification kit. Paired-end sequencing was performed using Illumina NovaSeq PE250 (Illumina, San Diego, USA). After sequencing, QIIME (2020.2.0) was used to perform bioinformatics analysis on the 16S rDNA sequence data.

[0081] 2. Effect of synbiotic-loaded emulsion beads on the intestinal flora of DSS-induced ulcerative colitis rats

[0082] It can be seen from Figure 10It can be seen that the species richness of the control group was the highest. Compared with the model group, the species richness increased after the treatment with the mycelial pellet group. The Venn diagram was used to analyze the similarities and differences of species among groups. There were 216 species in the control group, 145 species in the model group, 120 species in the free group, 179 species in the mycelial pellet group, and 134 species in the empty pellet group. Compared with the control group, the number of unique species in the model group decreased, and the number of species in the mycelial pellet group recovered. The species community diversity was represented by the Simpson index. Compared with the control group, the species diversity in the model group decreased significantly (P<0.05), indicating that the drug treatment reduced the diversity of the rat intestinal microbiota. Compared with the control group, the Chao1 index in the model group decreased. Compared with the model group, the Chao1 index in the mycelial pellet group increased significantly and was close to that of the control group. After the intervention with the mycelial pellet and the empty pellet, the decrease in the intestinal microbial abundance was restored, and the reversal effect in the mycelial pellet group was better. Beta diversity analysis allowed the comparison of sample differences from different subgroups. As shown in the PCA, the control group and the model group were well separated, and the intestinal flora structure of the rats in the model group changed significantly. The principal coordinate analysis (PCoA) based on the weighted UniFrac distance was used to evaluate the relationship between the intestinal flora of each group, which was consistent with the PCA. The microbial structure of the mycelial pellet treatment group was close to that of the control group, indicating that the mycelial pellet treatment improved the composition of the intestinal flora.

[0083] It can be seen that Figure 11 at the phylum level, the results showed that the six main bacteria with relatively high abundances in each group of samples were Firmicutes, Bacteroidota, Verrucomicrobiota, Desulfobacterota, Proteobacteria, and Actinobacteriota. Firmicutes and Bacteroidota were the dominant phyla in all samples. In the model group, the relative abundances of Bacteroidota and Firmicutes increased, and the relative abundance of the Verrucomicrobiota decreased. In this study, the mycelial pellet treatment reversed the increase in the Bacteroidota group. After the treatment with the mycelial pellet, the abundance of Firmicutes increased significantly, and the Verrucomicrobiota group decreased significantly. The results indicated that the mycelial pellet was beneficial for reducing the aggregation of Verrucomicrobiota and restoring intestinal function.

[0084] It can be seen that Figure 12It can be seen that at the genus level, the intestinal flora of rats is mainly composed of Lactobacillus (Muribaculaceae), Lachnospiraceae_unclassified, Lachnospiraceae_NK4A136_group, Akkermansia, Prevotellaceae_NK3B31_group, Oscillospiraceae_unclassified, Roseburia, and Lactobacillus. Compared with the control group, the relative abundances of Muribaculaceae, Lachnospiraceae_NK4A136_group, and Oscillospiraceae_unclassified in the model group increased, while the relative abundances of Lachnospiraceae_unclassified, Akkermansia, and Bacteroides decreased. After the intervention with the bacterial beads, the abundances of Muribaculaceae and Prevotellaceae_NK3B31_group decreased, while the abundance of Lachnospiraceae_unclassified increased. As a beneficial bacterium, Lachnospiraceae is one of the indicators of intestinal health. The results show that the emulsion beads play a synergistic effect and can regulate the intestinal microflora.

[0085] In summary, the emulsion beads can alleviate the shortening of the colon in rats with ulcerative colitis, reduce the levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum, increase the level of the anti-inflammatory factor IL-10, increase the expression of tight junction proteins ZO-1, Claudin-1, and Occludin, significantly alleviate the inflammatory damage of the colonic mucosa, and can regulate the intestinal flora structure.

[0086] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A method for preparing pH-responsive emulsion beads loaded with synbiotics, characterized in that: The following steps are involved: S1: Under anaerobic conditions and a temperature of 30-40°C, a single colony of the probiotic strain was activated and cultured in a culture medium for two consecutive 12-24 hours, and then centrifuged at a temperature of 0-10°C and a speed of 1000-5000 rpm for 2-15 minutes to obtain a probiotic slurry; S2: Probiotic slurry (1.0×10 8 ~1.0×10 12 CFU / mL) and oligofructose with a mass concentration of 2-5% were dispersed in sterile distilled water and vortexed to form a W1 aqueous phase; polyglycerol polyricinoleate (PGPR) was fully stirred and dissolved in corn oil as a surfactant to obtain an oil phase; the oil phase was mixed and stirred with the W1 aqueous phase at a speed of 8000-12000 rpm to prepare a W1 / O emulsion; S3: fully dissolving the zein powder in a 70-80% ethanol aqueous solution under stirring at 20-30° C. to obtain a zein alcohol solution; Dissolve the apple pectin powder in deionized water at 20-30°C and stir for 2-3 h to obtain an apple pectin aqueous solution; The zein alcohol solution was mixed with the apple pectin aqueous solution, wherein the mass ratio of zein to apple pectin was 2:1, and then stirred at 7000 rpm for 5-8 minutes; finally, the zein-apple pectin nanoparticle mixed solution was obtained by rotary evaporation at 45°C; and the W2 phase aqueous solution was obtained after the pH of the solution was adjusted to 4.0 with 1M HCl; S4: dispersing the W1 / O emulsion in the W2 phase aqueous solution, with the volume ratio of the W2 phase aqueous solution to the W1 / O emulsion being 4:6; homogenizing at a speed of 5000 rpm for 1 to 5 minutes to obtain a W1 / O / W2 double emulsion loaded with synbiotics; S5: Take 5 mL of W1 / O / W2 double emulsion, cross-link it with 0.1% poly-lysine under stirring at 150 rpm, then add 2 mL of 2% sodium alginate to mix, and inject the mixed emulsion into 1.5% CaCl2 solution to prepare emulsion beads.

2. The method for preparing emulsion beads according to claim 1, characterized in that: The probiotic strain in step S1 is one of Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium thermophilum.

3. The method for preparing emulsion beads according to claim 1, characterized in that: The mass concentration of polyglycerol ricinoleate in corn oil in step S2 is 0.5-4%.

4. The method for preparing emulsion beads according to claim 1, characterized in that: The mass concentration of the zein-apple pectin mixed nanoparticles in the W2 phase solution in step S3 is 1-5%.

5. Use of the emulsion beads prepared according to the preparation method of the emulsion beads according to any one of claims 1 to 4 in targeted improvement of colitis.

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