Multi-component composite microcapsule based on programmed targeted release technology as well as preparation method and application of multi-component composite microcapsule

Abstract

The invention discloses a multi-component composite microcapsule based on a programmed targeted release technology and a preparation method and application thereof, and belongs to the technical field of biologication.The preparation method comprises the steps that probiotics are subjected to double-layer coating through a single cell encapsulation technology, and then the probiotics and anthocyanin are jointly encapsulated in the microcapsule based on a complex coacervation method. Mixing the probiotics with chitosan oligosaccharide and pectin in proportion in sequence, forming a silkworm cocoon-shaped double-layer coating through self-assembly, realizing layer-by-layer assembly of the coating on the surfaces of the probiotics, then encapsulating the chitosan oligosaccharide / pectin double-layer coating bacteria and anthocyanin in a composite agglomerate formed by soybean polysaccharide and whey protein isolate, and finally preparing the composite agglomerate. And freeze-drying to obtain the microcapsule. The microcapsule prepared by the invention not only can improve the survival rate and tolerance of the probiotics under adverse environmental conditions, but also can realize programmed targeted release of the anthocyanin and the probiotics in the small intestine and the colon respectively.

Description

Technical Field

[0001] This patent relates to the field of biotechnology, specifically to the preparation and application of a multi-component composite microcapsule. Background Art

[0002] With the rapid development of modern food science and nutrition, research has shifted from analyzing the efficacy of single functional factors to exploring the synergistic interactions of multiple bioactive ingredients. Notably, the co-encapsulation of probiotics with prebiotics such as anthocyanins to create a synbiotic system not only triggers a bidirectional "microbiome-metabolite" regulatory cascade for synergistic nutrient supply but also creates a physical-chemical barrier to enhance bacterial viability. However, the extreme sensitivity of probiotics to gastric acid, bile salts, and digestive enzymes results in an oral survival rate of less than 1%. Furthermore, anthocyanins, due to the chemical instability of their molecular structure, are susceptible to degradation in the gastrointestinal environment. Currently developed food-grade co-delivery systems primarily focus on single-target delivery in the gastrointestinal tract, with few reports on the multi-target release of different functional factors in the gastrointestinal tract. While traditional delivery systems can provide some protection, they struggle to address the differences in physicochemical properties and delivery requirements between the two substances. Furthermore, they lack the ability to respond to different intestinal compartments, hindering the synergistic effects of the different functional factors.

[0003] In recent years, microcapsules prepared based on the complex coacervation method have attracted much attention due to their unique advantages. This technology spontaneously forms complexes with responsive characteristics through the electrostatic interaction of oppositely charged biopolymers under specific pH conditions. Compared with synthetic polymer materials, complex coacervates of natural origin not only have excellent biocompatibility and degradability, but their structural parameters can be precisely controlled by adjusting the phase separation conditions, providing an ideal platform for the construction of responsive delivery carriers. Therefore, the construction of a co-delivery system of probiotics and anthocyanins based on microencapsulation technology, which achieves physical isolation of probiotics and anthocyanins and colon-targeted spatiotemporal controllable release by precisely controlling the colloidal microenvironment, is a key node for breaking through the gastric acid and bile salt barrier, protecting active ingredients, and ultimately synergistically regulating the homeostasis of the intestinal microenvironment.

[0004] As shown in the following patent: Application Number: CN202310857567.X, Publication Number: CN116869970A, Invention Name: "A Highly Active Microencapsulated Probiotic and Its Preparation Method," discloses a method for preparing highly active microencapsulated probiotics. This method uses an encapsulation solution (Ophiopogon oligosaccharides, betaine, and whey protein) along with calcium alginate and carboxymethyl chitosan to encapsulate Lactobacillus plantarum to produce secondary microcapsules. The highly active microcapsules prepared by this method can effectively improve the freeze-drying survival rate and stability of calcium alginate-encapsulated probiotics, but are limited to encapsulation of probiotics and do not involve the co-delivery of probiotics with other functional factors. The multi-component composite microcapsules prepared in this patent can co-encapsulate Lactobacillus plantarum and anthocyanins.

[0005] As shown in the following patent: Application number: CN202411118890.6, publication number: CN118985881A, invention name: "A method for preparing a multi-level co-delivery composite emulsion using glycosylated walnut protein fiber", a method for preparing a multi-level co-delivery composite emulsion using glycosylated walnut protein fiber is disclosed. This method uses glycosylated walnut protein fiber as an emulsifier and a special oil as the oil phase to load fat-soluble substances. The resulting emulsion has good in vivo antioxidant activity and embedding and release properties. However, the emulsion system is a liquid system, which is not stable enough and is not convenient for storage and transportation. The multi-component composite microcapsules prepared by this patent are solid systems, which have more advantages in storage and transportation.

[0006] Article "Co-encapsulation of probiotic bacteria L. rhamnosus GG and β-carotene by a novel biphasic encapsulation technique: Stability and in vivo The article "Anti-inflammatory Properties" discloses a method for co-encapsulating β-carotene and Lactobacillus rhamnosus GG by encapsulating β-carotene in liposomes and spray-drying them together with the probiotic Lactobacillus rhamnosus GG to obtain a biphasic structure containing two functional ingredients. However, the co-delivery system obtained by this method does not achieve the release of β-carotene and Lactobacillus rhamnosus GG in different sections of the gastrointestinal tract. The multi-component composite microcapsules prepared in this patent can achieve programmed targeted release of anthocyanins and Lactobacillus plantarum in the small intestine and colon, respectively. Summary of the Invention

[0007] In order to overcome the shortcomings of the existing technology and the difficulties faced by probiotics and anthocyanins in the gastrointestinal environment, the present invention uses microencapsulation technology to provide a multi-component composite microcapsule that can program the targeted release of probiotics and anthocyanins.

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned multi-component composite microcapsules.

[0009] The third object of the present invention is to provide applications of the above multi-component composite microcapsules.

[0010] To achieve the above purpose, the probiotics are taken as an example of Lactobacillus plantarum, and the technical solution adopted by the present invention is as follows: 1) Inoculate Lactobacillus plantarum into MRS medium and activate it using 40% glycerol to prepare a glycerol culture. Add 500 μL of the glycerol culture to 25 mL of sterilized MRS broth and incubate in a 37°C biochemical incubator for 16 hours. 2) Centrifuge the culture prepared in step 1) twice (7000 rpm, 10 min, 4°C), wash the precipitate, disperse the precipitated bacterial slurry in 5 mL of sterile deionized water, and vortex for 30 s to obtain a Lactobacillus plantarum suspension; 3) Dilute the plant lactobacillus suspension prepared in step 2) 10-fold with sterile deionized water at pH 4.0 to a concentration of 10 10 ~10 11 CFU / mL, vortex for 30 s to evenly disperse Lactobacillus plantarum; 4) Weigh a certain amount of coating material and dissolve it in deionized water. Stir at 700 rpm for 2 hours at a fixed temperature to ensure complete dissolution. Then adjust the pH of the solution to 4.0 with 0.1 M NaOH and 0.1 M HCl and store at 4°C. 5) The Lactobacillus plantarum suspension obtained in step 3) was mixed with the coating material solution obtained in step 4) in a certain proportion, stirred for 30 minutes, and cultured on a shaker for 10 minutes; 6) Centrifuge the mixed solution prepared in step 5) twice (7000 rpm, 10 min, 4°C), remove the supernatant, and resuspend the precipitate in sterile deionized water of the corresponding pH to obtain a monolayer coating bacterial suspension; 7) Mixing the Lactobacillus plantarum suspension coated with a single layer of coating obtained in step 6) with another coating material solution in a certain proportion, stirring for 30 minutes, and shaking and culturing for 10 minutes; 8) Centrifuge the mixed solution prepared in step 7) twice (7000 rpm, 10 min, 4°C), remove the supernatant, and resuspend the precipitate in sterile deionized water of the corresponding pH to obtain a double-layer coated bacterial suspension.

[0011] 9) A certain amount of soy polysaccharide and whey protein isolate were weighed and dissolved in deionized water. The mixture was stirred at 700 rpm for 2 h at a fixed temperature to ensure complete dissolution. The pH of the solution was then adjusted to a fixed value with 0.1 M NaOH and 0.1 M HCl and stored at 4°C.

[0012] 10) Mix the double-coated bacterial suspension prepared in step 8) with a certain amount of anthocyanin and stir until uniformly distributed. Add the mixed solution to the whey protein isolate solution prepared in step 9).

[0013] 11) Subsequently, the mixed solution prepared in step 10) and the soybean polysaccharide solution prepared in step 9) are mixed and stirred until uniform, and the solution is adjusted to a fixed pH to obtain a soybean polysaccharide / whey protein isolate complex coacervate containing double-layer coated bacteria and anthocyanins.

[0014] 12) The composite coacervate prepared in step 11) was placed in a freeze dryer and freeze-dried for 36 hours to obtain multi-component composite microcapsules containing double-layer coated bacteria and anthocyanins.

[0015] Preferably, in step 4), the coating material is selected from one of chitosan oligosaccharide (COS), whey protein isolate (WPI) and gelatin (GE).

[0016] More preferably, the coating material is selected from chitosan oligosaccharide, and the concentration of the chitosan oligosaccharide solution is 2 mg / mL. Preferably, in step 5), the volume ratio of the Lactobacillus plantarum suspension to the chitosan oligosaccharide solution is 1:5.

[0017] Preferably, in step 7), the coating material is pectin (PE), the concentration of the pectin solution is 2 mg / mL, and the volume ratio of the Lactobacillus plantarum suspension to the pectin solution is 1:5.

[0018] Preferably, in step 9), the concentration of soybean polysaccharide (SPSS) and whey protein isolate (WPI) is 20 mg / mL, and the pH of the solution is 6.0.

[0019] Preferably, the concentration of anthocyanins (ACNs) in step 10) is 0.3 mg / mL.

[0020] Preferably, in step 11), the volume ratio of soybean polysaccharide to whey protein isolate is 1:3, and the pH of the solution is 4.0.

[0021] Compared with the prior art, the beneficial effects and advantages of the present invention are: (1) The coating materials and wall materials used in the present invention have the advantages of low cost, non-toxicity, biocompatibility, and biodegradability, providing an ideal platform for constructing responsive delivery carriers.

[0022] (2) The present invention utilizes single-cell encapsulation technology to prepare a single-layer coating carrier, thereby achieving protection for probiotics. Simultaneously, a double-layer coating bacteria is constructed through layer-by-layer assembly technology, further improving the survival rate and tolerance of probiotics.

[0023] (3) This invention is the first to construct a co-delivery system with programmed sequential release performance, successfully achieving the programmed targeted release of Lactobacillus plantarum and anthocyanins in different sections of the gastrointestinal tract.

[0024] (4) The microcapsules prepared by freeze-drying technology in the present invention not only significantly enhance the physicochemical stability of the co-delivery system, but also animal experiments have shown that the programmed targeted delivery mechanism can effectively alleviate the pathological symptoms of DSS-induced colitis in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the potential value of soybean polysaccharide and whey protein isolate at pH 2.0~8.0.

[0026] Figure 2 The turbidity of soybean polysaccharide / whey protein isolate complex coacervate at different ratios.

[0027] Figure 3 Optical microscopic images of soybean polysaccharide / whey protein isolate complex coacervates at different ratios.

[0028] Figure 4 (A) Circular dichroism spectra and (B) secondary structure contents of soybean polysaccharide / whey protein isolate complex coacervates at different ratios.

[0029] Figure 5 This figure shows the effect of different anthocyanin concentrations on the survival rate of probiotics.

[0030] Figure 6 Viable counts of probiotics in free PL, PL@COS / PE, SW-P and SW-PA before and after pasteurization.

[0031] Figure 7 Viable counts of probiotics in free PL, PL@COS / PE, SW-P, and SW-PA before and after freeze-thaw cycles.

[0032] Figure 8 Figure 2 shows the viable counts of probiotics in free PL, PL@COS / PE, SW-P, and SW-PA before and after 28 days of storage at 4°C.

[0033] Figure 9 To determine the viable bacterial counts of free PL, PL@COS / PE, SW-P and SW-PA during in vitro simulated gastrointestinal digestion.

[0034] Figure 10 The antioxidant activity of anthocyanins in SW-PA after (A) pasteurization, (B) three freeze-thaw cycles, and (C) storage at 4°C for one week.

[0035] Figure 11 To simulate the retention rate of free ACNs and ACNs in SW-PA during gastrointestinal digestion in vitro.

[0036] Figure 12In vitro release of (A) probiotics and (B) anthocyanins from PL@COS / PE+ACNs and SW-PA.

[0037] Figure 13 SEM images of (A) free freeze-dried bacterial powder, (B) microcapsule MP, (C) microcapsule MSW-P, and (D) MSW-PA.

[0038] Figure 14 FTIR of free bacterial freeze-dried powder and three types of microcapsules.

[0039] Figure 15 XRD patterns of (A) freeze-dried free bacterial powder, three types of microcapsules, and (B) wall materials.

[0040] Figure 16 The DSC graphs of free bacterial freeze-dried powder and three types of microcapsules.

[0041] Figure 17 The figure shows the viable bacterial counts of free-living freeze-dried bacterial powder and probiotics in three microcapsules during 28 days of storage at 25°C.

[0042] Figure 18 (A) Schematic diagram of the experimental design and (B) body weight changes of mice in each group during the experiment.

[0043] Figure 19 The DAI scores of mice in each group were calculated during the experiment.

[0044] Figure 20 The bloody stools of mice in different groups on the 7th day.

[0045] Figure 21 (A) Colon appearance and (B) colon length of mice in each group.

[0046] Figure 22 Representative H&E-stained colon tissue sections.

[0047] Figure 23 The concentrations of (A) TNF-α, (B) IL-1β, and (C) IL-10 in the serum of each group. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the examples. It should be noted that the description of these embodiments is intended to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0049] The experimental methods used in the present invention are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0050] Example 1: Preparation of soybean polysaccharide-whey protein isolate complex coacervate 1) Determination of optimal pH SPSS and WPI solutions with a concentration of 1 mg / mL were prepared, and their pH values ​​were adjusted to 2.0-8.0, respectively. The zeta potentials of SPSS and WPI at different pH values ​​were measured using a Nano ZS90 particle size analyzer.

[0051] The results are as follows Figure 1 As shown, SPSS is negatively charged within the pH range of 3.0 to 8.0. Its zeta potential decreases with increasing pH, from 5.28 ± 1.02 mV at pH 2.0 to -39.37 ± 1.93 mV at pH 8.0. In contrast, the zeta potential of WPI exhibits significant variations with pH. Within the pH range of 2.0 to 4.0, WPI is positively charged, with its zeta potential decreasing from 10.94 ± 1.53 mV at pH 2.0 to 9.90 ± 1.02 mV at pH 4.0. When the pH exceeds its isoelectric point, WPI begins to acquire a negative charge, reaching -26.37 ± 0.84 mV at pH 8.0. Therefore, at pH 3.0 and 4.0, the oppositely charged SPSS and WPI can associate through electrostatic interactions to form complex coacervates. Considering that the preparation condition of chitosan oligosaccharide / pectin double-layer coated bacteria is pH 4.0, and that this pH can provide moderate electrostatic attraction between SPSS and WPI while avoiding excessive aggregation caused by excessive charge density, 4.0 was selected as the optimal preparation pH for the complex coacervation of SPSS and WPI.

[0052] 2) Determination of the optimal SPSS / WPI ratio 20 mg / mL solutions of SPSS and WPI were prepared, respectively, and their pH was adjusted to 6.0. SPSS and WPI were then mixed and stirred at volume ratios of 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively, and the pH was adjusted to 4.0 to prepare SPSS / WPI complex coacervates. The turbidity and morphology of the complex coacervates were observed. Turbidity was measured using a UV spectrophotometer, with deionized water as a blank control. The morphology of the SPSS / WPI complex coacervates prepared at different ratios was observed using an optical microscope. Furthermore, the prepared complex coacervates were diluted to 0.2 mg / mL with deionized water at pH 4.0 and analyzed by circular dichroism at wavelengths of 190–260 nm. The scan rate was set at 100 nm / min, and the response time was 1 s. The secondary structure of the protein was calculated using analysis software.

[0053] The results are as follows Figure 2 As shown in the figure, as the WPI ratio increases, the turbidity of the complex coacervate increases. Among them, when the SPSS / WPI ratio is 1:3, the turbidity of the complex coacervate reaches the maximum value, indicating that the yield of the complex coacervate is the highest under this ratio. When the WPI ratio increases, the electrostatic interaction between it and the negatively charged SPSS is enhanced, thereby promoting the formation of complex coacervate. Figure 3 As can be seen in the results, the complex coacervates formed at different SPSS / WPI ratios all exhibited an irregular network structure. Notably, the volume of the complex coacervates increased when the SPSS / WPI ratio exceeded 1:2. In contrast, the complex coacervates formed at SPSS / WPI ratios of 2:1 and 1:1 were smaller. Therefore, the optimal SPSS / WPI ratio at pH 4.0 was determined to be 1:3. This ratio not only exhibited the highest turbidity (i.e., the highest yield) but also formed a moderate network structure, providing an ideal material foundation for subsequent microcapsule preparation.

[0054] The results are as follows Figure 4 As shown in Figure A, the α-helical structure is reflected by the positive peak at 192 nm and the negative peaks at 208 nm and 222 nm, while the broad peak between 205 and 220 nm represents the abundant β-sheet structure in WPI. The significant changes in peak position and peak height indicate that the addition of SPSS has a significant effect on the secondary structure of WPI. Figure 4 As shown in Figure B, 5.35% α-helices, 28.5% β-sheets, 13.7% β-turns, and 52.4% random coils were present in WPI (control). The addition of SPSS altered the protein's secondary structure. The maximum α-helical structure (8.6%) was achieved at an SPSS:WPI ratio of 1:3, while the random coil structure decreased.

[0055] Example 2: Effects of different anthocyanin concentrations on the survival rate of probiotics Lactobacillus plantarum was activated by inoculating it into MRS medium. Glycerol cultures were prepared using 40% glycerol and stored at -80°C. 500 μL of the glycerol culture was added to 25 mL of sterilized MRS broth and incubated in a 37°C biochemical incubator for 16 h. The culture was then centrifuged at 7000 rpm for 10 min twice. The precipitate was washed with sterile deionized water, and the resulting bacterial slurry was dispersed in 5 mL of sterile deionized water and vortexed for 30 s to obtain a Lactobacillus plantarum suspension. The suspension was then diluted 10-fold and mixed with various concentrations of ACNs (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL). The cultures were then incubated at 4°C for 6 days. After 6 days, the viable counts were determined to investigate the effect of ACN concentration on the survival rate of the probiotic.

[0056] like Figure 5 As shown, at an ACNs concentration of 0 mg / mL (control group), the viable count of probiotics was 10.89 ± 0.06 log CFU / mL. As the ACNs concentration increased, the viable count of probiotics initially stabilized and then decreased: when the ACNs concentration ranged from 0.1 to 0.3 mg / mL, the viable counts were 10.90 ± 0.03 log CFU / mL, 10.89 ± 0.01 log CFU / mL, and 10.92 ± 0.01 log CFU / mL, respectively, which were not significantly different from the control group. When the concentration increased to 0.4 mg / mL and 0.5 mg / mL, the viable count of probiotics decreased to 10.85 ± 0.01 log CFU / mL and 10.84 ± 0.08 log CFU / mL, respectively. These results indicate that low concentrations of ACNs (0.1–0.3 mg / mL) slightly enhance the survival of probiotics, with viable counts remaining stable or increasing slightly with increasing ACN concentration. Therefore, 0.3 mg / mL was selected as the optimal ACNs concentration for subsequent experiments. This concentration allows ACNs to exert their potential prebiotic effects while also avoiding the potential inhibitory effects of higher concentrations, providing reliable experimental conditions for the subsequent synergistic effects of probiotics and ACNs.

[0057] Example 3: Preparation and characterization of complex coacervates of encapsulated coated probiotics PL@COS / PE and co-encapsulated coated probiotics PL@COS / PE and anthocyanidins COS and PE were used as coating materials and added to sterile deionized water at room temperature and stirred at 700 rpm for 2 h to prepare a 2 mg / mL aqueous solution. The pH of the above solution was then adjusted to 4.0. The Lactobacillus plantarum suspension was then diluted 10-fold with sterile deionized water at pH 4.0 to a concentration of 10 10 ~10 11 CFU / mL. The diluted Lactobacillus plantarum suspension was mixed with COS solution at a ratio of 1:5 (v / v) and stirred for 30 minutes, incubated on a shaker for 10 minutes, and then centrifuged twice at 4°C (7000 rpm, 10 minutes). The supernatant was discarded, and the precipitate was resuspended in sterile deionized water (pH 4.0) to prepare chitosan oligosaccharide-coated bacteria, PL@COS. Subsequently, the PL@COS suspension was mixed with PE solution at a ratio of 1:5 (v / v), stirred at room temperature for 30 minutes, incubated on a shaker for 10 minutes, and centrifuged twice at 7000 rpm to obtain a bacterial slurry. Finally, the slurry was dispersed in sterile deionized water (pH 4.0) to obtain a chitosan oligosaccharide / pectin double-coated bacteria, named PL@COS / PE. Subsequently, 20 mg / mL SPSS and WPI solutions were prepared, respectively, and the pH was adjusted to 6.0. 100 μL of the double-coated PL@COS / PE was mixed with 4.5 mL of WPI solution. The above solution was then mixed with 1.5 mL of SPSS solution (volume ratio of SPSS to WPI:1:3) and stirred until homogeneous. The pH was adjusted to 4.0 to obtain SPSS / WPI complex coacervates loaded with PL@COS / PE, referred to as SW-P. The preparation method for complex coacervates co-encapsulating PL@COS / PE and ACNs was similar to the above description. 100 μL of double-coated PL@COS / PE and 1.35 mg of ACNs (concentration: 0.3 mg / mL) were mixed with 4.5 mL of WPI solution. This solution was then mixed with 1.5 mL of SPSS solution and stirred until homogeneous. The pH was adjusted to 4.0 to obtain SPSS / WPI complex coacervates loaded with PL@COS / PE and ACNs, referred to as SW-PA.

[0058] 1) Evaluation of pasteurization stability of probiotics in complex coacervates Free bacteria, double-coated PL@COS / PE bacteria, and complex coacervates SW-P and SW-PA were placed at 63°C for 30 minutes. After 30 minutes, the samples were removed and cooled in ice water. Following cooling, the pH of SW-P and SW-PA was adjusted to 6.0 to dissolve the complex coacervates, facilitating subsequent plate counting and studying changes in probiotic activity in the samples.

[0059] like Figure 6As shown in the results, after 30 min of heating, the viable counts of probiotics in free bacteria, PL@COS / PE, SW-P, and SW-PA decreased from 10.89±0.03 log CFU / mL, 10.85±0.16 log CFU / mL, 10.83±0.05 log CFU / mL, and 10.85±0.06 log CFU / mL to 4.95±0.21 log CFU / mL, 6.01±0.22 log CFU / mL, 7.39±0.07 log CFU / mL, and 7.39±0.02 log CFU / mL, respectively. These results indicate that the thermal stability of probiotics in the composite coacervates was significantly improved compared to free bacteria and double-layer-coated PL@COS / PE. In addition, the thermal stabilities of the complex coacervates SW-P and SW-PA were comparable, indicating that the addition of ACNs did not negatively affect the thermal protection of the complex coacervates, further verifying the possibility of co-encapsulation of ACNs and PL@COS / PE.

[0060] 2) Evaluation of freeze-thaw stability of probiotics in complex coacervates Using free bacteria and double-coated PL@COS / PE as controls, the complex coacervates SW-P and SW-PA were stored at -20°C for 22 hours and then thawed in a 30°C water bath for 2 hours, repeated three times. After three freeze-thaw cycles, the samples were removed, and the pH of SW-P and SW-PA was adjusted to 6.0 to dissolve the complex coacervates, facilitating subsequent plating and counting. Changes in the viability of the probiotics in the samples were then investigated.

[0061] The results are as follows Figure 7 As shown, the viable bacterial counts of free bacteria and double-coated PL@COS / PE decreased most significantly during three freeze-thaw cycles, with cumulative reductions of approximately 2.30 log CFU / mL and 1.65 log CFU / mL, respectively. In contrast, the viable bacterial counts of probiotics in the composite coacervates SW-P and SW-PA decreased by only approximately 0.18 log CFU / mL after three freeze-thaw cycles, significantly lower than those of free bacteria and PL@COS / PE. Notably, there was no significant difference in the freeze-thaw stability of the composite coacervates SW-P and SW-PA, further confirming that the addition of ACNs did not affect the protective properties of the composite coacervates.

[0062] 3) Storage stability evaluation of probiotics in complex coacervates The storage viability of complex coacervates (SW-P) and (SW-PA) was studied over 28 days (4°C), using free bacteria and double-coated bacteria (PL@COS / PE) as controls. Samples were removed after 28 days for viable bacterial counts. The pH of SW-P and SW-PA was adjusted to 6.0 to dissolve the complex coacervates, facilitating subsequent plate counts and studying changes in probiotic activity within the samples.

[0063] The results are as follows Figure 8 As shown, the survival rate of probiotics in all samples decreased with prolonged storage. The viability of free bacteria and double-coated PL@COS / PE decreased significantly, decreasing by approximately 4.79 log CFU / mL and 2.89 log CFU / mL compared to day 0. The viable counts of probiotics in the composite coacervates SW-P and SW-PA after 28 days of storage were 10.66±0.04 log CFU / mL and 10.64±0.03 log CFU / mL, respectively, representing decreases of approximately 0.22 log CFU / mL and 0.26 log CFU / mL. Compared with free bacteria and double-coated PL@COS / PE, the composite coacervates significantly improved the storage stability of probiotics. There was no significant difference in the storage stability of the composite coacervates SW-P and SW-PA, confirming that the addition of ACNs did not affect the protective ability of the composite coacervates for probiotics.

[0064] 4) Evaluation of the in vitro simulated digestion stability of probiotics in complex coacervates Using free bacteria and double-coated PL@COS / PE as controls, the activity of probiotics encapsulated in complex coacervates (SW-P) and SW-PA was investigated during simulated in vitro digestion. Simulated gastrointestinal fluid (SGF) was prepared as follows: Sterile deionized water containing sodium chloride (2 mg / mL) and pepsin (1.6 mg / mL) was used to prepare simulated gastrointestinal fluid (SGF), and the pH was adjusted to 2.0 with 1 M HCl. The sample was mixed with 20 mL of SGF solution, and the pH of the mixture was adjusted to 2.0 to initiate gastric digestion. The mixture was then incubated in a 37°C waterbath for 2 hours. After 2 hours, the pH of the SGF was adjusted to 6.8 to terminate gastric digestion. Subsequently, simulated intestinal fluid (SIF) was prepared by dissolving CaCl2 (5 mM), porcine bile salts (5 mg / mL), and pancreatin (1.6 mg / mL) in sterile deionized water and adjusting the pH to 6.8. An equal volume of SIF was then added to the digestive fluid for small intestinal digestion. After incubation in small intestinal fluid for 2 hours, an equal amount of simulated colonic fluid (SCF) was added. Simulated colonic fluid, made from sterile PBS buffer at a pH of 7.4, was then digested for 4 hours. During digestion, the temperature was maintained at 37°C. Samples were collected at 0, 2, 4, 6, and 8 hours, and the activity of the probiotic cells was calculated using MRS agar.

[0065] from Figure 9As can be seen, after 2 h of gastric digestion, the viable counts of probiotics in free bacteria, double-coated PL@COS / PE, and complex coacervates SW-P and SW-PA decreased to 5.11±0.08 log CFU / mL, 5.88±0.02 log CFU / mL, 6.40±0.03 log CFU / mL, and 6.67±0.04 log CFU / mL, respectively. The low gastric pH significantly reduced the activity of probiotics in all samples. To further investigate the protective effect of complex coacervates on probiotics, digestion in SGF was followed by digestion in SIF and SCF. The results showed that the viability of probiotics in all samples digested in SIF for 2 h and SCF for 4 h was not significantly different from that in SGF for 2 h, which is due to the high resistance of probiotics to bile salts. After 4 hours of colonic digestion, the viable counts of probiotics in the complex coacervates SW-P and SW-PA increased by approximately 1.63 log CFU / mL and 1.61 log CFU / mL, respectively, compared to the double-coated PL@COS / PE. These results demonstrate that the presence of the complex coacervates effectively protects the encapsulated probiotics from damage under simulated gastrointestinal conditions and confirms the stability of the complex coacervates in the gastrointestinal system.

[0066] 5) Stability evaluation of anthocyanins in complex coacervates The thermal stability of ACNs in the complex coacervates (SW-PA) was investigated by heating free ACNs at 63°C for 30 minutes, using free ACNs as a control. The freeze-thaw stability of the ACNs was investigated by storing the free ACNs and complex coacervates at -20°C for 22 hours and then thawing them in a 30°C water bath for 2 hours, a freeze-thaw cycle repeated three times. Finally, the storage stability of the free ACNs and complex coacervates (SW-PA) was investigated by storing them at 4°C for one week. The antioxidant activity of the ACNs in the free ACNs and complex coacervates (SW-PA) after these treatments was then evaluated. The antioxidant activity of the ACNs reflects the effects of the complex coacervates on the thermal stability, freeze-thaw thermal stability, and storage thermal stability of the ACNs. The antioxidant activity of ACNs was tested as follows: a 7 mmol / L stock solution of 2,2'-hydrazino(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was prepared. An equal volume of 2.45 mmol / L potassium persulfate solution was added to the solution. The mixture was thoroughly mixed and allowed to stand at room temperature in the dark for 16 hours to prepare the ABTS stock solution. The ABTS stock solution was then diluted to an absorbance of 0.7 ± 0.02 at 734 nm before use. Subsequently, 1 mL of the sample was diluted appropriately and mixed with 4 mL of the ABTS stock solution. A control solution of 1 mL of distilled water was used as a control. The mixture was reacted at room temperature in the dark for 10 minutes. The absorbance at 734 nm was measured using a UV spectrophotometer, and the ABTS free radical scavenging rate was calculated according to the following formula.

[0067] ; Where: Ac is the absorbance of the reaction solution of free ACNs and ABTS, and As is the absorbance of the reaction solution of anthocyanins in the complex coacervate and ABTS.

[0068] like Figure 10 As shown, the ABTS radical scavenging rates of free ACNs after heating at 63°C for 30 minutes, undergoing three freeze-thaw cycles, and storing for one week were 66.69%, 72.37%, and 64.52%, respectively. In contrast, the antioxidant activity of ACNs in the complex coacervates SW-PA was 74.53%, 86.49%, and 75.06%, respectively, representing increases of approximately 7.84%, 14.12%, and 10.54% compared to free ACNs. This is because the antioxidant activity is highly correlated with the ACN content. Compared to free ACNs, the complex coacervates formed by SPSS and WPI can protect ACNs against various adverse environmental conditions during heat, freeze-thaw cycles, and storage, reducing ACN degradation during processing.

[0069] 6) Evaluation of the in vitro simulated digestion stability of anthocyanins in complex coacervates Using an unencapsulated ACNs solution as a control, the SW-PA solution and simulated digestive fluid were mixed in a ratio of 1:4. The specific method and procedures for preparing simulated gastrointestinal fluid were the same as above. 1 mL of sample was removed at 0, 2, 4, 6, and 8 h into the digestion process. Subsequently, the sample solution was centrifuged at 12,000 rpm for 10 min, and the supernatant and precipitate were collected. The ACNs in the precipitate were dissolved with 70% ethanol. The ACNs content in the supernatant and precipitate was determined using a pH differential method.

[0070] Subsequently, the retention rate of ACNs was calculated according to the following formula.

[0071] ; Where, M is the amount of ACNs initially added (mg), M 1 is the content of ACNs in the supernatant after centrifugation (mg), M 2 is the content of ACNs in the precipitate (mg).

[0072] Depend on Figure 11 After 2 h of gastric digestion, the retention rates of ACNs in the SW-PA composite coacervate and free ACNs were 70.11% and 58.26%, respectively, indicating that the composite coacervate plays a positive role in the stability of ACNs in gastric fluid. The retention rate of ACNs gradually decreased with time, due to the instability of ACNs in a high pH environment and their accelerated degradation. Notably, after 4 h, the retention rate of free ACNs was 40.29%, while that of ACNs in the SW-PA composite coacervate was 52.03%, indicating that the composite coacervate significantly protected ACNs during digestion. After 8 h, there was no significant difference in the retention rates between the two composite coacervates. This is because the structure of the SW-PA composite coacervate gradually disintegrated under the action of digestive fluid with continued digestion time, resulting in the continuous release of the encapsulated ACNs into the intestinal environment until equilibrium was reached.

[0073] 7) In vitro programmed release of probiotics and anthocyanins from complex coacervates The in vitro programmed release of PL@COS / PE and ACNs encapsulated in the complex coacervate (SW-PA) was investigated, using a probiotic and ACNs blend (PL@COS / PE+ACNs) as a control. The simulated gastrointestinal fluid preparation and procedures were the same as above. Samples were collected every 2 hours during digestion, and 1 mL of the sample was centrifuged (10,000 rpm, 10 min) before the ACNs release was determined by pH differential analysis. The probiotic release rate was quantified using the plate colony count method in a 0.5 mL sample. The cumulative release of the probiotics and ACNs at different times was expressed as a percentage of the initial addition amount.

[0074] Depend on Figure 12 As shown in Figure 5, after 2 h of SGF digestion, the release rate of probiotics from the PL@COS / PE+ACNs system was 36.89%, significantly higher than the 17.53% from the SW-PA system. This result demonstrates that the electrostatic interaction between the amphiphilic biopolymers in the SW-PA complex effectively maintains the structural stability of the delivery system in an acidic environment. Notably, while the coating in the PL@COS / PE+ACNs system provides a certain degree of protection to the microorganisms, delamination of the coating still occurs under gastric acid conditions, resulting in a higher release of probiotics. Upon entering the simulated small intestine, the release kinetics of the two systems exhibit significant differences. The cumulative release rate of probiotics from the SW-PA system only increased to 26.59%, while that from the PL@COS / PE+ACNs system rapidly reached 65.90%. Under the neutral pH of the small intestine, the electrostatic interaction between SPSS and WPI weakens, leading to microcapsule disintegration, but the released coating material continues to provide a protective barrier for the microorganisms. In contrast, in PL@COS / PE+ACNs, the coating had already sustained partial damage in the stomach, leading to the rapid release of probiotics under the synergistic effects of bile salts, pancreatic enzymes, and other substances in the intestinal fluid. Finally, after 8 hours of simulated colonic digestion, the cumulative release rates of probiotics in SW-PA and PL@COS / PE+ACNs were 85.55% and 80.68%, respectively. This indicates that after entering the colon, the coated bacteria completely disintegrate under the action of digestive enzymes, allowing the probiotics to be rapidly released, achieving a high cumulative release rate. In summary, the SW-PA system exhibits superior targeting properties throughout the digestive process: it not only effectively protects the probiotics from gastric acid but also delays their release in the small intestine, ultimately achieving colon targeting.

[0075] The release of ACNs during the simulated digestion process is shown in Figure 2. Figure 12As shown in Figure B, within 2 hours of SGF digestion, the release rate of ACNs from SW-PA was only 21.15%, significantly lower than that of the PL@COS / PE+ACNs system (62.10%). This suggests that the composite structure of SW-PA effectively protects the active ingredients from the acidic environment of the stomach. Upon entering the small intestine, the increased pH induced dissociation of the complex coacervates, prompting rapid release of ACNs from SW-PA, with a cumulative release rate of 56.03%. After 4 hours of colonic digestion, a final cumulative release rate of 85.60% was achieved. For the PL@COS / PE+ACNs system, premature release of ACNs in the stomach due to loss of carrier protection prevented the effective release of the functional ingredients. Combined with the targeted release of probiotics, SW-PA demonstrated excellent programmed targeting properties, successfully achieving the orderly, coordinated release of ACNs and probiotics within the digestive tract. This not only significantly enhanced the stability of both functional factors but also maximized their health benefits through synergistic action.

[0076] Example 4: Preparation and characterization of microcapsules loaded with coated probiotics PL@COS / PE and microcapsules co-loaded with coated probiotics PL@COS / PE and anthocyanins Free probiotic suspensions, double-layer coated PL@COS / PE suspensions, and complex coacervates SW-P and SW-PA were pre-frozen at -80°C for 2 h and then freeze-dried in a freeze dryer for 36 h to prepare freeze-dried free bacterial powder and three types of microcapsules. The microcapsules were designated as probiotic-loaded chitosan oligosaccharide / pectin microcapsules (MP), PL@COS / PE-loaded soybean polysaccharide / whey protein isolate microcapsules (MSW-P), and PL@COS / PE and ACN-loaded soybean polysaccharide / whey protein isolate microcapsules (MSW-PA).

[0077] 1) Scanning electron microscopy (SEM) observation of microcapsules The free bacterial freeze-dried powder and microcapsule samples were adhered to a double-sided conductive carbon tape, and then the sample surface was gold-sprayed. The microstructure of the samples was observed using a scanning electron microscope at an accelerating voltage of 5.00 KV.

[0078] The microstructure of free freeze-dried bacterial powder and microcapsule MP is as follows Figure 13 A and 13B. As can be seen from the figure, the free bacteria present a typical long rod morphology with a smooth surface and intact structure. In contrast, although the microcapsule MP maintains a typical long rod morphology, its surface is obviously attached with wall material and there is obvious adhesion. In addition, Figure 13As can be seen in Figures C and 13D, the microcapsules MSW-P and MSW-PA exhibit irregular, amorphous, and broken glass or flake structures, which are typical characteristics of freeze-dried microcapsules. It is worth noting that the surfaces of the microcapsules MSW-P and MSW-PA are smooth, without obvious cracks or wrinkles.

[0079] 2) Texture characteristics of microcapsules A cylindrical probe with a diameter of 30 mm mounted on a 100 N force sensor was used. During the test, the sample was compressed twice at a speed of 0.1 mm / s, with a maximum deformation of 40% each time. The five indicators of hardness, adhesion, elasticity, cohesion and chewiness were obtained using the default software.

[0080] Table 1 shows that there were no significant differences in hardness and adhesion among all samples. However, as the probiotics were gradually encapsulated into double-layer coated microcapsules and composite coacervation microcapsules, the hardness of the samples gradually decreased. Furthermore, microcapsules formed by SPSS / WPI composite coacervation exhibited lower hardness than those formed by a single double-layer coating. Despite modification with COS and PE, the MP microcapsules maintained a relatively dense structure, with a hardness intermediate between that of the free freeze-dried bacterial powder and the MSW-P and MSW-PA microcapsules. While adhesion did not change significantly, the free freeze-dried bacterial powder exhibited the highest adhesion. However, due to the exposed carboxyl groups of the pectin, the MP microcapsules retained a certain degree of polarity, resulting in lower adhesion than that of the free bacterial powder but higher than those of the MSW-P and MSW-PA microcapsules. SPSS and WPI surface modification of the MSW-P and MSW-PA microcapsules resulted in lower adhesion. Cohesion also exhibited a similar downward trend. Furthermore, the elasticity results indicate that the free-freeze-dried bacterial powder exhibited higher elasticity, while the elasticity of the microcapsules MP was superior to that of the microcapsules MSW-P and MSW-PA, but lower than that of the free-freeze-dried bacterial powder. The elasticity of the microcapsules MSW-P and MSW-PA was significantly lower than that of the free-freeze-dried bacterial powder and microcapsules MP. The chewability data in the table indicate that the addition of SPSS and WPI to the microcapsules MSW-P and MSW-PA reduced their hardness and, consequently, their chewability. Furthermore, there were no significant differences in the textural properties of the microcapsules MSW-P and MSW-PA. The results indicate that the hardness, adhesion, elasticity, cohesion, and chewiness of the microcapsules MSW-P and MSW-PA were all lower than those of the free-free bacterial powder and microcapsules MP. This suggests that the flexible network structure formed by WPI and SPSS improves the mechanical properties of the microcapsules, thereby achieving the desired texture and making the microcapsules easier to digest and absorb in powder form.

[0081] Table 1 Texture characteristics of freeze-dried free bacterial powder and three types of microcapsules

[0082] 3) Fourier transform infrared spectroscopy of microcapsules The chemical structure and interactions of the monolayer-coated bacteria were analyzed using Fourier transform infrared spectroscopy. The sample powder was mixed with potassium bromide at a mass ratio of 1:100, ground thoroughly, and pressed into a thin sheet. The sheet containing the sample was loaded to obtain a wavelength range of 500-4000 cm -1 The spectral signal recorded was averaged over 64 scans with a resolution of 4 cm -1 .

[0083] from Figure 14 It can be seen that WPI is at 1537 cm -1 The characteristic peak at 1647 cm is caused by amide II related to intermolecular β-sheet aggregation. -1 The characteristic peak at 3384 cm is the C=O stretching vibration of amide I, which confirms the existence of antiparallel β-sheets in WPI. -1 (O-H stretching vibration) and 1631 cm -1 (COO- stretching). The characteristic peaks of the infrared spectra of these two wall materials also appear in the microcapsules MSW-P and MSW-PA, but there is a certain shift in the wave number. In the microcapsules MSW-P and MSW-PA, the peaks are between 3600 and 3200 cm -1 A broad and strong absorption band appeared at both ends, which represents the stretching vibration of -OH between molecules, and its characteristic peak moved to 3298 cm -1 and 3296 cm -1 This suggests that hydrogen bonding may play an important role in the formation of microcapsules. A shift to 2933 cm-1 was also observed in the microcapsules MSW-P and MSW-PA. -1 The characteristic peak at 1647 cm (methylene C-H asymmetric stretching vibration) is a common feature in FTIR of proteins, indicating that hydrophobic interaction is also involved in the formation of microcapsules. -1 and 1541 cm -1 The peak at 1396 cm indicates that electrostatic interaction also exists in the microcapsules. In the fingerprint region, the microcapsules MSW-P and MSW-PA have a peak at 1396 cm -1 、1242 cm -1 and 1039 cm -1 The characteristic peaks on the left and right represent the bending vibrations of N-H and -NH2 respectively. The peaks of ACNs at 3392 cm -1 The characteristic peak of is caused by the -OH stretching vibration of its phenolic hydroxyl structure, which is at 1631 cm -1 , 1520 cm -1 and 1454 cm -1The peak at 820 cm is attributed to the benzene ring structure and is the characteristic absorption peak of ACNs. -1 、762 cm -1 and 706 cm -1 The absorption peak at is caused by the out-of-plane bending vibration of the CH- benzene ring. The figure shows that the spectrum of the microcapsule MSW-PA is not a simple superposition of the wall material and the ACNs, indicating that there is an interaction between SPSS and WPI and the ACNs. Furthermore, no characteristic absorption peaks of ACNs were detected in the microcapsule MSW-PA, indicating that the ACNs are completely embedded in the microcapsule wall material.

[0084] 4) X-ray diffraction (XRD) of microcapsules X-ray diffraction patterns of the free, freeze-dried bacterial powder, microcapsules, and wall materials were obtained using X-ray diffraction (XRD). Approximately 0.2 g of sample was placed on a material plate and scanned continuously from 5° to 60° (2θ) at a scan rate of 5° / min under Cu Ka radiation, with an operating voltage of 40 kV and a current of 40 mA.

[0085] from Figure 15 The probiotics exhibit no crystalline peaks. Similarly, broad amorphous peaks were observed in microcapsules MP, MSW-P, and MSW-PA, indicating the presence of probiotics within the microcapsules. COS and PE exhibited typical amorphous and crystalline structures, respectively. Significant characteristic peaks were observed for PE at 15°, 20°, and 25°, indicating that PE exhibited characteristics of a crystalline structure. However, no characteristic peaks were observed for PE in microcapsules MP, MSW-P, and MSW-PA, indicating that the PE was in an amorphous form. This suggests that interaction between the COS and PE layers in the double-layer coating resulted in the transformation of PE from a crystalline to an amorphous form. Furthermore, no significant differences were observed in the XRD spectra of the free freeze-dried bacterial powder and the three microcapsules MP, MSW-P, and MSW-PA, indicating that the crystallinity of the microcapsules formed by encapsulation with different wall materials remained unchanged. However, the amorphous peaks observed in microcapsules MSW-P and MSW-PA shifted slightly, with the peak around 15° for MP shifting to around 13° for microcapsules MSW-P and MSW-PA, respectively. This indicates that a complex is formed between the microcapsule wall material WPI and SPSS.

[0086] 5) Differential Scanning Calorimetry (DSC) of Microcapsules Differential scanning calorimetry was used to determine the thermodynamic characteristics of the microcapsules. 5 mg of each sample was weighed and sealed in an aluminum crucible, using an empty crucible as a control. Under nitrogen, the temperature was increased from 25°C to 200°C at a heating rate of 10°C / min and a flow rate of 30 mL / min.

[0087] from Figure 16As shown in the results, the melting enthalpy of the freeze-dried powder of free bacteria is 150.5 J / g, and the endothermic peak is 99.7°C. The lower melting enthalpy and endothermic peak indicate that the freeze-dried powder of free bacteria has poor thermal stability. This may be because the free bacteria lack a protective wall material, making them more susceptible to thermal degradation during heating. The melting enthalpy of the microcapsules MP is 204.3 J / g, and the endothermic peak is 105.6°C, both higher than those of the freeze-dried powder of free bacteria. This indicates that the double-layer coating structure of the MP microcapsules improves the thermal stability of the bacteria to a certain extent. The COS and PE coatings on the MP outer layer, as natural polysaccharides, have excellent film-forming properties and thermal stability, effectively blocking heat transfer and delaying thermal degradation of the bacteria. The melting enthalpy of the microcapsules MSW-P is 180.1 J / g, and the endothermic peak is 109.9°C. Compared with MP, the melting enthalpy of MSW-P is lower, but the endothermic peak is higher. However, the higher endothermic peak indicates that the thermal stability of the microcapsules MSW-P is superior to that of MP. The melting enthalpy of the MSW-PA microcapsules was 198.1 J / g, and the endothermic peak was 114.7°C, both higher than those of the MSW-P microcapsules. This indicates that the addition of anthocyanins further improved the thermal stability of the microcapsules. Furthermore, the synergistic effect between anthocyanins and the wall material may have enhanced the structural stability of the microcapsules, providing effective thermal protection for the microorganisms and delaying thermal degradation.

[0088] 6) Evaluation of storage stability of probiotics in microcapsules The storage viability of probiotics in microcapsules (MP, MSW-P, and MSW-PA) was studied over 28 days (25°C), using free freeze-dried bacterial powder as a control. Samples were taken every 7 days to determine viable bacterial counts. A certain amount of sample was taken each time and reconstituted in sterile deionized water. Subsequently, the pH of the MSW-P and MSW-PA microcapsules was adjusted to 6.0 to dissolve the complex aggregates within the microcapsules, facilitating subsequent plate counting.

[0089] from Figure 17The initial viable cell counts of the freeze-dried powder and microencapsulated MP on day 0 were 8.79 ± 0.08 log CFU / mL and 8.99 ± 0.11 log CFU / mL, respectively. However, the initial viable cell counts of the microencapsulated MSW-P and MSW-PA were significantly higher than those of the freeze-dried powder and microencapsulated MP, reaching 10.40 log CFU / mL and 10.32 log CFU / mL, respectively. It can be seen that the initial viable cell counts of both the freeze-dried powder and microencapsulated MP decreased significantly. After 7 days of storage, the viable cell count of the freeze-dried powder decreased significantly, by approximately 0.44 log CFU / mL, while the viable cell counts of the microencapsulated MP, MSW-P, and MSW-PA decreased by only approximately 0.28 log CFU / mL, 0.22 log CFU / mL, and 0.29 log CFU / mL, respectively. Furthermore, as shown in the figure, after 28 days of storage, the viable bacterial counts of both the free-living freeze-dried powder and the microcapsules MP showed a significant decrease, falling to 7.81±0.03 log CFU / mL and 8.33±0.03 log CFU / mL, respectively, representing decreases of approximately 0.97 log CFU / mL and 0.66 log CFU / mL from the initial values. The microcapsules MSW-P and MSW-PA, on the other hand, have a network structure formed by SPSS and WPI on the outside of their double-layer coating, which better isolates them from adverse external environmental conditions. Specifically, after 28 days of storage, the viable bacterial counts of the probiotics in the microcapsules MSW-P and MSW-PA only decreased by approximately 0.44 log CFU / mL and 0.30 log CFU / mL, respectively. Notably, both the free-living freeze-dried powder and microcapsules in solid form exhibited higher viable bacterial counts compared to the liquid probiotic samples.

[0090] Example 5: Alleviating effect of microcapsules containing probiotics PL@COS / PE and anthocyanins on colitis in mice After one week of adaptive feeding, the mice were randomly divided into six groups, each with eight mice. These groups included the control group (Control group), the DSS group, the MP group, the ACNs group, the M-P + ACNs group, and the MSW-PA group. The mice in the control group were given free access to water for six days, while the mice in the other groups were given free access to 2.5% DSS for six days to induce acute colitis. At the same time, different preparations were administered to each group via gavage over the six days: the Control group and the DSS group were gavaged with 200 μL of sterile saline daily; the MP group was gavaged with 200 μL of an aqueous solution of microencapsulated MP (1×10 9 CFU / mL); the ACNs group was gavaged with 200 μL of ACNs aqueous solution (1.35 mg / mL) every day; the M-P+ACNs group was gavaged with 200 μL of MP aqueous solution (1×109 CFU / mL); the MSW-PA group was gavaged daily with 200 μL of an aqueous solution of microcapsules MSW-PA (containing 1×10 9 CFU / mL of bacteria and 1.35 mg / mL of ACNs). The experimental design is as follows Figure 18 As shown in A.

[0091] 1) Weight changes During the entire modeling and gavage process, the mice were weighed using an electronic scale at the same time every day, the changes in their weight were detected, and a curve of the changes in their weight over time was drawn.

[0092] Figure 18 Figure B shows the changes in weight of mice in each group throughout the experiment. As can be seen from the figure, each group showed varying degrees of weight change as the days progressed. The weight of mice in the control group did not change significantly, with a slight increase. The other five groups all experienced significant weight loss due to oral administration of DSS. On day 7, the weight of mice in the DSS group decreased by 14.42%. The weight loss trend in the MP, ACNs, M-P+ACNs, and MSW-PA groups was more gradual than that in the DSS group. Among them, the weight change of mice in the microencapsulated MSW-PA group was the smallest of the five groups, decreasing by only 10.31%.

[0093] 2) Pathological index statistics (DAI score) Mouse body weight (0: no weight loss; 1: less than 5% weight loss; 2: 5%-10% weight loss; 3: 11%-15% weight loss; 4: >15% weight loss), diarrhea (0: normal; 2: loose stools; 4: diarrhea), hematochezia (0: normal; 2: positive fecal occult blood; 4: gross bleeding), activity status, and mortality were recorded daily. The DAI score was calculated as follows: ; Where, A , B , C They represent weight score, diarrhea score and bloody stool score respectively.

[0094] from Figure 19 As can be seen in the figure, DAI scores and body weight changes showed opposite trends over time. During the experiment, mice orally administered with 2.5% DSS showed significant weight loss and severe diarrhea, and in some cases, a certain degree of blood in the stool was observed ( Figure 20The DAI scores in the DSS group were significantly higher than those in the control group. In contrast, the daily DAI scores in the MP, ACNs, M-P + ACNs, and MSW-PA groups were significantly lower than those in the DSS group. The DAI scores in the M-P + ACNs and MSW-PA groups were even lower. Furthermore, compared to the M-P + ACNs group, the microencapsulated MSW-PA group significantly improved weight loss, diarrhea, and bloody stools in the mice.

[0095] 3) Colon morphology observation and length measurement After the experiment ended on the 7th day, the mice were killed, and colon tissue samples were taken. After removing fat and connective tissue, the colon length of each mouse was measured and photographed for record.

[0096] from Figure 21 The results showed that the colon length of healthy mice (control group) was 6.77±0.06 cm, with a smooth colonic surface and no obvious pathological changes. However, the colon length of mice treated with 2.5% DSS (DSS group) was significantly shortened, decreasing to 3.7±0.20 cm. Compared with the control group, the colonic surface of mice in the DSS group was significantly congested, edematous, and ulcerated, indicating that DSS successfully induced a colitis model in mice. The MP, ACNs, M-P+ACNs, and MSW-PA groups showed varying degrees of improvement in DSS-induced colon shortening. The colon lengths of the MP and ACNs groups were 4.67±0.29 cm and 4.47±0.29 cm, respectively, significantly longer than those in the DSS group. M-P+ACNs and MSW-PA demonstrated a stronger alleviating effect on DSS-induced colon shortening in mice. The colon length of mice in the MSW-PA group was 5.43±0.40 cm, which was approximately 0.33 cm longer than that in the M-P+ACNs group and close to the colon length of healthy mice, indicating that MSW-PA had the best effect on alleviating colitis.

[0097] 4) Colonic Histomorphological Analysis Colonic tissue samples removed after dissection were washed twice with normal saline and then placed in a centrifuge tube containing 8 mL of 4% paraformaldehyde tissue fixative. The samples were then dehydrated with 95% alcohol and absolute ethanol and embedded in paraffin. After embedding, sections were cut at a thickness of 10 μm and stained with hematoxylin and eosin (H&E) for 15 minutes. After staining, the stain was rinsed with deionized water, followed by dehydration and sealing. Images were captured under a motorized fluorescence microscope at 40x and 200x magnifications, respectively.

[0098] The results are as follows Figure 22It can be seen that the colon tissue of the Control group was smooth, with intact epithelial cells and clear crypt structure. Severe inflammatory lesions could be observed in the DSS group, showing typical pathological characteristics of colitis, such as a decrease in goblet cells, disappearance of crypts, and infiltration of inflammatory cells. Compared with the DSS group, the colon tissue structure of the MP group and the ACNs group showed a slight recovery, the number of goblet cells rebounded, the crypt structure was partially rebuilt, and colitis was alleviated. However, a large area of ​​inflammatory cell infiltration was still observed, and tissue repair was incomplete. In addition, the M-P+ACNs group was better than the MP group and the ACNs group, and the ulcer area was further reduced, indicating that the two substances had a better synergistic therapeutic effect on colitis. It is worth noting that the crypt structure of mice in the MSW-PA group was clearer and more complete than that in the M-P+ACNs group, the number of goblet cells was close to normal levels, the inflammatory cell infiltration was significantly reduced, and the integrity of the colon tissue was improved.

[0099] 5) Determination of serum inflammatory cytokine levels The eyeballs of mice were removed and blood was collected. After standing for 30 min, the blood was centrifuged at 3000 rpm for 15 min to obtain serum. The levels of tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and interleukin 10 (IL-10) in the serum were determined by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0100] from Figure 23As can be seen, compared with the control group, the expression levels of the pro-inflammatory cytokines TNF-α and IL-1β in mice treated with DSS were significantly increased, while the expression level of the anti-inflammatory cytokine IL-10 was significantly decreased. In contrast, the TNF-α and IL-1β levels in the MP and ACNs groups were similar and significantly lower than those in the DSS group, while IL-10 levels were significantly higher than those in the DSS group. However, the effect of ACNs alone was comparable to that of microencapsulated MP, and the effectiveness of both in suppressing inflammation needs to be improved. In the MP-P + ACNs group, the combined use of MP and ACNs exhibited a synergistic effect, more pronounced than either MP or ACNs alone, and more effectively inhibited the inflammatory response. This may be because the probiotics in MP enhance the bioavailability of ACNs by regulating the intestinal flora, while the antioxidant effects of ACNs may provide a more favorable environment for probiotic colonization. Compared with the other groups, the microencapsulated MSW-PA group showed significantly lower TNF-α and IL-1β levels and significantly increased IL-10 levels. This is because microencapsulation technology encapsulates the double-coated bacteria PL@COS / PE and ACNs through complex coacervation, improving their stability and enabling their beneficial effects in the intestine. Furthermore, the pH-responsive nature of the microcapsules enables the programmed release of probiotics and ACNs, and their sustained-release properties prolong the duration of their action, thereby more effectively suppressing inflammatory responses. Studies have confirmed that the microencapsulated MSW-PA alleviates intestinal inflammation through a synergistic mechanism of probiotic metabolic regulation and the anti-inflammatory effects of ACNs, and its programmed targeted release properties are also supported by in vivo experiments.

Claims

1. Application of soybean polysaccharides and whey protein isolate in the preparation of multi-component composite microcapsules.

2. A multi-component composite microcapsule based on programmed targeted release technology, characterized in that: The wall material is soybean polysaccharide and whey protein isolate, and the core material is chitosan oligosaccharide / pectin double-layer coating bacteria and anthocyanin.

3. A method for preparing multi-component composite microcapsules, characterized in that: The steps are to attract the probiotic suspension and the polysaccharide or protein solution in a specific ratio at a certain pH, and after stirring, oscillating, centrifuging, resuspending and other operations, prepare a double-layer coated bacteria, and then mix it with anthocyanins and encapsulate it together into microcapsules formed by complex coagulation and freeze-drying of soybean polysaccharides and whey protein isolate.

4. The method for preparing the multi-component composite microcapsule according to claim 3, characterized in that: The specific steps include: 1) Inoculate the probiotics into MRS medium for activation and prepare glycerol culture using 40% glycerol. Add 500 μL of the glycerol culture to 25 mL of sterilized MRS broth and incubate in a 37°C biochemical incubator for 16 h. 2) Centrifuge the culture prepared in step 1) twice, wash the precipitate, disperse the precipitated bacterial slurry in 5 mL of sterile deionized water, and vortex for 30 seconds to obtain a probiotic suspension; 3) The probiotic suspension prepared in step 2) was diluted 10 times with sterile deionized water of pH 4.0 to a concentration of 10 10 ~10 11 CFU / mL, vortex for 30 s to evenly disperse the probiotics; 4) Weigh a certain amount of coating material and dissolve it in deionized water. Stir at 700 rpm for 2 h at a fixed temperature to ensure complete dissolution. Then adjust the pH of the solution to 4.0 with 0.1 M NaOH and 0.1 M HCl and store at 4°C. 5) The probiotic suspension obtained in step 3) was mixed with the coating material solution obtained in step 4) in a certain proportion, stirred for 30 minutes, and cultured on a shaker for 10 minutes; 6) Centrifuging the mixed solution prepared in step 5) twice, removing the supernatant, and resuspending the precipitate in sterile deionized water of corresponding pH to obtain a monolayer coating bacterial suspension; 7) The probiotic suspension coated with a single layer of coating obtained in step 6) was mixed with another coating material solution in a certain proportion, stirred for 30 minutes, and cultured on a shaker for 10 minutes; 8) Centrifuging the mixed solution prepared in step 7) twice, removing the supernatant, and resuspending the precipitate in sterile deionized water of the corresponding pH to obtain a double-layer coated bacterial suspension; 9) Soybean polysaccharides and whey protein isolate (WPI) were weighed and dissolved in deionized water. The mixture was stirred at 700 rpm for 2 h at room temperature to ensure complete dissolution. The pH of the solution was then adjusted to a fixed value using 0.1 M NaOH and 0.1 M HCl and stored at 4°C. 10) The double-layer coated bacterial suspension prepared in step 8) is mixed with a certain amount of anthocyanin and stirred evenly; the mixed solution is added to the whey protein isolate solution prepared in step 9); 11) Subsequently, the mixed solution prepared in step 10) and the soybean polysaccharide solution prepared in step 9) are mixed and stirred until uniform, and the solution is adjusted to a fixed pH to obtain a soybean polysaccharide / whey protein isolate complex coacervate containing double-layer coated bacteria and anthocyanins; 12) The composite coacervate prepared in step 11) was placed in a freeze dryer and freeze-dried for 36 hours to obtain multi-component composite microcapsules containing double-layer coated bacteria and anthocyanins.

5. The method for preparing multi-component composite microcapsules according to claim 4, characterized in that: In step 4), the coating material is selected from one of chitosan oligosaccharide (COS), whey protein isolate (WPI) and gelatin (GE), and the concentration of the coating solution is 2 mg / mL.

6. The method for preparing multi-component composite microcapsules according to claim 4, characterized in that: In step 5), the volume ratio of the probiotic suspension to the coating solution is 1:

5.

7. The method for preparing multi-component composite microcapsules according to claim 4, characterized in that: In step 7), the coating material is pectin (PE), the concentration of the coating solution is 2 mg / mL, and the volume ratio of the probiotic suspension to the coating solution is 1:

5.

8. The method for preparing multi-component composite microcapsules according to claim 4, characterized in that: In step 9), the concentrations of soybean polysaccharide (SPSS) and whey protein isolate (WPI) were 20 mg / mL, and the pH of the solution was 6.

0.

9. The method for preparing multi-component composite microcapsules according to claim 4, wherein: In step 10), the concentration of anthocyanins (ACNs) is 0.3 mg / mL; in step 11), the volume ratio of soybean polysaccharides to whey protein isolate is 1:3, and the pH of the solution is 4.

0.

10. Use of the multi-component composite microcapsules prepared according to any one of the above claims in functional foods.

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