Lactobacillus acidophilus-epigallocatechin gallate double-layer multi-core microcapsule as well as preparation method and application thereof

Abstract

The invention relates to a lactobacillus acidophilus-epigallocatechin gallate double-layer multi-core microcapsule as well as a preparation method and application thereof, and belongs to the technical field of microcapsule preparation. The invention relates to a lactobacillus acidophilus-epigallocatechin gallate double-layer multi-core micro-capsule, which comprises an outer layer shell formed by sodium alginate, and epigallocatechin gallate and a plurality of inner layer micro-capsules which are distributed in the outer layer shell, the epigallocatechin gallate is filled in a gap between the outer layer shell and the inner layer micro-capsule; the inner layer microcapsule comprises an inner layer shell formed by sodium alginate and chitosan, and lactobacillus acidophilus wrapped by the inner layer shell. The double-layer multi-core microcapsule provided by the invention can effectively isolate the external environment and protect the activity of internal components; on the other hand, the influence on the activity of internal components due to direct contact can be effectively avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to a Lactobacillus acidophilus-epigallocatechin gallate double-layer multi-nuclear microcapsule and its preparation method and application, belonging to the technical field of microcapsule preparation. BACKGROUND

[0002] Avian pathogenic Escherichia coli as a conditional pathogen can infect various poultry, causing huge economic losses to the breeding industry. Due to the abuse and irrational use of antibiotics, the drug resistance problem of avian pathogenic Escherichia coli (APEC) has become a serious situation, which greatly reduces the therapeutic effect of antibiotics on chicken colibacillosis. Therefore, the research and development of alternative products have attracted great attention in the treatment of chicken colibacillosis and other aspects.

[0003] Previous studies have shown that microecological preparations and plant extracts can be used for the prevention and treatment of chicken colibacillosis, and the present research group disclosed in the patent CN 118593549 A that Lactobacillus acidophilus combined with epigallocatechin gallate (EGCG) can be used in the preparation of anti-livestock and poultry colibacillosis drugs. The invention discloses that the combination of Lactobacillus acidophilus and EGCG can produce a synergistic effect, which can improve production performance, enhance immune function, maintain intestinal flora homeostasis, and play a good role in preventing and treating colibacillosis in chicks. However, both are easily affected by external environmental changes (temperature, pH, gastrointestinal fluid, etc.), resulting in low bioavailability and difficulty in achieving full effect of oral administration. In addition, the present research group found that EGCG can inhibit the activity of Lactobacillus acidophilus during the combination process, resulting in poor prevention and treatment effect of colibacillosis in chicks. Therefore, it is necessary to develop a delivery system that can protect the activity of Lactobacillus acidophilus and EGCG and allow them to interact with each other, thereby significantly improving their bioavailability and therapeutic effect. SUMMARY

[0004] To solve the above problems, the present application provides a Lactobacillus acidophilus-epigallocatechin gallate double-layer multi-nuclear microcapsule and its preparation method and application, specifically providing a Lactobacillus acidophilus-epigallocatechin gallate (LA-EGCG) double-layer multi-nuclear microcapsule and its preparation method and its application in the treatment and / or prevention of colibacillosis in chicks. The present application uses sodium alginate and chitosan as wall materials, and uses endogenous emulsification and extrusion method to layer-embed Lactobacillus acidophilus and EGCG, obtaining a double-layer multi-nuclear microcapsule with core-shell structure, which has good stability and sustained release, and the preparation method is simple and easy to operate, providing a new idea and method for the development of efficient new alternative drugs.

[0005] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions: The Lactobacillus acidophilus-epigallocatechin gallate double-layer multi-nuclear microcapsule comprises an outer shell formed by sodium alginate, and epigallocatechin gallate and a plurality of inner layer microcapsules distributed inside the outer shell, the epigallocatechin gallate being filled in the gap between the outer shell and the inner layer microcapsule; the inner layer microcapsule comprises an inner shell formed by sodium alginate and chitosan, and Lactobacillus acidophilus wrapped by the inner shell.

[0006] Further, the particle size of the double-layer multi-nuclear microcapsule is 315-2740 μm, the particle size of the inner layer microcapsule is 35.6-251 μm, the embedding rate of epigallocatechin gallate is 93-95%, and the embedding rate of Lactobacillus acidophilus is 78-80%.

[0007] Another object of the present application is to provide a preparation method of the double-layer multi-nuclear microcapsule. S1: the sodium alginate solution, the Lactobacillus acidophilus bacterial suspension and the calcium carbonate powder are mixed and then added into the soybean oil containing Tween 80 for stirring and emulsification, the glacial acetic acid is added for further emulsification, the distilled water containing Tween 80 is added after the stirring and emulsification is completed, the oil phase is absorbed to collect the precipitate, the chitosan solution is added, and the inner layer microcapsule is obtained after stirring, suction filtration and washing; S2: the inner layer microcapsule obtained above is mixed with the epigallocatechin gallate solution to obtain a mixed solution A, the mixed solution A is mixed with the sodium alginate solution to obtain a mixed solution B, and then the mixed solution B is added dropwise into the CaCl2 solution, stirred and solidified, centrifuged and washed.

[0008] In the above technical solution, in S1, the mass ratio of calcium carbonate, sodium alginate and glacial acetic acid is 1:4-8:4-8.

[0009] Further, the concentration of the sodium alginate solution is 0.5%-2.5%.

[0010] In the above technical solution, in S1, the volume ratio of the Lactobacillus acidophilus bacterial suspension to the sodium alginate solution containing the calcium carbonate powder is 1:4-8.

[0011] Further, the total number of viable bacteria in the Lactobacillus acidophilus bacterial suspension is 1x10 9 CFU / mL.

[0012] In the above technical solution, in S1, the volume ratio of the solution obtained by mixing the sodium alginate solution, the Lactobacillus acidophilus bacterial suspension and the calcium carbonate powder to the soybean oil containing Tween 80 is 1:3-7.

[0013] In the above technical solution, the volume ratio of the precipitate to the chitosan solution is 1:1-2.

[0014] Further, the concentration of the chitosan solution is 0.2%-1.0%.

[0015] In the S2, the volume ratio of the epigallocatechin gallate solution to the inner layer microcapsule is 1:1.

[0016] Further, the concentration of the epigallocatechin gallate solution is 1-9 mg / mL.

[0017] In the S2, the volume ratio of the mixed solution A to the sodium alginate solution is 1:2-6.

[0018] Further, the concentration of the sodium alginate solution is 0.5%-2.5%.

[0019] Further, the concentration of the CaCl2 solution is 1%-4%.

[0020] In the above technical solution, the distilled water containing Tween 80 is used to make the emulsion stratify, and the specific amount is determined according to the situation.

[0021] Preferably, in the S1, the sodium alginate solution, the L. acidophilus bacterial suspension and the calcium carbonate powder are mixed and then added into the soybean oil containing 1% Tween 80 to be stirred and emulsified at 200-1000 rpm for 15 min, and then the glacial acetic acid is added to continue emulsification for 10 min, and then the distilled water containing 1% Tween 80 is added, the oil phase is absorbed to collect the precipitate, the chitosan solution is added, and stirring is carried out at 100 rpm for 10 min, and then the inner layer microcapsule is obtained after filtration and washing three times, and the inner layer microcapsule is stored at 4°C for standby.

[0022] Preferably, in the S2, the mixed solution B is added dropwise into the CaCl2 solution through a needle head by using a peristaltic pump, and stirring is carried out at 400 rpm for 0.5-2.5 h to solidify.

[0023] Another purpose of the present application is to provide the use of the above-mentioned L. acidophilus-epigallocatechin gallate double-layer multi-nuclear microcapsule in the preparation of a medicine or feed for preventing and / or treating E. coli disease of livestock and poultry.

[0024] Further, the livestock and poultry are chicks.

[0025] The present application has the following beneficial effects: (1) The LA-EGCG double-layer multi-nuclear microcapsule prepared by the present application has a smooth nuclear shell structure, which can effectively isolate the external environment and protect the activity of the internal components L. acidophilus and EGCG; on the other hand, through the layering embedding technology, the interaction between EGCG and L. acidophilus can be effectively avoided, thereby reducing the activity of L. acidophilus.

[0026] (2) The average embedding rate of the microcapsule obtained by the present application to EGCG is 94%, the average embedding rate to Lactobacillus acidophilus is 79%, the average particle size is 1320 μm, the whole has good pH, temperature and storage stability, has good resistance to gastric juice, and can be completely released in intestinal juice; in addition, it also has low hemolytic activity and significant anti-E. coli effect.

[0027] (3) The LA-EGCG double-layer multi-nuclear microcapsule obtained by the present application can more effectively play a role in preventing and / or treating E. coli disease of chicks by inhibiting the proliferation of E. coli, improving the immune function of chicks, reducing inflammatory reaction and improving intestinal microbial population, etc. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a result graph of the influence of different factors on the embedding rate of Lactobacillus acidophilus in the inner layer microcapsule in Example 2, wherein a is the concentration of sodium alginate solution, b is the concentration of chitosan solution, c is the stirring speed, d is the water / oil volume ratio, e is the ratio of calcium carbonate to glacial acetic acid, f is the mass ratio of calcium carbonate to sodium alginate, and g is the ratio of bacteria to glue.

[0029] Figure 2 It is a response surface stereogram of the embedding rate of Lactobacillus acidophilus in the inner layer microcapsule in Example 2.

[0030] Figure 3 It is a result graph of the influence of different factors on the embedding rate of EGCG in the double-layer multi-nuclear microcapsule in Example 3, wherein a is the concentration of sodium alginate, b is the concentration of chitosan, c is the concentration of CaCl2, d is the volume ratio of wall to core, e is the solidification time, and f is the concentration of EGCG.

[0031] Figure 4 It is a response surface stereogram of the embedding rate of EGCG in the double-layer multi-nuclear microcapsule in Example 3.

[0032] Figure 5 It is a particle size distribution graph of the double-layer multi-nuclear microcapsule obtained in Example 1, wherein a is the inner core microcapsule and b is the double-layer multi-nuclear microcapsule.

[0033] Figure 6 It is a surface morphology graph of the double-layer multi-nuclear microcapsule under a scanning electron microscope obtained in Example 1.

[0034] Figure 7 It is an X-ray spectroscopy diffraction graph of the double-layer multi-nuclear microcapsule obtained in Example 1.

[0035] Figure 8 It is an infrared spectroscopy graph of the double-layer multi-nuclear microcapsule obtained in Example 1.

[0036] Figure 9 It is a differential scanning calorimetry analysis result graph of the double-layer multi-nuclear microcapsule obtained in Example 1.

[0037] Figure 10 A graph of the results of thermal gravimetric analysis of the double-layered multi-core microcapsules obtained in Example 1.

[0038] Figure 11 A graph of the release rate of the double-layered multi-core microcapsules obtained in Example 1 at different temperatures.

[0039] Figure 12 A graph of the release rate of the double-layered multi-core microcapsules obtained in Example 1 at different pHs.

[0040] Figure 13 A graph of the release of the double-layered multi-core microcapsules obtained in Example 1 in simulated gastric juice.

[0041] Figure 14 A graph of the release of the double-layered multi-core microcapsules obtained in Example 1 in simulated intestinal juice.

[0042] Figure 15 A graph of the entrapment efficiency of the double-layered multi-core microcapsules obtained in Example 1 under different storage environments.

[0043] Figure 16 A graph of the hemolytic activity of the double-layered multi-core microcapsules obtained in Example 1.

[0044] Figure 17 A graph of the inhibitory ability of the double-layered multi-core microcapsules obtained in Example 1 on E. coli.

[0045] Figure 18 A graph of the incidence of disease in chicks after prophylactic administration of the double-layered multi-core microcapsules obtained in Example 1.

[0046] Figure 19 A graph of the TNF-α content in the small intestine tissue of chicks (n=10) after administration of the double-layered multi-core microcapsules obtained in Example 1.

[0047] Figure 20 A graph of the IL-1β content in the small intestine tissue of chicks after administration of the double-layered multi-core microcapsules obtained in Example 1.

[0048] Figure 21 A graph of the IL-10 content in the small intestine tissue of chicks after administration of the double-layered multi-core microcapsules obtained in Example 1.

[0049] Figure 22 A graph of the intestinal pathological changes in chicks of 14 days of age in each group (100x), wherein a is a blank control group, b is a model group, c is an empty microcapsule group, d is a low-dose double-layered multi-core microcapsule group, e is a medium-dose double-layered multi-core microcapsule group, f is a high-dose double-layered multi-core microcapsule group, g is a free LA+EGCG group, and h is a polymyxin sulfate group.

[0050] Figure 23The intestinal pathological changes of 21-day-old chicks in each group (100x) are shown in the figure, wherein a is the blank control group, b is the model group, c is the empty microcapsule group, d is the low-dose double-layer multi-nuclear microcapsule group, e is the medium-dose double-layer multi-nuclear microcapsule group, f is the high-dose double-layer multi-nuclear microcapsule group, g is the free LA+EGCG group, and h is the polymyxin sulfate group.

[0051] Figure 24 The effect of the double-layer multi-nuclear microcapsule obtained in Example 1 on the relative expression of genes in E. coli in cecal contents is shown in the figure. 16s rRNA

[0052] Figure 25 The effect of the double-layer multi-nuclear microcapsule obtained in Example 1 on the relative expression of genes in L. acidophilus in cecal contents is shown in the figure. 16s rRNA DETAILED DESCRIPTION

[0053] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but in no way limit the present application.

[0054] In the following examples, the test methods described are conventional methods unless otherwise specified; and the reagents and materials described are commercially available unless otherwise specified.

[0055] Example 1 The preparation method of the LA-EGCG double-layer multi-nuclear microcapsule includes the following steps: (1) Preparation of inner layer microcapsule Take 10 mL of 1.5% sodium alginate, 2 mL of L. acidophilus (1x10 9 CFU / mL), and 37.5 mg of calcium carbonate powder, mix to obtain a mixed solution, add to 60 mL of soybean oil containing 1% Tween 80, stir on a magnetic stirrer at 600 rpm for 15 min; add 150 μL of glacial acetic acid and continue to emulsify for 10 min, after emulsification, add distilled water containing 1% Tween 80 to separate the emulsion, collect the inner layer microcapsule and add to 100 mL of 0.4% chitosan solution, stir on a magnetic stirrer at 100 rpm for 10 min, collect by suction filtration, wash three times, and store at 4°C for standby.

[0056] (2) Preparation of double-layer multi-nuclear microcapsule Take 5 mL of the inner core microcapsule obtained in step (1) and mix with 5 mL of EGCG (5 mg / mL), mix the mixed solution with 1.5% sodium alginate at a ratio of 1:1, use a peristaltic pump to add the mixed solution to a 2% CaCl2 solution through a needle, stir at 400 rpm for 2 h, centrifuge and wash to obtain LA-EGCG double-layer multi-nuclear microcapsule. ​​

[0057] The content of Lactobacillus acidophilus and EGCG in the double-layer multi-core microcapsules obtained in Example 1 was determined: 1 g of the double-layer multi-core microcapsules was taken and added to 9 mL of the microcapsule breaking solution, and placed in a constant temperature shaking incubator at 37°C for shaking to promote complete lysis of the microcapsules. The content of Lactobacillus acidophilus was determined by plate colony counting method, and the content of EGCG was determined by ultraviolet spectrophotometry. It was determined that the content of Lactobacillus acidophilus in the double-layer multi-core microcapsules obtained in Example 1 was 1×10 8 CFU / g, and the content of EGCG was 50 mg / g.

[0058] Example 2 Single-factor investigation and response surface optimization experiment of the inner-layer microcapsules in the LA-EGCG double-layer multi-core microcapsules: In order to explore the optimal factor level of the inner-layer microcapsules, seven factors including the concentration of sodium alginate (0.5%, 1%, 1.5%, 2% and 2.5%), the mass ratio of calcium carbonate to sodium alginate (1:4, 1:5, 1:6, 1:7 and 1:8), the volume ratio of water to oil (1:3, 1:4, 1:5, 1:6 and 1:7), the stirring speed (200, 400, 600, 800 and 1000 rpm), the concentration of chitosan (0.2%, 0.4%, 0.6%, 0.8% and 1.0%), the molar ratio of calcium carbonate to glacial acetic acid (1:4, 1:5, 1:6, 1:7 and 1:8), and the ratio of bacteria to glue (1:4, 1:5, 1:6, 1:7 and 1:8) were investigated, and the evaluation index was the embedding rate of Lactobacillus acidophilus in the inner-layer microcapsules. Among them, the water phase in the water-oil ratio refers to the mixture of sodium alginate solution, Lactobacillus acidophilus suspension and calcium carbonate powder, and the oil phase refers to soybean oil containing Tween 80; the ratio of bacteria to glue refers to the volume ratio of Lactobacillus acidophilus solution to sodium alginate solution containing calcium carbonate powder. The Design Expert 13 software was used for experimental design and data analysis. According to the single-factor experiment results, the three levels of the four factors of sodium alginate concentration, chitosan concentration, stirring speed and water-oil volume ratio were determined, and the results are shown in Table 1 and Table 2. Figure 1 and Figure 2 .

[0059] From Figure 1 and Figure 2The results showed that the sodium alginate concentration, chitosan concentration and stirring speed had significant effects on the embedding rate of the inner microcapsules (P < 0.0001). The interactions between the sodium alginate concentration and chitosan concentration (P = 0.0002), the sodium alginate concentration and stirring speed (P = 0.0022), the sodium alginate concentration and the oil-water volume ratio (P < 0.0001), the chitosan concentration and stirring speed (P < 0.0001), the chitosan concentration and the oil-water volume ratio (P < 0.0001), and the stirring speed and the oil-water volume ratio (P < 0.0001) significantly affected the embedding rate of L. acidophilus. The embedding rate of L. acidophilus in the inner microcapsules reached the maximum value only when the sodium alginate concentration was 1.44%, the chitosan concentration was 0.44%, the stirring speed was 800 rpm, and the oil-water volume ratio was 6.68 (v:v).

[0060] Example 3 Single-factor investigation and response surface optimization of LA-EGCG double-layer multi-nuclear microcapsules: To explore the optimal factor level of the double-layer multi-nuclear microcapsules, the sodium alginate concentration (0.5%, 1%, 1.5%, 2%, and 2.5%), the chitosan concentration (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%), the solidification time (0.5, 1.0, 1.5, 2.0, and 2.5 h), the CaCl2 concentration (1%, 2%, 3%, 4%, and 5%), the EGCG concentration (1, 3, 5, 7, and 9 mg / mL), and the wall material-core material volume ratio (2:1, 3:1, 4:1, 5:1, and 6:1) were investigated, and the evaluation index was the embedding rate of EGCG in the double-layer multi-nuclear microcapsules. In the wall material-core material volume ratio, the wall material was the sodium alginate solution, and the core material was the epigallocatechin gallate solution mixed with the inner microcapsule mixture. The Design Expert 13 software was used for experimental design and data analysis. According to the single-factor test results, the three levels of the four factors, i.e., the sodium alginate concentration, the chitosan concentration, the calcium chloride concentration, and the wall-core volume ratio, were determined, and the results are shown in Table 2. Figure 3 and Figure 4 .

[0061] From Figure 3 and Figure 4It was found that the sodium alginate concentration, chitosan concentration, calcium chloride concentration and the volume ratio of wall to core had significant effects on the embedding rate of the double-layer multi-nuclear microcapsules (P<0.0001). The interactions between the sodium alginate concentration and the calcium chloride concentration (P<0.0001), the sodium alginate concentration and the volume ratio of wall to core (P=0.0406), the calcium chloride concentration and the chitosan concentration (P=0.0039), and the chitosan concentration and the volume ratio of wall to core (P<0.0001) significantly affected the embedding rate of the double-layer multi-nuclear microcapsules. The embedding rate of the double-layer multi-nuclear microcapsules reached the maximum value only when each variable was in the optimal condition (sodium alginate concentration 0.9%, chitosan concentration 0.45%, calcium chloride concentration 3.4%, and the volume ratio of wall to core 3.88:1 (v:v)).

[0062] Example 4 Properties of the double-layer multi-nuclear microcapsules obtained in Example 1 were studied. (1) Physical properties of the double-layer multi-nuclear microcapsules, and the results are shown in Table 1.

[0063] A certain amount of the double-layer multi-nuclear microcapsule lyophilized powder was loaded into a measuring cylinder, and the measuring cylinder was horizontally shaken at a uniform speed to allow the product to naturally settle. The volume of the sample at this time was measured, and the bulk density was calculated according to the formula ρ1=m1 / v. On the basis of the bulk density, the measuring cylinder was gently tapped to allow the double-layer multi-nuclear microcapsules to reach a compact state, and the volume of the sample at this time was recorded, and the compact density was calculated according to the formula ρ2=m2 / v. The flowability of the double-layer multi-nuclear microcapsules was evaluated in combination with the angle of repose, the Carr index (CI), and the Hausner ratio (HR). The angle of repose was measured by the injection method: the double-layer multi-nuclear microcapsule lyophilized powder was slowly added above the funnel, allowed to naturally fall into the funnel, and stacked on the circular plate. The angle of repose of the sample was calculated.

[0064] The bulk density and the compact density are important parameters for measuring the flowability of the microcapsules and evaluating the production cost. The bulk density of the double-layer multi-nuclear microcapsules was 0.35 g / mL, and the compact density was 0.38 g / mL, which was at a normal level, which was conducive to improving the utilization rate of storage space and reducing transportation and storage costs. The flowability of the microcapsules was usually evaluated by the angle of repose, and the angle of repose of the double-layer multi-nuclear microcapsules was 25.63°, the CI measured by the bulk density and the compact density was 7.89, and the HR was 1.08. It can be concluded that the flowability of the lyophilized double-layer multi-nuclear microcapsules is excellent, which indicates that the surface of the double-layer multi-nuclear microcapsules is smooth, and the friction between them is small.

[0065] Table 1 Basic physical properties of the double-layer multi-nuclear microcapsules

[0066] (2) Morphological observation of the double-layer multi-nuclear microcapsules After fixing the microcapsule sample on the object table with double-sided tape, the unabsorbed residual powder was removed with an ear bulb, and the double-layer multi-core microcapsule was placed in the sample chamber after 30 s vacuum gold spraying treatment. The microstructure of four types of samples (inner layer empty microcapsule, inner layer microcapsule, double-layer empty microcapsule and double-layer multi-core microcapsule) was characterized by scanning electron microscopy (SEM), respectively. The surface morphology of the selected clear, uniform and non-overlapping area was observed, and the results are shown in Figure 5 and Figure 6 .

[0067] As can be seen from Figure 5 , the average particle size of the inner layer microcapsule is 154.18 μm, and the average particle size of the double-layer multi-core microcapsule is about 1.32 mm. The particle size of the microcapsule obtained by extrusion method is related to many factors, such as the straight-line distance between the needle and the liquid surface, the pore size of the needle, etc.

[0068] As can be seen from Figure 6 , the double-layer multi-core microcapsule has a good spherical shape, obvious concave, but the structure is compact, no broken hole phenomenon, the surface has long wrinkles, and the pore is small (d1, d2); compared with the double-layer multi-core microcapsule, the double-layer empty microcapsule has more concave, and the shrinkage is more obvious, which should be related to the higher water evaporation. Due to the existence of the core material of the double-layer multi-core microcapsule, the water evaporation rate is reduced, and the shape is relatively complete. When the magnification is 1000x, 2000x, it is observed that the surface is uneven due to many convex rods, which form a dense microcapsule wall, which proves that the microcapsule forming property is good.

[0069] (3) Analysis of Fourier transform infrared spectroscopy determination results of double-layer multi-core microcapsule The infrared absorption spectra of EGCG, sodium alginate, chitosan and double-layer multi-core microcapsule were determined by Fourier infrared spectrometer combined with KBr pressing method, and the scanning analysis was carried out in the wavelength range of 4000-400 cm -1 , the embedding effect of sodium alginate and chitosan on EGCG was discussed, each sample was repeated three times, and the results are shown in Figure 7 . According to the FTIR spectrum of the core material EGCG, the main absorption peak is at 3356.33 cm -1 , which is the -OH peak, and the peak is strong without association. The characteristic peak vibration is mainly concentrated in the range of 427-1691 cm -1 . The four absorption peaks of 1527.9, 1467.3, 1446.7 and 1376.5 cm -1 are benzene ring C=C absorption peaks. The main stretching vibration of alcohol and phenol C-O group in EGCG is 1013-1292 cm -1 . The FTIR spectrum of double-layer multi-core microcapsule shows that the absorption peak of -OH is at 3432.66 cm-1 FTIR spectra of double-layer multi-core microcapsules showed that the wave number of -OH absorption peak was shifted to 3432.48 cm -1 , and the characteristic absorption peak intensity of the core material EGCG was significantly weakened, indicating that EGCG was wrapped inside the double-layer multi-core microcapsules.

[0070] (4) Analysis of double-layer multi-core microcapsule X-ray diffraction measurement results An appropriate amount of sample was weighed, ground and laid on the surface smooth X-ray diffraction glass plane to obtain a rectangular powder, which was then placed on the operation table for detection. The measurement conditions were: current was set to 30 mA, voltage was set to 40 kV, Cu was used as X-ray source (λ = 1.54), scanning rate was 2° / min, scanning range was 5°~60° (2θ), and the results are shown in Figure 8 .

[0071] Sodium alginate was found to have crystalline peaks at 2θ = 13.102°, 13.627°, 21.176°. Chitosan had crystalline peaks at 2θ = 8.295°, 8.896°, 14.566°, 25.081°. Double-layer multi-core microcapsules had crystalline peaks at 2θ = 11.525°, 12.125°, 20.049°, 28.192°, 29.137°. The characteristic peaks of EGCG at 2θ = 5.136°, 8.508°, 10.307°, 12.180°, 15.477°, 16.976°, 20.536°, 21.435°, 24.545°, 25.819°, 28.067°, 28.979°, 29.416°, 36.760° indicated that it had high crystallinity. These characteristic peaks were also observed in double-layer multi-core microcapsules, indicating that EGCG also existed in a crystalline state in the microcapsules, but some characteristic peaks were missing and the intensity was reduced, indicating that EGCG could be well wrapped in double-layer multi-core microcapsules.

[0072] (5) Analysis of double-layer multi-core microcapsule thermal stability measurement results A standard aluminum crucible was used to package 2.0 mg of sample, and differential scanning calorimetry analysis was performed under nitrogen protection (flow rate 50 mL / min). The dynamic thermal analysis program was set to linear temperature rise mode (10℃ / min), and the thermodynamic characterization interval covered 25~500℃. A blank reference cell was used to correct the baseline drift to ensure data reliability, and the results are shown in Figure 9 .

[0073] Differential scanning calorimetry (DSC) reveals the physicochemical transformation characteristics of materials by detecting the heat changes during the phase transition process. When the double-layer multi-nuclear microcapsule is in a specific environment, temperature fluctuations will trigger the phase transition of the capsule wall material, and then regulate the permeability of the capsule membrane to achieve the sustained-release function of the core material. The results show that EGCG presents a single characteristic endothermic peak at 226.0℃, while the double-layer multi-nuclear microcapsule system exhibits higher thermal stability: the glass transition starts at 236℃ (peak at 254℃), and completely melts at 306℃. This phenomenon indicates that the multi-layer structure of the double-layer multi-nuclear microcapsule effectively improves the core material protection performance and can maintain structural integrity under conventional thermal processing conditions.

[0074] (6) Thermal gravimetric analysis of double-layer multi-nuclear microcapsules Thermodynamic characterization was performed under a nitrogen atmosphere (50 mL / min constant flow). The experimental configuration was a linear temperature program (10℃ / min) covering the thermal analysis range of 30~600℃, with a baseline correction program implemented simultaneously. The heat flow-mass change response signals were monitored in real time by a high-sensitivity sensor, and the material thermal conversion behavior characteristics spectrum was recorded completely, as shown in Figure 10 .

[0075] Thermogravimetric analysis can reflect the change of sample mass with temperature under program-controlled temperature, and can be used to evaluate the thermal stability of microcapsules. As can be seen from Figure 10 , the weight loss of sodium alginate is divided into two stages: 25~220℃ and 220~600℃. The weight loss of chitosan is divided into two stages: 25~253℃ and 253~600℃. The first stage loses 16%, and in the second stage (161~600°C), the weight decreases rapidly and continuously, and at 600℃, it has lost 60%, which is 47% lower than the first stage, 2.94 times the first stage. The first stage (25~205℃) of the double-layer multi-nuclear microcapsule loses 13.4%, and the second stage (205~600℃) starts to level off at 600℃, with a mass loss of 3.85 times that of the first stage. That is, the weight loss rate of the double-layer multi-nuclear microcapsule is lower than that of sodium alginate and chitosan in the range of 30~600℃. The reason for the weight loss of the three in the first stage is that the sample has a certain hygroscopicity, and the evaporation of water causes weight loss. As can be seen from Figure 10 , when the sample undergoes severe degradation, the order of the severity of mass loss of the three is: double-layer multi-nuclear microcapsule < chitosan < sodium alginate.

[0076] (7) Stability analysis of double-layer multi-nuclear microcapsules Accurately take 0.1 g of double-layer multi-nuclear microcapsules 7 parts, add 9 mL of PBS (pH = 7.2) to it, and place it in a water bath at a temperature of 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃, respectively, and incubate for 15 min. Then, lyse the double-layer multi-nuclear microcapsules, and measure the EGCG content using an ultraviolet spectrophotometer. Repeat the detection three times in parallel, and the results are shown in Table 1. Figure 11 As can be seen, the stability of the double-layer multi-nuclear microcapsules is basically unchanged at 20℃ and 30℃, and the stability rapidly decreases as the temperature increases. This is probably because the increase in temperature causes the decomposition of the complex structure formed by sodium alginate and chitosan, causing the release of EGCG in the microcapsules, which cannot protect EGCG well.

[0077] Accurately take 0.1 g of double-layer multi-nuclear microcapsules and add it to PBS with different pH values of 2, 4, 6, 8, and 10, respectively. Place it in a constant temperature shaking incubator at 37℃ and 200 r / min. After 1 h, take it out and detect the remaining EGCG content, calculate the EGCG release rate, and the results are shown in Table 2. Figure 12 As can be seen, when the pH is 6.0 and 8.0, the stability of the double-layer multi-nuclear microcapsules does not change significantly, and when the pH is lower than 6.0 or higher than 8.0, the stability of the double-layer multi-nuclear microcapsules decreases significantly.

[0078] Accurately take 0.1 g of double-layer multi-nuclear microcapsules and add it to 9 mL of simulated gastric juice. Place it in a constant temperature shaking incubator at 37℃ and 200 r / min. Every 0.5 h, take out one tube and detect the remaining EGCG content in the microcapsules. Draw a time release rate curve, and the results are shown in Table 3. Figure 13 As can be seen, the double-layer multi-nuclear microcapsules gradually release the internal EGCG in the simulated gastric juice within 0~3 h, and the release rate slows down and tends to be stable after 3 h. This shows that the double-layer multi-nuclear microcapsules have certain resistance to gastric juice and can effectively avoid the premature exposure of the core material to the gastric juice.

[0079] Accurately take 0.1 g of double-layer multi-nuclear microcapsules and add it to 9 mL of simulated intestinal juice. Place it in a constant temperature shaking incubator at 37℃ and 200 r / min. Every 0.5 h, take out one tube and detect the remaining EGCG content in the microcapsules. Draw a time release rate curve, and the results are shown in Table 4. Figure 14 As can be seen, in the simulated intestinal juice, the release rate of the double-layer multi-nuclear microcapsules gradually increases and can reach complete release. This shows that the simulated intestinal juice can completely dissolve the double-layer multi-nuclear microcapsules, so that the core material can play a role in the intestinal tract.

[0080] Accurately weigh 5 g of double-layer multi-nuclear microcapsules and divide them into sterile centrifuge tubes. Store them at 4°C and 25°C. On the 1st, 3rd, 7th, 14th, and 28th days, take an appropriate amount of sample, crush it in the solution, and check the remaining EGCG content and Lactobacillus acidophilus count in the microcapsules. Draw a time-release rate curve. Use free bacterial suspension and EGCG as controls. The results are shown in Figure 15 It can be seen that, as the storage time increases, the double-layer multi-nuclear microcapsule embedding rate shows a decreasing trend. In the same time, the free drug shows a more obvious decreasing trend than the double-layer multi-nuclear microcapsule. The microencapsulation process effectively improves the storage stability of EGCG.

[0081] (8) Hemolyticity of double-layer multi-nuclear microcapsules The experiment is divided into 5 groups. The specific grouping and treatment are shown in Table 2. Accurately weigh the double-layer multi-nuclear microcapsules, set three replicates for each group, add an equal volume of red blood cell suspension, and incubate at 37°C for 2 h. Centrifuge at 3000 rpm for 10 min. Take pure water and 0.9% physiological saline, add an equal volume of red blood cell suspension, and use them as positive and negative controls. Take the supernatant and measure the absorbance value at 540 nm to calculate the hemolysis rate. The results are shown in Figure 16 Compared with the blank control group, the hemolysis rate of the low-dose double-layer multi-nuclear microcapsule is not significantly different, and the hemolysis rates of the medium and high-dose groups are significantly different. The 2020 edition of Chinese Pharmacopoeia specifies that the hemolysis rate of the sample should not be higher than 5%. In this experiment, the hemolysis rates of the low, medium, and high-dose groups are 0.11%, 0.94%, and 3.65%, respectively, all of which meet the requirements, indicating that the double-layer multi-nuclear microcapsule has low hemolyticity and good safety.

[0082] Table 2: Test grouping and treatment

[0083] (9) Detection and analysis of the bacteriostatic ability of double-layer multi-nuclear microcapsules The experiment is divided into 6 groups, namely the model group, empty microcapsule group, low, medium, and high-dose double-layer multi-nuclear microcapsule groups, and free LA+EGCG group. Escherichia coli CVCC 1569 is cultured in a system containing 4 mL of MRS broth to OD 600 = 0.6, and 12.5 g of empty microcapsules, free LA+EGCG (1.25×10 6 CFU+625 μg), 3.125 mg of double-layer multi-nuclear microcapsules, 6.25 mg of double-layer multi-nuclear microcapsules, and 12.5 mg of double-layer multi-nuclear microcapsules (containing 1.25×10 6 CFU of Lactobacillus acidophilus and 625 μg of EGCG) are added to each group, respectively. Incubate in a 37°C incubator. Count the colonies of Escherichia coli in the co-culture system every 6 h. Test for 30 h. The results are shown in Figure 17 .Figure 17 It was found that compared with the free LA+EGCG group, the bilayer multinucleated microcapsules showed a weaker inhibitory effect on Escherichia coli at 6 h, possibly because the microcapsules could not completely release the encapsulated drug. However, at 24 h, the bilayer multinucleated microcapsules showed a significant inhibitory effect on E. coli, but high-dose bilayer multinucleated microcapsules still did not achieve the same effect as the free LA+EGCG group. In conclusion, the bilayer multinucleated microcapsules can inhibit the proliferation of E. coli CVCC 1569, but due to the sustained-release effect, the in vitro inhibitory effect on E. coli CVCC 1569 is weaker than that of the LA+EGCG group.

[0084] Example 5 Application of LA-EGCG bilayer multinucleated microcapsules obtained in Example 1 in the prevention or treatment of Escherichia coli infection in chicks: (1) Establishment of an Escherichia coli infection model in chicks From 1 to 7 days of age, birds had free access to food and water. At 7 days of age, they were randomly divided into 5 groups of 10 birds each. One group was administered 0.2 mL of physiological saline, and the other group was administered 2 × 10 mL of saline solution. 7 2×10 8 2×10 9 and 2×10 10 CFU / feather of *E. coli*. Observe and record morbidity and survival within 7 days of infection, and calculate morbidity and survival rates. Necropsy necropsy was performed on dead chicks, and preliminary bacterial isolation and identification were performed on lesions using MacConkey agar. On day 7 post-infection, all remaining chicks were culled and necropsy performed; necropsy results were recorded (see Table 3). The subsequent experimental infection dose was determined based on morbidity and mortality.

[0085] Table 3. Statistical results of morbidity, mortality and necropsy findings in chicks after challenge (n=9)

[0086] (2) Formal experimental grouping and treatment The experiment consisted of eight groups: a blank control group, a model group, low-, medium-, and high-dose double-layer multinucleated microcapsule groups, a free Lactobacillus acidophilus + EGCG group, and a polymyxin group, with 30 birds in each group. Treatment was as shown in Table 4. The empty microcapsule group was fed 2 g / kg bw; the low-dose double-layer multinucleated microcapsule group was fed 0.5 g / kg bw (equivalent to a Lactobacillus acidophilus dose of 5 × 10⁻⁶). 7 CFU / kg bw, EGCG dose was 25 mg / kg bw; the medium-dose bilayer multinucleated microcapsule group was fed with a dose of 1 g / kg bw (equivalent to 1×10 Lactobacillus acidophilus dose). 8CFU / kg b.w, EGCG dose was 50 mg / kg b.w.); high dose double-layer multi-nuclear micro-capsule group was fed at a dose of 2 g / kg b.w. (equivalent to Lactobacillus acidophilus dose of 2 x 10 8 CFU / kg b.w, EGCG dose was 50 mg / kg b.w.); high dose double-layer multi-nuclear micro-capsule group was fed at a dose of 2 g / kg b.w. (equivalent to Lactobacillus acidophilus dose of 2 x 10

[0087] Table 4 Test grouping and treatment

[0088] (3) Determination of the growth performance of the chicks From 3 days of age, the body weight of the chicks in each group was recorded every 2 days, and after the challenge at 7 days of age, the diarrhea and death of each group was recorded every day, and the average daily gain (ADG) and the morbidity of each group were calculated, the results are shown in Table 5 and Table 6. Figure 18 .

[0089]

[0090] Table 5 Average daily gain of the chicks after the administration of the double-layer multi-nuclear micro-capsule (g)

[0091] From Table 5: the average daily weight gain of each group of chicks aged 3-7 days was not significantly different, indicating that feeding Lactobacillus acidophilus and EGCG did not affect the body weight of chicks aged 3-7 days. Compared with the blank control group, the average daily weight gain of chicks in the model group and the empty microcapsule group was significantly reduced, and the average daily weight gain of chicks in the low-dose microcapsule group, the free Lactobacillus acidophilus + EGCG group and the polymyxin group was not significantly different. It is indicated that E. coli infection can affect the normal development of chicks, causing slow weight gain, and the wall material of the empty microcapsule cannot effectively alleviate E. coli infection. The average daily weight gain of chicks in the medium and high dose microcapsule groups was significantly higher than that in the blank control group and the free drug group, indicating that adding medium and high dose microcapsule to the diet of chicks can alleviate the impact of E. coli, and under the same dose, the effect of microcapsule drug is better than that of free drug, indicating that microcapsule can maintain drug activity and better promote the growth and development of chicks. Compared with the blank control group, the average daily weight gain of chicks in the model group and the empty microcapsule group was significantly reduced, and the medium and high dose microcapsule groups could significantly increase the average daily weight gain of chicks. The body weight of chicks in the medium dose microcapsule group was significantly higher than that in the free drug group, indicating that the application of Lactobacillus acidophilus and EGCG in the form of microcapsule can alleviate the slow weight gain caused by E. coli infection, promote the growth and development of chicks, and improve the body weight of chicks.

[0092] From Figure 18 It can be seen that on the first day after challenge (8 days old), clinical symptoms began to appear in each group except the blank control group, with yellowish or yellowish green sticky and watery feces. Within 7 days of challenge, the incidence of the model group was 84.38% (25 / 30), the incidence of the empty microcapsule group was 81.25% (24 / 30), the incidence of the low, medium and high dose groups was 30.38% (9 / 30), 15.66% (5 / 30) and 12.5% (4 / 30) respectively, the incidence of the free drug group was 21.18% (6 / 30), and the incidence of the polymyxin group was 25% (8 / 30).

[0093] The above test results show that Lactobacillus acidophilus and EGCG can reduce the incidence of E. coli disease in chicks. And when Lactobacillus acidophilus and EGCG are made into double-layer multi-nuclear microcapsules, the effect shows a dose-dependent manner, and when the dosage is consistent, the preventive effect of double-layer multi-nuclear microcapsules is better than that of directly feeding Lactobacillus acidophilus and EGCG.

[0094] (4) Measurement of immune organ index of chicks The immune organs (thymus, spleen, bursa of Fabricius) of 14 and 21-day-old chicks were weighed, and the immune organ index was calculated according to the following formula, and the results are shown in Table 6:

[0095] Table 6 Immune organ index of chicks (g / kg)

[0096] From Table 6, it can be seen that at 14 days of age, compared with the blank control group, the thymus and bursa of fabricius indexes of the model group were significantly reduced, and the spleen index was significantly increased. Compared with the model group, the thymus index and bursa of fabricius index of the low, medium and high dose microcapsule groups, the free LA+EGCG group and the polymyxin group were significantly increased, and the spleen index was significantly decreased. At 21 days of age, the thymus index of the medium and high dose microcapsule groups, the free LA+EGCG group and the polymyxin group, and the bursa of fabricius index of the low, medium and high dose microcapsule groups, the free LA+EGCG group and the polymyxin group were significantly increased, and the spleen index was significantly decreased. Compared with the free LA+EGCG group, at 14 days of age, the thymus index and bursa of fabricius index of the medium dose double-layer multi-nuclear microcapsule group were significantly increased, and the spleen index had no significant difference. At 21 days of age, the thymus index, bursa of fabricius index and spleen index had no significant difference.

[0097] The above experiments show that E. coli infection can affect the development of immune organs of chicks and affect the immune function of chicks. Adding double-layer multi-nuclear microcapsules to the diet of chicks to 21 days of age can improve the immune organ indexes of infected chicks and restore them to normal levels. And under the same drug dose, double-layer multi-nuclear microcapsules can better improve the immune organ indexes of chicks.

[0098] (5) Determination of blood physiological indexes of chicks The blood of 14 and 21 days old chicks was collected in 1.5 mL anticoagulant tubes, inverted up and down, and mixed gently. The blood physiological indexes were detected using a blood analyzer. The white blood cell count (WBC), lymphocyte percentage (LYM), monocyte percentage (MON), neutrophil percentage (GRA), hemoglobin content (HGB) and red blood cell count (RBC) were recorded, and the results are shown in Tables 7 and 8.

[0099] Table 7 Blood physiological indexes of 14-day-old chicks

[0100] Table 8 Blood physiological indexes of 21-day-old chicks

[0101] From Tables 7 and 8, it can be seen that at 14 days of age and 21 days of age, compared with the blank control group, the HGB of the model group chicks was significantly reduced, and the rest of the indexes were not significantly different. The experimental data showed that E. coli infection could cause inflammation in chicks. Adding double-layer multi-nuclear microcapsules and polymyxin to the diet of chicks could restore the physiological indexes to normal physiological levels.

[0102] (6) Detection of small intestine tissue inflammatory factor content of chicks The levels of IL-1β, TNF-α, and IL-10 in the supernatant of small intestinal tissue were measured strictly according to the ELISA kit instructions, and the OD was measured using a microplate reader. 450 Values ​​were used to calculate the concentrations of IL-1β, TNF-α, and IL-10 in small intestinal tissue. The results are shown in […]. Figure 19~Figure 21 .

[0103] Depend on Figure 19 , 20 As shown in Figure 21, at 14 days of age, compared with the blank control group, the levels of TNF-α and IL-1β in the small intestine tissue of chicks in the model group were significantly increased, and the level of IL-10 was significantly decreased. Compared with the model group, the levels of TNF-α, IL-1β, and IL-10 in the small intestine tissue of chicks in the empty microcapsule group were not significantly different. The level of TNF-α in the small intestine tissue of chicks in the low-dose microcapsule group was significantly decreased, while the levels of TNF-α and IL-1β in the small intestine tissue of chicks in the medium- and high-dose microcapsule groups, the free LA+EGCG group, and the polymyxin group were significantly decreased, and the level of IL-10 in the medium- and high-dose microcapsule groups and the free LA+EGCG group was significantly increased. Compared with the polymyxin group, the level of TNF-α in the small intestine tissue of chicks in the high-dose microcapsule group and the free LA+EGCG group was significantly decreased, the level of IL-1β in the medium- and high-dose group was significantly decreased, and the level of IL-10 in the medium- and high-dose microcapsule group and the free LA+EGCG group was significantly increased. Compared with the free LA+EGCG group, the IL-1β content in the small intestine tissue of chicks in the medium-dose microcapsule group was decreased, while the IL-10 and TNF-α contents did not differ significantly. At 21 days of age, compared with the blank control group, the TNF-α content in the model group chicks was significantly increased, the IL-10 content was significantly decreased, and the IL-1β content did not differ significantly. Compared with the model group, the TNF-α, IL-1β, and IL-10 contents in the small intestine tissue of chicks in the empty capsule group did not change significantly. The TNF-α content in the small intestine tissue of chicks in the low, medium, and high-dose microcapsule groups, the free LA+EGCG group, and the polymyxin group was significantly decreased, while the IL-10 content was significantly increased. The IL-1β content in the small intestine tissue of chicks in the high-dose microcapsule group was significantly decreased. Compared with the polymyxin group, the TNF-α content in the small intestine tissue of chicks in the high-dose microcapsule group was significantly decreased, while the IL-10 content in the low, medium, and high-dose microcapsule groups was significantly increased. Compared with the free LA+EGCG group, the IL-10 content in the small intestine tissue of chicks in the medium-dose microcapsule group with the same drug content was significantly increased.

[0104] The above results indicate that *E. coli* infection can induce intestinal inflammation in chicks. LA-EGCG bilayer multinucleated microcapsules significantly reduced the content of pro-inflammatory factors and significantly increased the content of anti-inflammatory factors in the small intestinal tissue of chicks, thereby alleviating the intestinal inflammation. Furthermore, the effect was superior to the combined use of uncoated LA and EGCG.

[0105] (7) Observation of the morphology and structure of the small intestine of chicks Small intestinal samples were fixed with 4% paraformaldehyde, followed by dehydration, clearing, paraffin permeation, embedding, paraffin trimming, and sectioning. After hematoxylin-eosin (HE) staining and mounting, the samples were observed under a microscope for histopathological examination. Severely affected areas were marked and recorded. Results are shown in [Figure number missing]. Figure 22 and Figure 23 .

[0106] Depend on Figure 22 and Figure 23 It was found that in the control group of 14-day-old chicks, the intestinal villi of the small intestine were intact, neatly arranged, and without hemorrhage. Compared with the control group, the model group showed atrophy and loss of intestinal villi, thickened intestinal mucosa, obvious hemorrhages, and extensive neutrophil infiltration. Compared with the model group, there was no significant difference in the morphology and structure of the intestinal villi of chicks in the empty microcapsule group. However, the morphology and structural integrity of the intestinal villi in the mixed-feed double-layer multinucleated microcapsule prevention group were significantly restored, and the intestinal tissue of the high-dose microcapsule group tended to be normal, without hemorrhages or obvious pathological damage. By 21 days of age, the degree of intestinal lesions was significantly reduced due to the gradual recovery of the chicks' intestinal immune function.

[0107] (8) qRT-PCR was used to detect the content of Lactobacillus acidophilus and Escherichia coli in the cecal contents of chicks in each experimental group. Using cecal contents genomic DNA as a template, and bacterial universal... 16s rRNA Using the internal reference genes, and employing the reaction conditions and system used in establishing the standard curve described above, Escherichia coli and Lactobacillus acidophilus were detected in cecal contents samples from each group. 16s rRNA Relative content, results are shown in Figure 24 and Figure 25 .

[0108] Depend on Figure 24 and Figure 25 It can be seen that, compared with the blank control group, the cecal contents of chicks in the 14- and 21-day-old model groups contained more *E. coli*. 16S rRNA The relative content of Lactobacillus acidophilus increased significantly. 16s rRNA The relative levels were significantly reduced. This indicates that infection with *E. coli* in chicks leads to an increase in *E. coli* levels and a decrease in *Lactobacillus acidophilus* levels in the cecum, resulting in cecal flora imbalance. Compared with the model group, the levels of *E. coli* in the cecum of chicks in the low-, medium-, and high-dose double-layer multinucleated microcapsule groups and the free LA+EGCG group at 14 and 21 days of age were significantly lower. 16s rRNA The relative content was significantly lower, similar to the polymyxin group. Compared with the free LA+EGCG group, the high-dose bilayer multinucleated microcapsule group showed a higher concentration of E. coli in the cecum of chicks. 16s rRNA The relative content was significantly reduced. Compared with the 21-day-old polymyxin group, the medium-dose double-layer multinucleated microcapsule group showed a higher concentration of Escherichia coli in the cecum of chicks. 16s rRNAThe relative content was significantly reduced. Compared with the model group, the relative content of Lactobacillus acidophilus in the cecum of 14 and 21-day-old chickens in the high-dose double-layer multi-nuclear microcapsule group and the free LA+EGCG group was significantly reduced 16s rRNA The relative content was significantly increased. Compared with the 21-day-old polymyxin group, the relative content of Lactobacillus acidophilus in the cecum of chickens in the low, medium and high dose double-layer multi-nuclear microcapsule group was significantly increased 16s rRNA The relative content was significantly increased.

Claims

1. A Lactobacillus acidophilus-epigallocatechin gallate bilayer multinucleated microcapsule, characterized in that: The bilayer multinucleated microcapsule comprises an outer shell formed of sodium alginate, epigallocatechin gallate distributed inside the outer shell, and several inner microcapsules, with the epigallocatechin gallate filling the gap between the outer shell and the inner microcapsules; the inner microcapsule comprises an inner shell formed of sodium alginate and chitosan, and Lactobacillus acidophilus encapsulated in the inner shell.

2. The bilayer multinucleated microcapsule according to claim 1, characterized in that: The bilayer multinucleated microcapsules have a particle size of 315~2740 μm, and the inner layer microcapsules have a particle size of 35.6~251 μm. The encapsulation rate of epigallocatechin gallate is 93%~95%, and the encapsulation rate of Lactobacillus acidophilus is 78%~80%.

3. The method for preparing the bilayer multinucleated microcapsules according to claim 1 or 2, characterized in that: Includes the following steps: S1: Sodium alginate solution, Lactobacillus acidophilus suspension and calcium carbonate powder are mixed and added to soybean oil containing Tween 80 for stirring and emulsification. Glacial acetic acid is added to continue emulsification. After the emulsification is completed, distilled water containing Tween 80 is added, the oil phase is removed and the precipitate is collected. Chitosan solution is added, and after stirring, filtration and washing, the inner microcapsules are obtained. S2: Mix the inner microcapsules obtained above with epigallocatechin gallate solution to obtain mixed solution A, then mix it with sodium alginate solution to obtain mixed solution B, then add it dropwise to CaCl2 solution, stir to solidify, centrifuge, wash, and obtain the final product.

4. The preparation method according to claim 3, characterized in that: In S1, The mass ratio of calcium carbonate, sodium alginate, and glacial acetic acid is 1:4~8:4~8; The volume ratio of the Lactobacillus acidophilus suspension to the sodium alginate solution containing calcium carbonate powder is 1:4~8; The volume ratio of the solution obtained by mixing sodium alginate solution, Lactobacillus acidophilus suspension and calcium carbonate powder to soybean oil containing Tween 80 is 1:3~7. The volume ratio of the precipitate to the chitosan solution is 1:1~2.

5. The preparation method according to claim 3, characterized in that: In S2, The volume ratio of the epigallocatechin gallate solution to the inner microcapsules is 1:1; The volume ratio of the mixed solution A to the sodium alginate solution is 1:2~6.

6. The preparation method according to claim 3, characterized in that: In S1, the total number of viable bacteria in the Lactobacillus acidophilus suspension is 1×10⁻⁶. 9 CFU / mL; sodium alginate solution concentration: 0.5%~2.5%; chitosan solution concentration: 0.2%~1.0%; In S2, the concentration of epigallocatechin gallate solution is 1~9 mg / mL; the concentration of sodium alginate solution is 0.5%~2.5%; and the concentration of CaCl2 solution is 1%~4%.

7. The preparation method according to claim 3, characterized in that: In step S1, sodium alginate solution, Lactobacillus acidophilus suspension and calcium carbonate powder are mixed and added to soybean oil containing 1% Tween 80. The mixture is stirred and emulsified at 200-1000 rpm for 15 min. Glacial acetic acid is added and emulsification is continued for 10 min. After the emulsification is completed, distilled water containing 1% Tween 80 is added, the oil phase is removed and the precipitate is collected. Chitosan solution is added, and the mixture is stirred at 100 rpm for 10 min. After filtration and washing three times, the inner microcapsules are obtained and stored at 4°C for later use.

8. The preparation method according to claim 3, characterized in that: In step S2, a peristaltic pump is used to add the mixed solution B dropwise into the CaCl2 solution through a needle, and the mixture is stirred at 400 rpm for 0.5 to 2.5 hours to solidify.

9. The use of the bilayer multinucleated microcapsules according to claim 1 or 2, or the bilayer multinucleated microcapsules prepared by the method according to any one of claims 3 to 8, in the preparation of drugs or feed for the prevention and / or treatment of Escherichia coli infection in livestock and poultry.

10. The application according to claim 9, characterized in that: The livestock and poultry mentioned are chicks.

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