A method for preparing a bi-nuclear microcapsule

Abstract

The present application relates to the field of food biotechnology, and in particular to a preparation method of double-core microcapsules. The method comprises the following steps: uniformly mixing sulfhydrylated radix codonopsis polysaccharide and sodium alginate solution; uniformly mixing the mixture of sulfhydrylated radix codonopsis polysaccharide and sodium alginate with a first core material to form a suspension, extruding into small droplets, dropping into CaCl2 to obtain first microcapsules; mixing xanthan gum or pectin with a second core material, extruding into small droplets, dropping into CaCl2 to obtain second microcapsules; respectively injecting the first microcapsules and the second microcapsules into a coaxial double-channel injection pump to form droplets, dropping into a CaCl2 solution, and solidifying; washing, filtering, freeze-drying to obtain double-core microcapsules. The double-core microcapsules prepared by the method have no interference between core materials, are spherical and uniform, and have an average particle size of 1-3 mm. The embedding of different wall materials in the inner layer achieves site-specific release, and the outer layer embedding forms a more compact protective structure, thereby improving the storage property of the microcapsules.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, specifically a method for preparing dual-core microcapsules.

[0002] Background Introduction

[0003] Microcapsules are a technology that uses natural materials or synthetic polymers as wall materials and liquid, solid, or gaseous substances as core materials, encapsulating them into microparticles through physical and chemical methods. Microcapsules come in various shapes, including spherical, grape-like, or irregular shapes. The diameter of microcapsules typically ranges from 1 to 1000 μm, and their morphology and size vary depending on the wall material, core material, and encapsulation method. A key advantage of microcapsules is that they allow for controlled release of the encapsulated substance at different rates under specific conditions, enabling it to exert its beneficial physiological properties.

[0004] The essence of probiotic microcapsules is to maintain a certain number of live probiotics and colonize them in specific sites to exert their beneficial properties in the human body. However, currently, some researchers have only focused on the preparation of mononuclear microcapsules and the exploration of their tolerance to simulated gastrointestinal fluids in vitro. There is limited research on the preparation of binuclear microcapsules with non-interfering, targeted release of probiotics and their survival and release in the gastrointestinal tract. Furthermore, live probiotics are affected by gastric acid and bile salts in the digestive tract, leading to a decrease in the number of live bacteria and affecting the biological activity of the live bacteria reaching the colon. Moreover, the viability of probiotic products decreases significantly during transportation and storage, necessitating a suitable method to protect the live bacteria. This requires a method that can encapsulate two strains of bacteria requiring different culture environments together to prevent interference, while also enabling layered release and targeted delivery through different wall materials.

[0005] Publicly available literature also reports some wall materials and preparation methods for binuclear microcapsules. For example, in application number 201410028452.0, the applicant, South China University of Technology, discloses a binuclear microcapsule and its preparation method and application: During preparation, melamine and formaldehyde solutions are stirred and mixed, the pH of the system is adjusted to 8-9, the melamine-formaldehyde mixture is heated and stirred in a water bath at 65-70℃, and distilled water is added to react. A water-soluble melamine-formaldehyde prepolymer was obtained. Then, the melamine-formaldehyde prepolymer was mixed with an emulsifier and stirred until completely dissolved. The pH was adjusted, and a first core material was added. The reaction yielded melamine-formaldehyde resin microcapsules with a particle size of 0.1–300 μm. These microcapsules, along with a second core material, were added to a chitosan solution. Droplets were formed using a syringe pump and collected in a CaCl2 solution. The solution was then cured for 0.5–3 hours, resulting in spherical, binuclear microcapsules with a particle size of 0.1–1000 μm. The binuclear microcapsules of this invention significantly reduce the release rate of the active core material, and the interaction between the core materials is minimal.

[0006] No other reports on dual-core microcapsules have been found to date. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing dual-core microcapsules. This method utilizes a two-stage injection pump extrusion process, ensuring that the core materials do not interfere with each other, and enabling targeted release and layer-by-layer delivery of the dual cores. The wall material, thiol-containing Codonopsis pilosula polysaccharide, combines with cysteine ​​residues in intestinal mucus and on the surface of bacteria to form stable disulfide bonds, acting as an intermediate bridge connecting probiotics and intestinal mucus. This enhances the intestinal adhesion of probiotics, promoting their intestinal adhesion, colonization, and proliferation. Furthermore, to avoid the threats posed by extreme gastrointestinal environments to probiotics, the method enhances their colon-targeted adhesion and colonization. The dual-core microcapsules of this invention can be directly applied as a feed additive in animal husbandry.

[0008] This invention is achieved through the following technical solution: a method for preparing dual-core microcapsules, comprising the following steps:

[0009] (1) Preparation of a mixture of thiolized Codonopsis pilosula polysaccharide and sodium alginate: The thiolized Codonopsis pilosula polysaccharide and sodium alginate solution were mixed evenly and stirred evenly under heating conditions;

[0010] (2) Preparation of microcapsules: The mixture of thiolized Codonopsis pilosula polysaccharide and sodium alginate is mixed evenly with the first core material to form a viscous suspension. The suspension is squeezed into small droplets by a single-channel injection pump and dropped into CaCl2 to solidify and obtain the first microcapsule. Xanthan gum or pectin is mixed evenly with the second core material. The suspension is squeezed into small droplets by a single-channel injection pump and dropped into CaCl2 to solidify and obtain the second microcapsule.

[0011] (3) Preparation process of dual-core microcapsules: The first microcapsule, the second microcapsule and 2% to 3% sodium alginate were injected into a coaxial dual-channel injection pump to form droplets, which were then dropped into CaCl2 solution and solidified; the microcapsules were washed with sterile water, filtered, and freeze-dried to obtain spherical dual-core microcapsules.

[0012] The above-mentioned method for preparing dual-core microcapsules, in step (1), the preparation method of thiolated Codonopsis pilosula polysaccharide includes the following steps: extracting Codonopsis pilosula polysaccharide, separating and purifying it, dissolving Codonopsis pilosula polysaccharide in water, adding alkaline solution, stirring, adding monochloroacetic acid to react, adjusting the pH to neutral after the reaction, vacuum drying the product obtained by dialysis to obtain carboxymethylated Codonopsis pilosula polysaccharide, dissolving carboxymethylated Codonopsis pilosula polysaccharide in distilled water, adding EDC, stirring, adding N-acetyl-L-cysteine ​​under nitrogen protection, and dialysis to obtain thiolated Codonopsis pilosula polysaccharide.

[0013] In the above-mentioned method for preparing a dual-core microcapsule, in step (2), the first core material and the second core material are: Bacillus or yeast.

[0014] In the above-mentioned method for preparing a dual-core microcapsule, in step (2), the spore-forming bacteria are Bacillus coagulans, Bacillus subtilis, or Bacillus licheniformis; and the yeast is Saccharomyces boulardii or Saccharomyces cerevisiae.

[0015] In the above-mentioned method for preparing a dual-core microcapsule, in step (2), the volume ratio of the mixture of thiolated Codonopsis pilosula polysaccharide and sodium alginate to the first core material is 1:1 to 1:1.5; the volume ratio of xanthan gum or pectin to the second core material is 1:1 to 1:1.5.

[0016] In the above-mentioned method for preparing a dual-core microcapsule, in step (2), the particle size of the first microcapsule is 0.1-0.5 mm, and the particle size of the second microcapsule is 0.1-0.5 mm.

[0017] In the above-mentioned method for preparing a dual-core microcapsule, the injection rate of the single-channel injection pump in step (2) is 0.1 ml / min to 1.7 ml / min.

[0018] In the above-mentioned method for preparing a dual-core microcapsule, in step (3), the particle size of the dual-core microcapsule is 1-3 mm.

[0019] In the above-mentioned method for preparing a dual-core microcapsule, step (3) involves freeze-drying, with an initial pre-freezing temperature of -20 to -40°C and a pre-freezing time of 2 to 3 hours, followed by freezing at -80°C for 20 to 24 hours.

[0020] A dual-core microcapsule is prepared by any of the above-described preparation methods.

[0021] The beneficial effects of this invention are:

[0022] 1. This invention uses thiolated Codonopsis pilosula polysaccharide as a prebiotic, adding it to probiotics to enhance their intestinal adhesion, promote intestinal colonization and proliferation. Codonopsis pilosula polysaccharide is the main active ingredient of Codonopsis pilosula and has multiple effects, widely used in the food and pharmaceutical industries. It has antioxidant, immune-enhancing, anti-inflammatory, and intestinal flora-regulating effects, promoting probiotic proliferation. Adding it to the microcapsule wall material not only promotes adhesion, colonization and proliferation, but also exerts anti-inflammatory and intestinal flora-regulating effects.

[0023] 2. The microcapsule core materials prepared by this invention do not interfere with each other, and the binuclear microcapsules are spherical and uniform with an average particle size of 1-3 mm. The embedding of different wall materials in the inner layer achieves targeted release, and the outer layer embedding forms a denser protective structure, which increases the number of live bacteria in the intestinal environment, allowing a sufficient number of probiotics to reach the colon, adhere and colonize, exert their effects, and improve the storability of the microcapsules.

[0024] 3. The dual-core microcapsules prepared by this invention can be directly used as a feed additive to regulate the animal gut in animal husbandry. Attached Figure Description

[0025] Figure 1 The microcapsules of Bacillus coagulans obtained in Example 1.

[0026] Figure 2 The microcapsules of Saccharomyces boulardii obtained in Example 1.

[0027] Figure 3 The dual-core microcapsule obtained in Example 1.

[0028] Figure 4 This is an optical microscope image of the single-nucleus microcapsule obtained in Example 1.

[0029] Figure 5 This is an optical microscope image of the dual-core microcapsule obtained in Example 1.

[0030] Figure 6 Venn plots are used to represent the level eigenvalues ​​of OTUs.

[0031] Figure 7 This is a two-dimensional ordination plot of the samples from the PCoA analysis.

[0032] Figure 8 The bacterial community structure at the phylum level is composed of 0 days and 30 days.

[0033] Figure 9 Species differences at the phylum level were considered for 0-day and 30-day observations (a: Firmicutes; b: Bacteroidetes; c: Spirochetes; d: Actinobacteria; e: Proteobacteria).

[0034] Figure 10 This diagram shows the bacterial community structure at the scientific level for different dose groups at 0 days and 30 days.

[0035] Figure 11 This diagram shows the bacterial community structure at the genus level for different dose groups at 0 days and 30 days.

[0036] Figure 12 The differences in fecal microbiota LEfSe between the control group and the experimental group at 0 days and 30 days were analyzed.

[0037] Figure 13 The differential function plots on KEGG Enzyme for the 0-day and 30-day control and experimental groups are shown. Detailed Implementation

[0038] To make the technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0039] Example 1: Preparation of Dual-Core Microcapsules

[0040] 1. Extraction of Codonopsis pilosula polysaccharides

[0041] Codonopsis pilosula was sliced ​​thinly and placed in a drying oven at 50℃ for 48 hours. The dried slices were then pulverized using an ultrafine pulverizer, and the dry powder was sieved through a 100-mesh sieve. The sieved powder was then treated with petroleum ether at room temperature for 6 hours to remove fat. The residue was then filtered and treated with 85% ethanol for 24 hours to remove pigments and small organic compounds. The residue was then filtered again and placed in an oven at 50℃ for 2 hours. Distilled water was then added at a ratio of 1:20 (w / v), and the mixture was extracted at 90℃ for 2 hours. The mixture was centrifuged at 10,000 r / min for 15 minutes, and the supernatant was collected. The resulting precipitate was then extracted three times using a hot water extraction process, and concentrated under reduced pressure at 70℃ to a suitable concentration. The volume of the resulting solution was precipitated with anhydrous ethanol at a ratio of 1:4 (v / v). After standing at 4°C for 12 hours, the precipitated solution was centrifuged at 4000 r / min for 10 minutes to obtain polysaccharide precipitate. The precipitate was reconstituted with distilled water, and protein was removed by the Sevag method. The protein-removed solution was then subjected to repeated freeze-thaw cycles at -80°C three times to remove the remaining small amount of protein. The solution was precipitated again with anhydrous ethanol at a ratio of 1:4 (v / v) for 12 hours. After centrifugation, the precipitate was reconstituted with distilled water. The resulting solution was dialyzed with running water in a dialysis bag (with a cutoff of 8000-14000 Da) for 48 hours, followed by dialyzed with distilled water for 48 hours. The dialyzed polysaccharide solution was then freeze-dried to obtain crude Codonopsis pilosula polysaccharide.

[0042] 2. Isolation and purification of Codonopsis pilosula polysaccharides

[0043] The crude polysaccharide solution was dissolved in distilled water at a certain ratio and filtered through a 0.45 μm aqueous filter membrane. The filtered polysaccharide solution was then separated using an agarose DEAE-52 anion exchange gel chromatography column (2.6 cm × 40 cm). Different concentrations of NaCl (0, 0.1, 0.2, 0.3, 0.4, and 0.5 M) were used as eluents, and gradient elution was performed at a flow rate of 1 mL / min. The eluent was collected in 5 min / tube, and 50 tubes were collected for each elution gradient. The polysaccharide content of each tube of eluent was determined using the phenol-sulfuric acid method, and elution curves were plotted. Elution peaks of the same eluent were combined, and the solution was dialyzed with distilled water in a dialysis bag (with a cutoff of 3500 Da) for 48 h. The dialysate was then lyophilized.

[0044] The separated polysaccharides were dissolved in distilled water at a certain concentration and filtered through a 0.45 μm aqueous filter membrane. The filtered polysaccharide solution was then separated using a dextran gel G-100 column (1.6 × 30 cm) with distilled water as the eluent. The eluent was collected at 5 min / tube, and 50 tubes were collected. The polysaccharide content of each tube of eluent was detected by the phenol-sulfuric acid method, and an elution curve was plotted. The elution peaks were combined and dialyzed with distilled water in a dialysis bag (with a cutoff of 3500 Da) for 48 h. The dialysate was then freeze-dried to obtain Codonopsis pilosula polysaccharide.

[0045] 3. Preparation of carboxymethyl Codonopsis pilosula polysaccharide

[0046] Dissolve 1g of Codonopsis pilosula polysaccharide in 50mL of deionized water. After complete dissolution, add 150mL of NaOH solution (3mol / L), stir for 10min, add 20g of monochloroacetic acid, react at 65℃ for 4h, cool to room temperature, adjust the pH to 7.0, and dialyze until the remaining reagents are eliminated. Vacuum dry the product obtained by dialyzing to obtain carboxymethylated Codonopsis pilosula polysaccharide. The vacuum degree of vacuum drying is 9Pa. First dry at -30℃ for 24h, and then heat to 40℃ for 12h.

[0047] 4. Preparation of thiol-modified Codonopsis pilosula polysaccharide

[0048] 100 mg of carboxymethyl Codonopsis pilosula polysaccharide was dissolved in 20 mL of distilled water. After complete dissolution, 166 mg of EDC was added to activate the carboxyl groups in the reaction system. The mixture was stirred at room temperature for 1 h. Then, 76 mg of N-acetyl-L-cysteine ​​(NAC) was added under nitrogen protection. The mixture was stirred for 24 h. The system was dialyzed with distilled water for 24 h to remove EDC and NAC. The mixture was then lyophilized to obtain thiol Codonopsis pilosula polysaccharide.

[0049] 5. Preparation of a mixture of thiolated Codonopsis pilosula polysaccharide and sodium alginate

[0050] Thiol-modified Codonopsis pilosula polysaccharide and sodium alginate solution were mixed evenly at a volume ratio of 0.5:2 to 1:3, and heated and stirred evenly in a water bath at 80-90℃. The mass fraction of sodium alginate was 2%-3%. When the volume ratio of thiolated Codonopsis pilosula polysaccharide to sodium alginate solution was 0.5:2, the high viscosity of the mixture led to extrusion difficulties, resulting in excessively large microcapsule sizes. When the volume ratio of thiolated Codonopsis pilosula polysaccharide to sodium alginate solution was 1:1, the microcapsules had smooth surfaces, intact morphology, and the highest encapsulation efficiency.

[0051] 6. Preparation of microcapsules:

[0052] Activation of Bacillus coagulans in the first core material: Bacillus coagulans preserved on test tube slant culture was inoculated into sterile MRS medium and cultured at 37℃ for 48 h to obtain primary seed culture; the primary seed culture was inoculated into sterile MRS medium at a 10% inoculum and cultured at 37℃ for 48 h to obtain secondary fermentation broth; the fermentation broth was centrifuged to collect bacterial sludge, which was washed three times with 0.85% physiological saline and resuspended in 10 ml of physiological saline to obtain concentrated Bacillus coagulans bacterial solution with a viable count of 2 × 10⁻⁶ cells / mL. 10 CFU / mL.

[0053] Activation of the second core material, *Saccharomyces boulardii*: *Saccharomyces boulardii* cells preserved on test tube slant agar were inoculated into sterilized YPD medium and cultured at 28°C for 48 hours to obtain the primary seed culture. The primary seed culture was then inoculated into sterilized YPD medium at a 10% inoculum size and cultured at 28°C for 48 hours to obtain the secondary fermentation broth. The fermentation broth was centrifuged to collect the bacterial sludge, which was washed three times with 0.85% physiological saline and resuspended in 10 mL of physiological saline to obtain a concentrated *Saccharomyces boulardii* bacterial solution with a viable count of 2 × 10⁻⁶ cells / mL. 10 CFU / mL.

[0054] A mixture of thiolated Codonopsis pilosula polysaccharide and sodium alginate as the wall material was mixed with a concentrated Bacillus coagulans solution as the first core material at a volume ratio of 1:1 to 1:1.5 to form a viscous suspension. This suspension was then agitated into small droplets using a single-channel injection pump and dropped into 2% CaCl2 solution. After solidification for 1-2 hours, Bacillus coagulans microcapsules with a particle size of 0.1-0.5 mm were obtained. Figure 1 As shown. 2% xanthan gum (wall material) and concentrated *Saccharomyces boulardii* culture (second core material) were mixed evenly at a volume ratio of 1:1 to 1:1.5. The suspension was then agitated into small droplets using a single-channel injection pump and dropped into 2% CaCl2. After curing for 1-2 hours, *Saccharomyces boulardii* microcapsules with a particle size of 0.1-0.5 mm were obtained. Figure 2 As shown.

[0055] The concentration of thiolated Codonopsis pilosula polysaccharide in the concentrated Bacillus coagulans culture was 0.5%–1%, and the mass fraction of sodium alginate was 2%–3%.

[0056] When the ratio of wall material to core material is 1:1, the wall material is too thick and cannot release the number of live bacteria in time, resulting in a lower encapsulation rate and the extruded microcapsules will exhibit a tailing phenomenon. However, when the ratio of wall material to core material is 1:1.5, the prepared microcapsules have a good morphology and are in the form of regular granules, and the encapsulation rate reaches its maximum at this time.

[0057] The injection rate of the single-channel infusion pump is 0.1 ml / min to 1.7 ml / min.

[0058] 7. Preparation process of dual-core microcapsules: Bacillus coagulans microcapsules and Saccharomyces boulardii microcapsules, along with 2% sodium alginate, were separately injected into a coaxial dual-channel injection pump to form droplets. These droplets were then collected in CaCl2 solution and solidified for 0.5-1 hour. After washing with sterile water, filtration, and freeze-drying, dual-core microcapsules with a spherical shape and a particle size of 1-3 mm were obtained. The outermost layer of the dual-core microcapsules is sodium alginate, containing two cores: microcapsules embedded with thiolized Codonopsis pilosula polysaccharide and microcapsules embedded with wall materials such as xanthan gum. Figure 3 and Figure 5 As shown.

[0059] In the coaxial dual-channel injection pump, the rate of the outer injection pump is 0.05 ml / min to 0.1 ml / min, and the rate of the inner injection pump is 0.1 mL / min to 1 mL / min.

[0060] Freeze-drying: the initial pre-freezing temperature is -20 to -40℃, the pre-freezing time is 2 to 3 hours, and then it is placed in an ultra-low temperature freeze-drying environment at -80℃ for 20 to 24 hours.

[0061] Example 2

[0062] The probiotic microcapsules prepared according to the method of the present invention were subjected to the following performance tests:

[0063] (1) Tolerance of microcapsules in simulated gastric fluid

[0064] 1g of the binuclear microcapsules prepared in Example 1 were added to 9mL preheated artificial gastric fluid test tubes and incubated at 37℃ with constant temperature shaking in a water bath at 100r / min. Samples were taken at 0h, 0.5h, 1h, 1.5h, and 2h. The microcapsules were washed 2-3 times with sterile distilled water and then lysed in the decapsulation fluid. The viable bacterial count was determined using the plate count method. The average value of three parallel tests was taken to obtain the final number of viable bacteria in the microcapsules. Under the same conditions, an unencapsulated mixed bacterial suspension was used as a control. After 2h of digestion in the stomach, the viable bacterial survival rate of Bacillus coagulans and Saccharomyces boulardii binuclear microcapsules was 88.63%.

[0065] Table 1. Tolerance (survival rate) of microcapsules in simulated gastric fluid.

[0066]

[0067] (2) Survival experiment of microcapsules in continuous simulated gastrointestinal fluid

[0068] 1g of the dual-core microcapsules prepared in Example 1 were added to 9mL of simulated gastric fluid and treated in a constant-temperature shaker at 37℃ and 100r / min for 3h. Samples were taken and counted at 0h, 1h, 2h, and 3h. The samples treated with simulated gastric fluid were centrifuged at 4℃ and 4000r / min for 5min, and the precipitate was collected and transferred to 9mL of simulated intestinal fluid and mixed thoroughly. The mixture was then shaken in a shaker for 3h under the same conditions. Samples were taken at 0h, 1h, 2h, and 3h, and colony counts were performed after serial dilution. Under the same conditions, a mixed free bacteria control was used for counting. The viable cell survival rate of the Bacillus coagulans and Saccharomyces boulardii dual-core microcapsules was 79.31%.

[0069] Calculation method:

[0070] Simulated gastric fluid survival rate (%) = (Number of viable bacteria per 1g sample after artificial gastrointestinal fluid treatment / (CFU / g) / Number of viable bacteria per 1g sample before artificial gastrointestinal fluid treatment / (CFU / g)) × 100%

[0071] Table 2 Survival rate of microcapsules in continuous simulated intestinal fluid.

[0072]

[0073]

[0074] Example 3: Application in animal husbandry as a feed additive for regulating animal gut health.

[0075] 1. Materials and Methods

[0076] 1.1 Experimental Animals and Grouping

[0077] The experiment was conducted at a dairy farm in Shenyang. Six Holstein cows of similar parity (2 calf), calving date, and milk yield, all in mid-lactation, were selected. They were randomly assigned to two groups: a blank control group and a microencapsulated preparation experimental group. Each group consisted of three cows. The control group was fed a basal diet, while the experimental group received 10g of microecological preparation added to their basal diet. The pre-trial period was 7 days, and the formal trial period was 28 days.

[0078] The experimental dairy cows were kept in the same barn using fixed troughs and tethered, and the feeding and management levels of each group were consistent.

[0079] 1.2 Measurement Indicators

[0080] Microbial diversity was measured in cow feces from both the control and experimental groups before and after the experiment. The control group was designated as C, and the experimental group as H.

[0081] 2. Experimental Results

[0082] 2.1 Venn diagram analysis of bacteria

[0083] like Figure 6The figure shows the Venn plot of OTU levels in the control and experimental groups at different feeding times. A total of 814 OTUs were found in all samples, with 40 specific OTUs in the experimental group. The experimental group had more specific OTUs after 30 days of feeding than after 0 days. This indicates that the addition of the compound microcapsule formulation can alter the composition of OTUs in the gut microbiota.

[0084] 2.2 PCA Analysis Based on OTU Level

[0085] The closer the β-diversity samples are, the more similar their species composition. Based on this analysis, the differences in fecal microbiota profiles between the control group and different dosage groups of dairy cows on days 0 and 30 were preliminarily distinguished. Figure 7 As shown, based on the Bray-Curtis distance algorithm and PCoA analysis, PC1 contributed 23.79%, and PC2 contributed 15.35%. The figure below shows the spatial separation of fecal microbiota in the control and experimental groups. On day 0, both control and experimental group samples were located above the horizontal axis, while on day 30, both control and experimental group samples were located below the horizontal axis. The large sample distance and significant differences at different feeding times indicate that feeding microencapsulated formulations can affect microbial distribution.

[0086] 2.3 Bacterial community structure at the phylum level

[0087] OTUs from six samples were classified and annotated to determine the common locations and relative abundances of the fecal microbiota. Classification was performed at the phylum level, such as... Figure 8As shown, the bacteria in the six samples belonged to the phyla Firmicutes, Bacteroides, Spirochactota, Actinobacteria, Proteobacteria, and Patescibacteria. On day 0, the bacterial composition of the control and experimental groups was basically similar, with Firmicutes and Bacteroides being the dominant phyla. After 30 days of feeding, the content of Firmicutes in the experimental group (62.91%) was higher than that in the control group (55.47%). Compared with day 0, the content of Firmicutes decreased after 30 days of feeding, with a decrease of 16.79% in the control group and a decrease of 9.27% ​​in the experimental group, a smaller decrease than that in the control group. The overall abundance of Bacteroides increased, with an 8.28% increase in the control group and a 1.73% increase in the experimental group compared to the control group. Both the low-dose and medium-dose groups showed slight increases. The abundance of Spirochactota decreased in the experimental group and increased in the control group. Proteobacteria increased by 1.23% in the control group and by 0.20% in the experimental group. This indicates that the addition of the dual-core microcapsule formulation can alter the abundance of the dominant gut microbiota. The overall decrease in Firmicutes may be due to the experiment being conducted in December, when colder weather led to a decrease in Firmicutes abundance.

[0088] 2.4 Bacterial community structure at the family level

[0089] At the scientific level, such as Figure 10As shown, the top 10 most abundant species in the six samples were Oscillospiraceae, Lachnospiraceae, Rikenellaceae, Peptostreptococcaceae, Spirochaetaceae, Prevotellaceae, Muribaculaceae, Bifidobacteriaceae, and UCG-010. The abundance of *Anaerovoracaceae* was significantly lower in the 0-day feeding group compared to the 30-day feeding group (P<0.05). The abundance of *Oscillospiraceae*, *Lachnospiraceae*, *Rikenellaceae*, and *Peptostreptococcaceae* species in the 30-day feeding group increased compared to the control group. However, the abundance of the pathogenic *Spirophycetes* species increased in the 30-day feeding group compared to the 0-day control group; the abundance decreased in the experimental group. This indicates that feeding with the dual-core microcapsule formulation can inhibit the increase of pathogenic bacteria.

[0090] 2.5 Bacterial community structure at the genus level

[0091] At the genus level, such as Figure 11 As shown, 291 genera and species were identified from 24 fecal samples. Compared with the 0-day experimental group, the average relative abundance of *Ruminococcus* (g__UCG-005), *Rikenellaceae* (g__Rikenellaceae_RC9_gut_group), unclassified *Lachnospiraceae* (g__unclassified_f__Lachnospiraceae), and unidentified *Muribaculaceae* (g__norank_f__Muribaculaceae) increased in the 30-day feeding group, increasing by 2.33%, 0.307%, 3.09%, and 3.909%, respectively, compared with the control group. The abundance of *Treponema* (g__Treponema) and *Paeniclostridium* (g__Paeniclostridium) decreased significantly in the 30-day feeding group compared with the control group.

[0092] 2.6 Analysis of LEfSe species in the gut microbiota of dairy cows

[0093] The results of differential microbial community screening, combined with the abundance changes of species at the phylum, family, and genus levels, revealed significant species differences between the experimental and control groups at day 0 and day 30 of feeding. Therefore, the control group at day 0 and the experimental group, as well as the control group and experimental group at day 30 of feeding, were selected for LEfSE analysis. Figure 12-13 As shown, a total of 27 differentially expressed bacterial groups were identified. An LDA threshold of 2 was selected, with classification levels ranging from phylum to genus. The experimental group fed for 30 days had 10 marker species: Bacteroidetes RF16 group (f__Bacteroidales_RF16_group), unranked Bacteroidetes RF16 group (g__norank_f__Bacteroidales_RF16_group), Ruminococcus, Oscillibacter, Elusimicrobium, o__Elusimicrobiales, c__Elusimicrobia, p__Elusimicrobiota, f__Elusimicrobiaceae, and Agathobacter, with Bacteroidetes having the largest marker count. The control group fed for 30 days had 3 marker species, including Succinivibrionaceae, Aeromonadales, and Succinivibrio.

[0094] The enzyme pathways of KEGG were analyzed in the control group and the experimental group after 0 days and 30 days of feeding using the Wilcoxon rank-sum test. There were significant differences in metabolic pathways between the experimental group after 0 days and the experimental group after 30 days of feeding (P = 0.2712). The main metabolic pathways with significant differences were 5, namely glutamate synthase (NADH), glutamate synthase (NADPH), tryptophan synthase, glycosidase, and N-acetylmuramoyl-L-alanine amidase.

[0095] In summary, the addition of binuclear microcapsule formulations can alter the composition and distribution of gut microbiota OTUs. At the phylum level, the experimental group showed a significant increase in Bacteroidetes abundance and inhibited the decline in Firmicutes compared to the control group. After 30 days of feeding, compared to day 0, the experimental group showed a significant decrease in the abundance of *Anaerovoracaceae* and a decrease in the abundance of pathogenic *Spiromelaceae*. At the genus level, the experimental group showed an increase in the abundance of *Ruminococcus*, *Rikabiformes*, uncultured *Trichophyton*, and unidentified *Rhizobium*, while the abundance of *Treponema* and *Clostridium* decreased. LEfSe analysis of species in the experimental group revealed 10 marker species, compared to only 3 in the control group. The binuclear microcapsule formulations prepared from *B. coagulans* SN-8 and *S. boulardii* SN-6 strains can increase beneficial gut bacteria and inhibit pathogenic bacteria, providing some ideas and theoretical references for future research on antibiotic alternatives for ruminants.

Claims

1. A method for preparing dual-core microcapsules, characterized in that, Includes the following steps: (1) Preparation of a mixture of thiolized Codonopsis pilosula polysaccharide and sodium alginate: The thiolized Codonopsis pilosula polysaccharide and sodium alginate solution were mixed evenly and stirred evenly under heating conditions; (2) Preparation of microcapsules: The mixture of thiolated Codonopsis pilosula polysaccharide and sodium alginate is mixed evenly with the first core material to form a viscous suspension. The suspension is squeezed into small droplets by a single-channel injection pump and dropped into CaCl2 to solidify and obtain the first microcapsule. Xanthan gum or pectin is mixed evenly with the second core material. The suspension is squeezed into small droplets by a single-channel injection pump and dropped into CaCl2 to solidify and obtain the second microcapsule. The particle size of the first microcapsule is 0.1~0.5mm, the particle size of the second microcapsule is 0.1~0.5mm, and the injection rate of the single-channel injection pump is 0.1ml / min~1.7ml / min. The first core material and the second core material are: Bacillus or yeast. The Bacillus is Bacillus coagulans, Bacillus subtilis or Bacillus licheniformis; the yeast is Saccharomyces boulardii or Saccharomyces cerevisiae. (3) Preparation process of dual-core microcapsules: The first microcapsule, the second microcapsule and 2%~3% sodium alginate were injected into a coaxial dual-channel injection pump to form droplets, which were then dropped into CaCl2 solution and solidified; washed with sterile water, filtered, and freeze-dried to obtain dual-core microcapsules with a spherical shape and a particle size of 1~3mm.

2. The method for preparing a dual-core microcapsule according to claim 1, characterized in that, In step (1), the preparation method of thiolized Codonopsis pilosula polysaccharide includes the following steps: extracting Codonopsis pilosula polysaccharide, separating and purifying it, dissolving Codonopsis pilosula polysaccharide in water, adding alkaline solution, stirring, adding monochloroacetic acid to react, adjusting the pH to neutral after the reaction, vacuum drying the product obtained by dialysis to obtain carboxymethylated Codonopsis pilosula polysaccharide, dissolving carboxymethylated Codonopsis pilosula polysaccharide in distilled water, adding EDC, stirring, adding N-acetyl-L-cysteine ​​under nitrogen protection, and dialysis to obtain thiolized Codonopsis pilosula polysaccharide.

3. The method for preparing a dual-core microcapsule according to claim 1, characterized in that, In step (2), the volume ratio of the mixture of thiolated Codonopsis pilosula polysaccharide and sodium alginate to the first core material is 1:1 to 1:1.5; the volume ratio of xanthan gum or pectin to the second core material is 1:1 to 1:1.

5.

4. The method for preparing a dual-core microcapsule according to claim 1, characterized in that, Step (3) involves freeze drying. The initial pre-freezing temperature is -20~-40℃, and the pre-freezing time is 2~3h. Then, the freezer is placed at -80℃ for 20~24h.

5. A dual-core microcapsule, characterized in that, It is prepared by the preparation method described in any one of claims 1-4.

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