A composite microecological preparation, a preparation method and application thereof

Abstract

The application discloses a kind of composite microecological preparation and preparation method and application, belong to microbial technical field.The composite microecological preparation is core-shell structure, the core-shell structure is divided into inside and shell layer, inside includes Bifidobacterium adolescentis, shell layer includes metal polyphenol network and ficoll polysaccharide;The metal polyphenol network includes the complex coating formed by pterostilbene and calcium ion.The core is BA, the shell is made of FCPS, PTE, Ca 2+ Network composition, can protect Bifidobacterium adolescentis to reach intestine successfully and colonize, directly inhibit inflammatory response, and relieve CDI-induced mouse colitis and oxidative stress damage by regulating intestinal flora, indole and the generation of indole derivatives, solve the pain point that traditional microecological preparation probiotic survival rate is low, poor targeting, limited effect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound microecological preparation, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] As a pillar industry for global food security, the livestock industry is facing severe challenges, including high incidence of diarrhea in young animals caused by Clostridium difficile infection (CDI), antibiotic resistance due to overuse, and antibiotic residues in meat products. While antibiotics are the primary treatment for CDI, their use in young animals such as calves is strictly regulated due to their unique physiological characteristics. Overuse can easily induce the spread of drug-resistant bacteria, disrupt the balance of intestinal flora, and threaten public health. Currently, using probiotics to prevent and treat intestinal infectious diseases is one of the important research directions for alternative antibiotic treatment. However, orally administered probiotics face severe challenges from gastric acid, bile salts, and digestive enzymes in the gastrointestinal tract, resulting in a significant reduction in their survival rate and colonization efficiency upon reaching the colon, severely limiting their practical application in preventing and treating Clostridium difficile and other intestinal infections.

[0004] To enhance the gastrointestinal tolerance of probiotics, various encapsulation strategies have been developed, such as using alginate, chitosan, and other biomacromolecules to encapsulate probiotics in single or multiple layers, or forming a protective coating on the bacterial surface through a metal polyphenol network. However, existing metal polyphenol network coatings are mostly constructed by combining water-soluble polyphenols such as tannic acid and gallic acid with metal ions such as iron and zinc ions, and their function is mainly focused on improving the physical barrier protection of probiotics. For the specific pathological environment of Clostridium difficile infection, the infection site has complex microenvironmental characteristics such as increased oxidative stress, aggravated inflammatory response, and microbial imbalance. Encapsulation technologies that simply provide physical protection are insufficient to synergistically exert multiple intervention effects such as anti-inflammatory, antioxidant, and microbial regulation. Therefore, how to design a composite formulation that combines targeted delivery protection of probiotics with active regulation of the infection microenvironment is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] In view of this, the present invention provides a compound microecological preparation, its preparation method and application.

[0006] In a first aspect, the present invention provides a composite microecological preparation, wherein the composite microecological preparation has a core-shell structure, the core-shell structure being divided into an interior and a shell layer, the interior comprising *Bifidobacterium adolescentis*, and the shell layer comprising a metal polyphenol network and fig polysaccharide; the metal polyphenol network comprising a complex coating formed by *Pterostilbene styracifolium* and calcium ions. Secondly, the present invention also provides a method for preparing the above-mentioned compound microecological preparation, comprising the following steps: A calcium ion solution was mixed with a pterostilbene solution and incubated to obtain a metal phenolic network premix; The suspension of Bifidobacterium adolescentis was added to the metallophenolic network premix solution to carry out the first reaction, and Bifidobacterium adolescentis encapsulated in the metallophenolic network was obtained. The *Bifidobacterium adolescentis* encapsulated in the metallophenolic network was mixed with a fig polysaccharide solution to carry out a second reaction, thereby obtaining the composite microecological preparation.

[0007] Preferably, the concentration of the calcium ion solution is 20-30 mM, the concentration of the pterostilbene solution is 15-25 mM, and the volume ratio of the calcium ion solution to the pterostilbene solution is 1:1.

[0008] Preferably, the mixed incubation conditions are: incubation at room temperature for 20-30 minutes under light-protected conditions.

[0009] Preferably, the concentration of the Bifidobacterium adolescentis suspension is 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

[0010] Preferably, the volume ratio of the Bifidobacterium adolescentis suspension to the metallophenolic network premix is ​​1:1 to 1:3.

[0011] Preferably, the temperature of the first reaction is 20-50 °C, more preferably 30-40 °C, and the time of the first reaction is 20-60 min, more preferably 30-40 min.

[0012] Preferably, the concentration of the fig polysaccharide solution is 10-30 mg / mL, and the volume ratio of the Bifidobacterium adolescentis suspension to the fig polysaccharide solution is 1:1 to 1:3.

[0013] Preferably, the temperature of the second reaction is 15-25 °C, and the time of the second reaction is 30-60 min.

[0014] Thirdly, the present invention also provides the application of the above-mentioned compound microecological preparation or the compound microecological preparation obtained by the above-mentioned preparation method in the preparation of drugs for treating Clostridium difficile infection.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) This invention adopts a core-shell structure design, with Bifidobacterium adolescentis as the core and the shell layer containing a metal polyphenol network formed by the combination of pterostilbene and calcium ions and fig polysaccharide. The synergistic effect between the components solves the technical problem of low survival rate of probiotics in the intestine infected by Clostridium difficile and the inability to actively intervene in the intestinal microecology. Specifically, firstly, pterostilbene, as a natural polyphenol compound, has its phenolic hydroxyl groups and calcium ions that coordinate to form a dense metal polyphenol network on the surface of the bacteria. This network acts as a physical barrier and can significantly resist the erosion of gastric acid, bile salts and digestive enzymes in the gastrointestinal tract, protecting the activity of Bifidobacterium adolescentis until it reaches the colon. At the same time, pterostilbene itself has antioxidant activity, and this network can form a local antioxidant microenvironment around the bacteria. Secondly, fig polysaccharide, as an outer layer modification, further stabilizes the metal polyphenol network structure through the steric hindrance and hydrogen bonding of polysaccharide molecules. On the other hand, fig polysaccharide itself has the functions of immunomodulation and promoting the colonization of probiotics. More importantly, the introduction of calcium ions not only participates in network construction but also influences network dissociation behavior in the colon through changes in local calcium ion concentration, facilitating the targeted release and accumulation of Bifidobacterium adolescentis in the colonic region. Therefore, this invention, through a hierarchical design of a core-shell structure, enables the composite microecological preparation to simultaneously achieve the triple functions of bacterial protection, colonic targeting, and regulation of the infection microenvironment during oral delivery. This significantly improves the survival rate, colonization ability, and anti-inflammatory and antioxidant effects of Bifidobacterium adolescentis in Clostridium difficile-infected colon, providing an effective antibiotic-free microecological preparation for the treatment of Clostridium difficile infection.

[0016] (2) The core of the core-shell structure design lies in using Bifidobacterium adolescentis as the internal active core, utilizing its inherent characteristics of regulating intestinal microecology and anti-inflammation to provide basic active support for the prevention and treatment of CDI; at the same time, through the double coating of the shell, the pain point of Bifidobacterium adolescentis being easily inactivated in the gastrointestinal environment is solved. The metallophenolic network is composed of a complex formed by pterostilbene and calcium ions. Its dense network structure can effectively isolate the adverse environment such as gastric acid and bile salts, protect the activity of Bifidobacterium adolescentis, and ensure that it can reach the target site in the colon smoothly; fig polysaccharide, as the outer shell, can not only further strengthen the coating structure and reduce the shedding of the metallophenolic network, but also utilize its own biocompatibility and anti-inflammatory properties to form a synergistic effect with Bifidobacterium adolescentis and the metallophenolic network.

[0017] (3) The complex coating of pterostilbene and calcium ions in the metal phenolic network not only plays a role in protecting probiotics, but pterostilbene itself has excellent anti-inflammatory and antioxidant activities, which can directly participate in alleviating intestinal inflammation and oxidative stress damage caused by CDI; the introduction of calcium ions can enhance the stability of the network structure, and at the same time regulate the ion balance in the intestine, and help improve the intestinal barrier function.

[0018] (4) The synergistic encapsulation of fig polysaccharide and metallophenolic network enables the compound microecological preparation to slowly release Bifidobacterium adolescentis and pterostilbene after reaching the colon, thus prolonging the duration of action. At the same time, fig polysaccharide can promote the colonization of probiotics in the colon and enhance the effect of probiotics in regulating the intestinal microecology. In turn, by regulating the intestinal flora structure and inhibiting the release of inflammatory factors, it can effectively prevent and control CDI, and finally solve the technical problem that existing antibiotic-free preparations cannot take into account the survival rate, targeting and prevention and control effect of probiotics. This provides a reliable technical solution for antibiotic-free prevention and control of CDI in animal husbandry.

[0019] (5) The compound microecological preparation showed good safety and strong antioxidant activity in the in vitro Caco-2 cell experiment, and had a certain tolerance to the in vitro simulated gastrointestinal environment, which can indeed protect Bifidobacterium adolescentis to successfully reach and colonize the colon. The compound microecological preparation can alleviate weight loss, colon shortening, colon tissue damage and expression of pro-inflammatory factors caused by CDI, and enhance the activity of antioxidant enzymes such as SOD and GSH-PX by activating the Nrf2 / Keap1 pathway, reducing lipid peroxidation product MDA, and improving the antioxidant defense capacity of intestinal tissue. The compound microecological preparation significantly alleviates the intestinal state caused by CDI by regulating the structure of intestinal flora and tryptophan metabolism, increasing the level of indole and indole derivatives, reducing the expression of pro-inflammatory factors, improving intestinal flora. Attached Figure Description

[0020] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0021] Figure 1 The images shown are scanning electron microscope (SEM) images and elemental mapping diagrams of BA_P_F prepared in Example 1 of this invention, where A is the SEM image and B is the elemental mapping diagram. Figure 2 In Embodiment 1 of this invention, the particle size, Zeta potential, and T-AOC of BA, BA_P, and BA_P_F are specified, where A is the particle size, B is the Zeta potential, and C is the T-AOC. Figure 3 To illustrate the effect of different concentrations of BA, BA_P, and BA_P_F on cell viability in Example 2, where A represents the CCK-8 cell viability assay with different concentrations of BA, BA_P, and BA_P_F, and B represents the cell viability assay according to... Figure 3 A represents the corresponding concentrations cultured for 24 h and 72 h, while C represents Calcein-AM / PI live / dead bacterial staining. Figure 4To evaluate the ability of the compound microecological preparation to scavenge ROS in Example 2, A is cell viability detection, B is intracellular ROS level, C is a histogram of intracellular ROS level detected by flow cytometry, and D is a representative image of intracellular ROS level detected by DCFH-DA fluorescence staining. Figure 5 To illustrate the application of in vivo and in vitro fluorescence imaging in healthy mice in Example 3, A represents in vivo fluorescence imaging, and B represents in vitro fluorescence imaging. Figure 6 The changes in body weight, survival rate, colon length, and colon tissue score of mice in Example 4 are shown in Figure 4. A is the curve of mouse body weight change, B is the survival curve of mice, C is a picture of mouse colon, D is the colon tissue score, and E is the statistical result of colon length. Figure 7 Example 4 shows HE staining of colon tissue (HE staining scale bar is 100 μm), where A is the HE staining image and B is the colon histological score; Figure 8 The expression levels of inflammatory factors mRNA in the colon in Example 5 are shown, where A is the mRNA expression level of IL-6, B is the mRNA expression level of TNF-α, and C is the mRNA expression level of IL-1β. Figure 9 The changes in the contents of SOD, GSH-PX, and MDA in the colon tissue of Example 5 are used as an example, where A is SOD, B is GSH-PX, and C is MDA. Figure 10 To illustrate the expression of Keap-1, Nrf2, and HO-1 proteins in the colon tissue of Example 5, A shows the Western Blot electrophoresis bands of Nrf2, Keap1, and HO-1 proteins, and B shows the grayscale analysis results of the relative expression levels of each protein. Figure 11 The effects of BA_P_F on gut microbiota diversity and community structure in CDI mice are shown in Figure 1. A represents α-diversity of gut microbiota, B represents β-diversity analysis, and C represents microbial composition. Figure 12 The effect of BA_P_F on the colonic contents of CDI mice is shown in the figure. A is a heatmap of microbial subpopulation abundance, B is a bar chart of metabolite functional classification, C is a bubble chart of KEGG pathway enrichment, and D is the core differential metabolites screened by VIP analysis. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] This invention provides a composite microecological preparation with a core-shell structure, comprising an inner layer and a shell layer. The inner layer contains *Bifidobacterium adolescentis* (BA), and the shell layer includes a metallopolyphenol network and fig polysaccharide (FCPS). The metallopolyphenol network is composed of a complex coating formed by the coordination of pterostilbene (PTE) and calcium ions. In this core-shell structure, the inner metallopolyphenol network assembles in situ on the bacterial surface through the dynamic coordination of calcium ions with the phenolic hydroxyl groups in pterostilbene, forming a barrier. The outer fig polysaccharide is adsorbed onto the surface of the metallopolyphenol network through hydrogen bonding and electrostatic interactions, further stabilizing the coating structure and providing additional functional activity. In the composite microecological preparation (BA_P_F) provided by this invention, the gastrointestinal tolerance, colonic targeted colonization ability, and antioxidant properties of *Bifidobacterium adolescentis* are significantly improved after double-layer encapsulation.

[0024] In this invention, the construction of the metal polyphenol network relies on the coordination chelation between the phenolic hydroxyl groups of pterostilbene and calcium ions. Pterostilbene, as a natural polyphenol compound, possesses antioxidant and anti-inflammatory activities. The metal polyphenol network formed by pterostilbene and calcium ions not only forms a protective layer on the surface of probiotics but also creates a local antioxidant microenvironment around the bacteria. The fig polysaccharide, as an outer layer modification material, enhances the structural stability of the metal polyphenol network through steric hindrance and hydrogen bonding, and also possesses immunomodulatory and probiotic colonization-promoting functions. This invention, through a hierarchical design of a core-shell structure, enables the composite microecological preparation to simultaneously achieve the triple functions of bacterial protection, colonic targeting, and infection microenvironment regulation during oral delivery.

[0025] In this invention, the *Bifidobacterium adolescentis* can be a commercially available strain, such as *Bifidobacterium adolescentis* strain CICC 6177 purchased from the China Industrial Microbial Culture Collection Center (CICC). The fig polysaccharide can be a crude extract or purified polysaccharide from figs, such as fig polysaccharide products from Yangling Feige Fig Industry Development Co., Ltd. The calcium ions can be derived from anhydrous calcium chloride, and the pterostilbene can be derived from conventional chemical reagents. This invention does not have any special limitations on the source of the above raw materials; any commercially available products in the art can be used.

[0026] This invention provides a method for preparing a compound microecological preparation, comprising the following steps: A calcium ion solution was mixed with a pterostilbene solution and incubated to obtain a metal phenolic network premix; The suspension of Bifidobacterium adolescentis was added to the metallophenolic network premixed solution to carry out the first reaction, and Bifidobacterium adolescentis (BA_P) encapsulated by metallophenolic network was obtained. The Bifidobacterium adolescentis encapsulated in the metallophenol network was mixed with a fig polysaccharide solution to carry out a second reaction, thereby obtaining the composite microecological preparation (BA_P_F).

[0027] In this invention, the concentration of the calcium ion solution can be 20-30 mM, preferably 25 mM; the concentration of the pterostilbene solution can be 15-25 mM, preferably 20 mM; and the volume ratio of the calcium ion solution to the pterostilbene solution is preferably 1:1. As an optional embodiment of this invention, the concentration of the calcium ion solution can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mM; and the concentration of the pterostilbene solution can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM. This invention optimizes the concentration range of calcium ions and pterostilbene to ensure that the metal polyphenol network can uniformly and densely coat the bacterial cell surface. If the concentration is too low, the network formation will be incomplete, and the coating efficiency will decrease; if the concentration is too high, it may lead to excessive aggregation on the bacterial cell surface, affecting bacterial activity. This invention does not specifically limit the preparation method of the pterostilbene solution and the calcium ion solution; conventional preparation methods in the art can be used. As an optional embodiment of the present invention, the solvent for the pterostilbene solution is anhydrous ethanol, and the solvent for the calcium ion solution is deionized water. The pterostilbene solution is prepared under light-protected conditions.

[0028] In this invention, the preferred conditions for the mixed incubation are: incubation at room temperature for 20-30 minutes under light-protected conditions. Pterostilbene is photosensitizing, and light protection prevents its degradation; room temperature conditions are mild and conducive to the full coordination of calcium ions with pterostilbene without damaging the cell activity. As an optional embodiment of this invention, the incubation time can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes.

[0029] In this invention, the concentration of the Bifidobacterium adolescentis suspension can be 1×10⁻⁶. 8 ~1×10 10 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL. As an optional embodiment of the present invention, the concentration of the bacterial suspension can be 1×10⁻⁶. 8 1×10 9 Or 1×10 10CFU / mL. This concentration range ensures a balance between encapsulation efficiency and the number of viable bacteria in the formulation. This invention does not specifically limit the preparation method of the *Bifidobacterium adolescentis* suspension; any conventional method in the art can be used. As an optional embodiment of this invention, the preparation method of the *Bifidobacterium adolescentis* suspension includes: culturing *Bifidobacterium adolescentis* in a culture medium, washing the *Bifidobacterium adolescentis*, and resuspending it to obtain the *Bifidobacterium adolescentis* suspension. This invention does not specifically limit the culture medium; any conventional culture medium suitable for the growth of *Bifidobacterium adolescentis* can be used. This invention preferably uses a liquid culture medium composed of fortified Clostridium perfringens medium (RCM) and sodium acetate; preferably, it uses a solid culture medium composed of fortified Clostridium perfringens medium (RCM), sodium acetate, and agar powder; the concentration of the fortified Clostridium perfringens medium (RCM) is 34.5 g / L, the concentration of the sodium acetate is 3 g / L, and the concentration of the agar powder is 15 g / L.

[0030] In this invention, the bacteria are preferably washed twice with 0.01 M PBS buffer at pH 7.2. After washing, the resulting Bifidobacterium adolescentis is resuspended. The resuspending is preferably performed with 0.01 M PBS buffer at pH 7.2, and the concentration is corrected to 1 × 10⁻⁶ using plate counting or turbidimetric methods. 8 ~1×10 10 CFU / mL.

[0031] In this invention, the preferred volume ratio of the *Bifidobacterium adolescentis* bacterial suspension to the metallophenolic network premix is ​​1:1 to 1:3. As an optional embodiment of this invention, the volume ratio can be 1:1, 1:2, or 1:3. The determination of this volume ratio range is based on the completeness of coating and the economy of the reaction system. If the bacterial suspension ratio is too high, some bacteria may not be completely coated; if the bacterial suspension ratio is too low, an excessively thick coating layer may affect the metabolic activity of the bacteria.

[0032] In this invention, the temperature of the first reaction is preferably 20-50 °C, more preferably 30-40 °C; the reaction time is preferably 20-60 min, more preferably 30-40 min. These mild conditions ensure that the in-situ assembly of the metal polyphenol network on the bacterial cell surface does not damage the activity of *Bifidobacterium adolescentis*. As an optional embodiment of this invention, the temperature of the first reaction can be 20, 30, 40, or 50 °C; the reaction time can be 20, 30, 40, 50, or 60 min. After the first reaction is completed, this invention preferably separates the bacterial cells from the reaction product and then washes the cells. This invention preferably centrifuges the reaction solution at 4 °C and 5000 rpm for 10 min, discards the supernatant, and washes 1-2 times with sterile PBS to remove unreacted calcium ions and pterostilbene to obtain BA_P.

[0033] In this invention, the concentration of the fig polysaccharide solution can be 10-30 mg / mL, preferably 20 mg / mL; the volume ratio of the *Bifidobacterium adolescentis* bacterial suspension (referring to the volume of the bacterial suspension used in the first reaction) to the fig polysaccharide solution is preferably 1:1 to 1:3. As an optional embodiment of this invention, the concentration of the fig polysaccharide solution can be 10, 12, 15, 18, 20, 22, 25, 28, or 30 mg / mL; the volume ratio can be 1:1, 1:2, or 1:3. This range ensures effective adsorption of fig polysaccharides on the surface of the metal-phenolic network without causing bacterial aggregation. If the polysaccharide concentration or volume ratio is too low, adsorption will be incomplete; if the concentration is too high, it may lead to cross-linking and aggregation of bacterial cells. This invention does not specifically limit the preparation method of the fig polysaccharide solution; conventional preparation methods in the art can be used. As an optional embodiment of this invention, the solvent for the fig polysaccharide solution is deionized water.

[0034] In this invention, the preferred temperature for the second reaction is 20-25 °C, and the preferred reaction time is 30-60 min. 20-25 °C facilitates the stable adsorption of fig polysaccharides onto the surface of the metal phenolic network via hydrogen bonding and electrostatic interactions, while maintaining bacterial cell activity. As an optional embodiment of this invention, the reaction time can be 30, 35, 40, 45, 50, 55, or 60 min. After the second reaction is completed, this invention preferably separates the bacterial cells from the reaction product and then washes them. This invention preferably centrifuges the reaction solution at 4 °C and 5000 rpm for 10 min, discards the supernatant, and washes 1-2 times with sterile PBS to remove excess impurities, obtaining BA_P_F.

[0035] This invention provides the application of the compound microecological preparation described above in the preparation of drugs for treating Clostridium difficile infection. This invention systematically evaluated the intervention effect of BA_P_F on CDI-induced colitis by constructing a mouse model of Clostridium difficile infection (CDI). The results showed that BA_P_F significantly reduced the mortality rate of CDI mice, alleviated weight loss and colonic shortening, and reduced the pathological damage score of colonic tissue. At the molecular level, BA_P_F significantly reduced the levels of inflammatory factors IL-6, TNF-α, and IL-1β in serum and colonic tissue, increased the activity of antioxidant enzymes SOD and GSH-PX, and reduced the content of the oxidative stress marker MDA. Furthermore, BA_P_F can regulate the intestinal flora structure, increase the relative abundance of Bifidobacterium, decrease the relative abundance of Escherichiae and Clostridium, and upregulate the levels of beneficial metabolites such as indole-3-acetic acid. These results indicate that the compound microecological preparation provided by this invention can effectively alleviate Clostridium difficile infection-induced colitis through multiple pathways, including anti-inflammatory, antioxidant, and intestinal microecological regulation.

[0036] To further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention. In the present invention, unless otherwise specified, other test materials and instruments are conventional test materials in the art and can be purchased through commercial channels.

[0037] Example 1: Preparation of the compound microecological preparation BA_P_F The *Bifidobacterium adolescentis* strain (CICC 6177) used in this embodiment was purchased from the China Industrial Microbial Culture Collection Center. The frozen *Bifidobacterium adolescentis* culture was rapidly thawed at room temperature, and 150 μL of the culture was injected into 10 mL of liquid culture medium (fortified Clostridium difficile medium RCM 34.5 g / L + sodium acetate 3 g / L), and anaerobically cultured at 37 ℃ for 24 h. The culture was then streaked onto the surface of sterile solid culture medium (RCM 34.5 g / L + sodium acetate 3 g / L + agar powder 15 g / L) and incubated in an anaerobic incubator for 24 h to complete the revival process. Well-formed single colonies were picked, inoculated into liquid culture medium, and anaerobically cultured for another 24 h to obtain the *Bifidobacterium adolescentis* culture. The culture of *Bifidobacterium adolescentis* cultured to the logarithmic growth phase was centrifuged at 5000 rpm for 10 min at 4 ℃, the supernatant was discarded, and the culture was washed twice with sterile PBS buffer (pH=7.2, 0.01 M), resuspended in sterile PBS buffer (pH=7.2, 0.01 M), and the bacterial concentration was corrected to 1×10⁻⁶ using the plate count method. 8 CFU / mL, for later use, denoted as BA.

[0038] Weigh 0.37 g of anhydrous calcium chloride and dissolve it in 100 mL of sterile deionized water to prepare a 25 mM CaCl2 stock solution. After sterilization via a 0.45 μm filter membrane, store at 4 °C for later use. Weigh 51.2 mg of pterostilbene and add it to 10 mL of anhydrous ethanol. Shake in the dark until completely dissolved to prepare a 20 mM pterostilbene stock solution, which is stored in the dark for later use. Thoroughly mix 2 mL of CaCl2 stock solution and 2 mL of pterostilbene stock solution and incubate at room temperature for 25 min in the dark to obtain a metallophenolic network premix.

[0039] Take 2 mL of the above metallophenolic network premix and add 1 mL of Bifidobacterium adolescentis bacterial suspension (bacterial suspension to premix volume ratio 1:2). React at 20 ℃ for 15 min to allow calcium ions and pterostilbene to assemble in situ on the bacterial surface to form a metallophenolic network coating layer. After the reaction, centrifuge the sample at 4 ℃ and 5000 rpm for 10 min, discard the supernatant, and wash twice with sterile PBS to obtain metallophenolic network-encapsulated Bifidobacterium adolescentis, denoted as BA_P.

[0040] Weigh 0.2 g of crude fig polysaccharide extract and add it to 10 mL of sterile deionized water. Stir magnetically at room temperature for 3 h until fully dissolved to prepare a 20 mg / mL fig polysaccharide stock solution. Mix the above BA_P with the fig polysaccharide stock solution at a volume ratio of 1:5 (i.e., add 5 mL of fig polysaccharide solution to 1 mL of BA_P) and react at 20 ℃ for 45 min. After the reaction, centrifuge at 5000 rpm for 10 min at 4 ℃, discard the supernatant, and wash twice with sterile PBS to obtain the compound microecological preparation, denoted as BA_P_F. Resuspend the obtained sample in an appropriate amount of sterile PBS and store at 4 ℃ for later use.

[0041] Application Example 1: Structural Characterization of the Compound Microecological Preparation BA_P_F (1) Scanning electron microscopy (SEM) analysis and elemental mapping analysis The structure of BA_P_F prepared in Example 1 was characterized. The sample was freeze-dried for 8 hours to obtain powder. A small amount of powder was uniformly dispersed on conductive adhesive, and excess sample was blown away with a syringe. After gold sputtering, it was observed in a scanning electron microscope (SEM). The target analysis area was selected in the SEM image, and the energy-dispersive X-ray spectrometer (EDS) was turned on. An appropriate acquisition time of 10 min was set, and the element acquisition program was started. The characteristic X-ray signals of each element in the area were collected simultaneously. The system automatically completed the element calibration, counting, and imaging, and generated a two-dimensional distribution mapping spectrum of the corresponding elements.

[0042] like Figure 1 As shown in Figure A, SEM characterization was performed on BA_P_F to observe the overall morphology of the engineered probiotics. Subsequently, mapping was performed on the elemental distribution of the BA_P_F surface to observe the distribution of different elements in the composite material. Figure 1 As shown in Figure B, the C, N, O, and Ca elements are uniformly distributed, indicating that Ca... 2+ The mediated metal-phenolic network has been successfully constructed and stably coated on the surface of probiotics.

[0043] (2) Determination of particle size and zeta potential The surface zeta potential and particle size of BA, BA_P, and BA_P_F prepared in Example 1 were quantitatively determined and analyzed using Dynamic Light Scattering (DLS) technology. The samples were prepared into liquids with ultrapure water at concentrations of 0.1–1 mg / mL and filtered through a 0.22 μm filter membrane. Two mL of the filtered sample dispersion was added to disposable polystyrene cuvettes and capillary sample cells, respectively. In the DLS software, the test temperature was set to 25 °C, the equilibration time to 120 s, and three replicates were performed. The average values ​​of the particle size and zeta potential data were calculated.

[0044] like Figure 2 As shown in A, the PTE and Ca in group BA_P 2+ Adsorption on the surface of the strain forms a complex layer, resulting in a larger overall particle size than the BA group. The BA_P_F group forms a bilayer structure, further increasing the particle size. For example... Figure 2 As shown in B, compared with the BA group, the absolute value of the Zeta potential in the BA_P_F group increased, as shown in Figure B. Figure 2 As shown in C, the antioxidant capacity of both the BA_P group and the BA_P_F group was better than that of the BA group. Compared with the BA_P group, the BA_P_F group had the best antioxidant capacity.

[0045] Application Example 2: Safety and antioxidant protective effect of BA_P_F on Caco-2 cells (1) Cell viability detection Cell viability and safety were assessed using the CCK8 cell viability assay, with cells at a density of 5 × 10⁶ cells / year. 4 Caco-2 cells / mL were seeded into two 96-well plates and cultured for 24 h. 100 μL of culture medium containing BA (250 μg / mL), BA_P (500 μg / mL), and BA_P_F (0, 125, 250, 500, 1000 μg / mL) was added to each well. After 24 h and 72 h of treatment, the culture medium was removed, and the Caco-2 cells were washed three times with PBS. 100 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader. The cell viability of each sample was calculated according to formula (1): (1) (2) Cell viability detection After treating Caco-2 cells with different concentrations of BA, BA_P, and BA_P_F (as per 2.1.2.7), the culture medium was removed and the cells were washed three times with PBS. 250 μL of working solution was added to a 96-well plate and incubated at 37 °C in the dark for 15 min. Observation and imaging were then performed using an inverted fluorescence microscope (OLYMPUS IX73). The working solution for fluorescence staining was prepared at a ratio of Calcein-AM:PI:buffer solution = 1:1:1000.

[0046] Figure 3 The results showed that for Caco-2 cells, the cell viability of each group remained at a high level and did not decrease significantly when cultured at concentrations of BA (250 μg / mL), BA_P (500 μg / mL), and BA_P_F (1000 μg / mL). Figure 3 B is to categorize its groups according to Figure 3 After culturing at the corresponding concentrations for 24 h and 72 h, the results showed that the cell viability of the BA group, BA_P group, and BA_P_F group was not significantly different from that of the NC group, indicating that BA_P_F has good time safety. Figure 3 As shown in Figure C, the BA, BA_P, and BA_P_F groups all exhibited predominantly green fluorescence, with only a very small amount of red fluorescence observed. There were no significant differences between the groups, and the cells maintained high activity, further verifying their safety.

[0047] (3) Establishment of H2O2-induced oxidative damage model: Three techniques were set up for each group to improve the accuracy of the results. Caco-2 cells with a density of 1×105 cells / mL were seeded into 96-well plates. 100 μL of LDM medium was added to the NC group, and 100 μL of medium solution containing H2O2 was added to the other four experimental groups. 100 μL of medium containing BA, BA_P, and BA_P_F were added respectively and cultured for 24 h. After that, 100 μL of 10% CCK-8 solution was added to each well, and the 96-well plate was incubated in a 37 ℃ incubator for 2 h. The absorbance value of each well was then detected at a wavelength of 450 nm using a microplate reader.

[0048] (4) Measurement of ROS content in Caco-2 cells Oxidative damage was induced in the experimental group cells, followed by culture. Intracellular ROS levels were measured using a ROS detection kit. 100 μL of 10 μmol / L DCFH-DA fluorescent probe working solution was added to each well. After 30 min, the remaining working solution in the wells was aspirated and the cells were washed three times with sterile PBS to thoroughly remove unloaded probes. The fluorescence intensity values ​​of each well were detected and recorded using a BD FACSAria III flow cytometer at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0049] Figure 4 A method used H2O2 to induce oxidative damage in Caco-2 cells, simulating the oxidative stress environment in intestinal inflammation. Compared with the NC group, the cell viability of the CDI group was significantly decreased, indicating that H2O2 successfully induced cell damage. The cell viability of the BA_P and BA_P_F groups was significantly higher than that of the CDI group, and the protective effect of BA_P_F was the most significant. Figure 4 The BC analysis showed that the ROS level was significantly higher in the CDI group, while the ROS levels in all other experimental sample treatment groups were significantly lower than those in the CDI group, with the ROS level in the BA_P_F group being extremely significantly lower than that in the CDI group. This indicates that BA_P_F alleviates oxidative stress by scavenging intracellular ROS.

[0050] Then, Figure 4 As shown in Figure D, intracellular ROS were detected using the 2,7-dichlorofluorescein diacetate (DCFH-DA) probe. The CDI group showed the strongest green fluorescence intensity, indicating the highest ROS level, while the BA_P_F group showed the weakest fluorescence.

[0051] Application Example 3: Gastrointestinal transport test of the compound probiotic preparation BA_P_F Nine 7-week-old male C57BL / 6J mice were randomly divided into three groups: BA, BA_P, and BA_P_F, with three mice in each group. Each group of mice was administered 1×10⁻⁶ ppm via gavage. 9 CFU containing DiR fluorescently labeled BA, BA_P, or BA_P_F. Mice were anesthetized with isoflurane at 2, 6, 12, 24, and 48 h after gavage, and fluorescence signals were collected using an in vivo imaging system. The colonization efficiency of BA, BA_P, and BA_P_F in the colon was assessed based on fluorescence intensity and localization.

[0052] Figure 5 The study demonstrated the differences in fluorescence intensity in healthy mice after oral administration of BA, BA_P, and BA_P_F to assess the distribution of BA_P_F in various organs. After 48 hours, the fluorescence signal of unencapsulated BA was almost undetectable, while the strong fluorescence signals of the BA_P and BA_P_F groups persisted in the colon, indicating significantly improved BA survival and colonization. Ex vivo imaging of intestinal tissue after 48 hours showed that BA_P exhibited strong fluorescence signals in both the cecum and colon, while the fluorescence of BA_P_F was mainly concentrated in the colon. This demonstrates that the compound probiotic preparation can precisely enrich areas of colonic inflammation.

[0053] Application Example 4: The therapeutic effect of BA_P_F on Clostridium difficile-infected colitis in mice. Antibiotic mixture preparation: Kanamycin 0.4 mg / mL + Gentamicin 0.035 mg / mL + Polymyxin 0.042 mg / mL + Metronidazole 0.216 mg / mL + Vancomycin 0.045 mg / mL. This antibiotic mixture is prepared by mixing multiple antibiotics with ddH2O provided by Shanghai Yuanye Biotechnology Co., Ltd.

[0054] The Clostridium difficile standard strain (ATCC 43593) used was purchased from the American Type Culture Collection (ATCC).

[0055] A mouse model of Clostridium difficile infection (CDI) was established: Thirty 7-week-old male C57 / BL mice were randomly divided into five groups of six each: NC group, CDI group, BA group, BA_P group, and BA_P_F group. After a 7-day acclimatization period, all mice were given a mixed antibiotic in their drinking water for three days. On the fourth day, they were injected with clindamycin (10 mg / kg) while drinking water normally. On the fifth day, the CDI, BA, BA_P, and BA_P_F groups were administered the same dose of Clostridium difficile (200 μL) by gavage to induce CDI. On the afternoon of the fifth day, the BA, BA_P, and BA_P_F groups were administered BA solution, BA_P solution, and BA_P_F solution by gavage for 5 days, after which the mice were sacrificed.

[0056] like Figure 6 As shown, compared to the NC group, CDI mice exhibited significant phenotypic characteristics such as decreased body weight, reduced survival rate, and shortened colon length. The BA, BA_P, and BA_P_F groups all improved mouse survival rate and alleviated CDI-induced weight loss, colon shortening, and increased colonic tissue damage scores, with the BA_P_F group showing the most significant improvement. These results indicate that BA_P_F can significantly alleviate CDI-induced weight loss, mortality, colon shortening, and colonic tissue damage.

[0057] like Figure 7 As shown, Figure 7 HE staining of A showed that the colonic tissue structure in the NC group was intact, with normal distribution of goblet cells in the glandular lumens and clear crypt structure. In contrast, the CDI group showed severe crypt damage, reduced goblet cells, and inflammatory cell infiltration in the colon. Compared with the CDI group, the BA, BA_P, and BA_P_F groups all reduced CDI-induced goblet cell damage, with the BA_P_F group showing the most significant allergic reaction. Figure 7 As shown in Figure B, compared with the NC group, the CDI group had a significantly higher colonic tissue damage score, which was significantly reduced after BA_P_F treatment. These results indicate that BA_P_F can effectively alleviate colonic lesions caused by CDI.

[0058] Application Example 5: Regulatory Effect of BA_P_F on Inflammatory Factors and Oxidative Stress Indicators in CDI Mice First, accurately weigh 200 mg of the tissue sample from Application Example 4, add 0.9 mL of physiological saline at a weight / volume ratio, and homogenize to obtain a 10% tissue homogenate. Then, centrifuge the homogenate at 4 ℃ and 3000 rpm for 10 min. Finally, collect the supernatant and determine its total antioxidant capacity using a UV-Vis spectrophotometer.

[0059] Depend on Figure 8 It was found that, compared with the NC group, the levels of inflammatory factors IL-6, TNF-α, and IL-1β were significantly increased in the CDI group. After BA_P_F intervention, compared with the CDI group, the BA_P_F group significantly reduced the mRNA expression levels of inflammatory factors IL-6, TNF-α, and IL-1β in colonic tissue. Compared with the BA group, the BA_P_F group significantly reduced the levels of inflammatory factors IL-6, TNF-α, and IL-1β. These results indicate that BA_P_F can effectively alleviate CDI-induced colonic inflammation, and its effect is superior to that of the BA and BA_P groups.

[0060] Figure 9 The results showed that, compared with the NC group, the CDI group induced a significant decrease in the levels of antioxidant enzymes SOD and GSH-PX. Meanwhile, MDA, a key indicator reflecting the degree of cellular lipid peroxidation damage, showed a highly significant increase in its content in the CDI group, suggesting severe cell damage. In the BA_P_F group, SOD and GSH-PX levels were significantly increased, while MDA levels showed a highly significant decrease. These results indicate that BA_P_F can significantly alleviate oxidative stress damage in colonic tissue.

[0061] Depend on Figure 10 Compared with the NC group, the CDI group significantly increased Keap-1 protein expression and significantly decreased HO-1 and Nrf2 protein expression levels. Compared with CDI, the BA_P_F group significantly increased HO-1 protein expression, significantly increased Nrf2 protein expression, and decreased Keap-1 protein expression. These results suggest that BA_P_F may activate the Nrf2 / Keap-1 / HO-1 pathway to alleviate CDI-induced oxidative damage.

[0062] Application Example 6: The regulatory effect of BA_P_F on gut microbiota and metabolites in CDI mice Fecal samples were collected from the mice in Application Example 4 for 16S and metabolomics assays.

[0063] like Figure 11As shown in Figure A, the ACE, Shannon, and Chao1 indices of the BA, BA_P, and BA_P_F groups were all higher than those of the CDI group, while the Simpson indices were lower. The BA_P_F group, however, exhibited gut microbiota richness and diversity levels approaching those of the NC group, indicating that BA_P_F can significantly improve gut microbiota richness and diversity. Figure 11 As shown in Figure B, BA_P_F showed the most significant effect on restoring the gut microbiota structure in CDI mice. It is evident that the BA, BA_P, and BA_P_F groups all improved gut microbiota diversity, with the BA_P_F group exhibiting the best improvement. This result further confirms that the synergistic effect of BA, PTE, and FCPs can significantly enhance the ability of the compound probiotic preparation to restore gut microbiota diversity.

[0064] like Figure 11 As shown in Figure C, the relative abundance distribution of each group of microorganisms is presented intuitively. At the genus level, the NC group is mainly enriched. Ligilactobacillus The CDI group is mainly enriched Escherichia-Shigella , Bacteroides The BA, BA_P, and BA_P_F groups are mainly enriched. Ligilactobacillus , Bifidobacterium , Akkermansia , Blautia This indicates that BA_P_F can regulate the abundance of key beneficial bacteria and improve the intestinal flora imbalance caused by CDI.

[0065] Depend on Figure 12 As shown, Figure 12 The heatmap of microbial subgroup abundance shows that the CDI group was dominated by high abundance of subgroups such as subcluster_1, while the BA_P_F group was dominated by high abundance of subgroups such as subcluster_3. This indicates that CDI significantly disrupted the structure of gut microbiota subgroups, while BA_P_F reshaped the abundance pattern of microbiota subgroups. Figure 12 The bar chart of metabolite function classification shows that the amino acid metabolic pathway is the functional category with the most concentrated differential metabolites, suggesting that the difference between the BA_P_F group and the CDI group is mainly reflected in the changes in intestinal regulation of intestinal metabolism. Figure 12 The KEGG pathway enrichment bubble plot of C shows that the tryptophan metabolic pathway is significantly enriched, indicating that the metabolic differences between CDI and BA_P_F intervention are mainly concentrated in the tryptophan pathway. The tryptophan metabolic pathway is usually closely related to intestinal inflammation and antioxidant function. Figure 12Among the core differentially expressed metabolites identified by VIP analysis in group D, three tryptophan metabolites—indole, indole-3-AceticAcid, and indole-3-Acetamide—were significantly upregulated in the BA_P_F group (VIP value > 1.5). This result confirms that BA_P_F exerts its anti-inflammatory and intestinal barrier protective effects by promoting tryptophan metabolism to produce indole and indole derivatives.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound microecological preparation, characterized in that, The compound microecological preparation has a core-shell structure, which is divided into an interior and a shell. The interior includes Bifidobacterium adolescentis, and the shell includes a metal polyphenol network and fig polysaccharide. The metal polyphenol network includes a complex coating formed by pterostilbene and calcium ions.

2. A method for preparing the compound microecological preparation according to claim 1, characterized in that, Includes the following steps: A calcium ion solution was mixed with a pterostilbene solution and incubated to obtain a metal phenolic network premix; The suspension of Bifidobacterium adolescentis was added to the metallophenolic network premix solution to carry out the first reaction, and Bifidobacterium adolescentis encapsulated in the metallophenolic network was obtained. The *Bifidobacterium adolescentis* encapsulated in the metallophenolic network was mixed with a fig polysaccharide solution to carry out a second reaction, thereby obtaining the composite microecological preparation.

3. The preparation method according to claim 2, characterized in that, The concentration of the calcium ion solution is 20-30 mM, the concentration of the pterostilbene solution is 15-25 mM, and the volume ratio of the calcium ion solution to the pterostilbene solution is 1:

1.

4. The preparation method according to claim 2, characterized in that, The conditions for mixed incubation are: incubation at room temperature for 20-30 minutes under dark conditions.

5. The preparation method according to claim 2, characterized in that, The concentration of the Bifidobacterium adolescentis suspension was 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

6. The preparation method according to claim 2, characterized in that, The volume ratio of the Bifidobacterium adolescentis suspension to the metallophenolic network premix is ​​1:1 to 1:

3.

7. The preparation method according to claim 2, characterized in that, The temperature of the first reaction is 20-50℃, more preferably 30-40℃, and the time of the first reaction is 20-60 min, more preferably 30-40 min.

8. The preparation method according to claim 2, characterized in that, The concentration of the fig polysaccharide solution is 10-30 mg / mL, and the volume ratio of the Bifidobacterium adolescentis suspension to the fig polysaccharide solution is 1:1 to 1:

3.

9. The preparation method according to claim 2, characterized in that, The temperature of the second reaction is 15-25℃, and the time of the second reaction is 30-60 min.

10. The use of the compound microecological preparation according to claim 1 or the compound microecological preparation prepared by any one of claims 2-9 in the preparation of drugs for treating Clostridium difficile infection.

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