Microorganism coated with metal-polyphenol secondary coordination nano-coating and preparation method and application thereof

Abstract

This invention belongs to the field of microbial technology and provides a microorganism coated with a metal-polyphenol secondary coordination nanocoating, its preparation method, and its applications. This invention involves the self-assembly of polyvinylpyrrolidone, metal ions, and polyphenols to form metal-polyphenol nanoparticles. Subsequently, a continuous nanocoating is rapidly deposited on the surface of the microorganism using the secondary coordination between the nanoparticles and the metal ions, resulting in a coating thickness of approximately 100 nm. The coating construction method of this invention is applicable to different types of microorganisms, and the coating has no significant impact on the normal growth of the microorganisms, exhibiting good biocompatibility. Simultaneously, this nanocoating can selectively promote the biofilm formation of Gram-negative probiotics (EcN). This promoting effect is maintained in a simulated intestinal mucus system. This invention can be used to improve the adhesion and colonization ability of probiotics and is applicable to the construction of probiotic preparations and microecological regulation systems.

Description

A microorganism coated with a metal-polyphenol secondary coordination nanocoating, its preparation method and application Technical Field

[0001] This invention belongs to the fields of materials and microbial technology, specifically relating to a microorganism coated with a metal-polyphenol secondary coordination nanocoating, its preparation method, and its application. Background Technology

[0002] Biofilms are three-dimensional structures formed by microorganisms at the interface through the secretion of extracellular polymers, playing a crucial role in the natural environment and microbial physiological activities. Compared to suspended bacterial cells, microorganisms in biofilms typically exhibit stronger adhesion, environmental tolerance, and resistance to chemical or physical stresses. For probiotics, biofilm formation not only facilitates their survival and colonization in the complex intestinal environment but also enhances their ability to resist various stressors such as gastrointestinal pH changes, bile salts, and mechanical shearing, thereby more effectively regulating the microecology, improving the intestinal barrier, and promoting host health. Therefore, promoting probiotic biofilm formation is considered an important means to improve the stability, activity, and biological function of probiotic preparations. However, different probiotics exhibit significant differences in cell wall structure, surface chemistry, and metabolic characteristics, making it difficult to achieve selective regulatory effects on specific types of probiotics (especially Gram-positive and Gram-negative bacteria). Therefore, exploring new strategies with broader applicability, controllability, and clear structure-function relationships is of great significance for promoting probiotic biofilm formation.

[0003] Existing research indicates that materials such as metal ions, natural polyphenols, and polymers can construct functional coatings on microbial surfaces through complexation, self-assembly, or interfacial deposition. Coatings formed using these materials can improve the tolerance of microorganisms to environmental stresses or enhance their specific biological behaviors, including promoting or inhibiting biofilm formation. However, systematic studies on the differences among probiotics are still limited. Simultaneously, research on constructing controllable structures on probiotic surfaces using nanomaterials and regulating their biofilm behavior is also relatively limited. Current technologies also have shortcomings in terms of coating structure uniformity, thickness control, continuity, and stability, limiting their further application in microbial engineering and probiotic formulations. Furthermore, a systematic evaluation of the specific relationship between coating structural parameters and probiotic biofilm formation is still lacking.

[0004] Therefore, there is an urgent need to develop a coating construction strategy that can build a continuous, thickness-controllable coating on the surface of microorganisms with specific functional effects, so as to effectively regulate the formation of biofilms of different types of probiotics. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for preparing microorganisms encapsulated with a metal-polyphenol secondary coordination nanocoating. This method overcomes the problems of traditional metal-polyphenol coordination systems, which can only form a thin layer of about 10 nm on the surface of microorganisms, requiring multiple cycles of deposition to thicken the layer, resulting in cumbersome operations and low efficiency. Furthermore, this coating is applicable to different types of microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and yeasts, exhibiting good biocompatibility and not affecting the normal growth of microorganisms. This preparation method achieves uniform encapsulation and stable protection of microorganisms, providing a safe and effective interfacial structure foundation for microbial engineering and probiotic preparations.

[0006] Another object of the present invention is to provide microorganisms coated with a metal-polyphenol secondary coordination nanocoating obtained by the above preparation method.

[0007] Another object of the present invention is to provide the application of the above-mentioned microorganisms coated with metal-polyphenol secondary coordination nanocoating and the preparation method thereof.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] A method for preparing microorganisms coated with a metal-polyphenol secondary coordination nanocoating includes the following steps:

[0010] (1) Dissolve polyvinylpyrrolidone (PVP) in MOPS buffer, then add biocompatible metal ions, then add polyphenols, adjust the pH of the system to 7.8-8.2, and self-assemble metal-polyphenol nanoparticles at room temperature. Separate the solid and liquid, wash the obtained solid, and obtain metal-polyphenol nanoparticles.

[0011] (2) Add biocompatible metal ions to the microorganisms, mix them evenly, add the metal-polyphenol nanoparticles obtained in step (1) and mix them evenly to obtain an assembly system. After coordination assembly, separate the solid and liquid, clean the obtained solid, and obtain a nano-coating on the surface of the carrier.

[0012] The polyvinylpyrrolidone mentioned in step (1) is preferably polyvinylpyrrolidone K30, with an average molecular weight of 40,000.

[0013] The MOPS buffer solution mentioned in step (1) is preferably a MOPS buffer solution with pH 7.8 to 8.2 and a strength of 90 to 110 mM; more preferably a MOPS buffer solution with pH 8.0 and a strength of 100 mM.

[0014] The metal ion mentioned in step (1) is preferably Cu. 2+ Zn 2+ and Fe 3+ At least one of the following; more preferably Cu2+ .

[0015] The Cu 2+ Preferably derived from copper chloride or its hydrate; more preferably derived from copper chloride or copper chloride dihydrate.

[0016] The Zn 2+ The preferred source is zinc chloride.

[0017] The Fe 3+ Preferably derived from ferric chloride or its hydrate; more preferably derived from ferric chloride or ferric chloride hexahydrate.

[0018] The polyphenols mentioned in step (1) are preferably tannic acid (TA).

[0019] The metal ions and polyphenols mentioned in step (1) are preferably in a molar ratio of 2:0.9 to 1.1; more preferably in a molar ratio of 2:1.

[0020] The pH value mentioned in step (1) is preferably 8.0.

[0021] The pH in step (1) is preferably adjusted using an aqueous NaOH solution; more preferably, it is adjusted using an aqueous NaOH solution with a concentration of 1 M.

[0022] The self-assembly time in step (1) is preferably 3 to 10 minutes; more preferably 5 to 10 minutes.

[0023] The solid-liquid separation method described in step (1) is preferably centrifugation; more preferably, centrifugation at 8000-12000g for 5-15min.

[0024] The cleaning solution used in step (1) is preferably deionized water or purified water.

[0025] The number of cleaning cycles in step (1) is preferably 2 to 3.

[0026] The assembly system described in step (2) is preferably obtained through the following steps: taking a concentration of OD 600 Centrifuge 1 mL of microbial culture medium with a concentration of 2.8–3.2, collect the bacterial cells, wash with PBS, and resuspend the cells in PBS. Add 0.19–0.21 mg of metal ions to the resulting bacterial suspension, mix well, and then add 40–60 μL of OD200. 600 A metal-polyphenol nanoparticle solution with a concentration of 1.1–1.3 was diluted to 1 mL with PBS; more preferably, it was obtained by the following steps: taking a concentration of OD 6001 mL of microbial culture medium with a concentration of 3.0 g / mL was centrifuged, and the bacterial cells were collected. The cells were washed with PBS and then resuspended in PBS. 0.20 mg of metal ions was added to the resulting bacterial suspension, and after thorough mixing, 50 μL of OD200 solution was added. 600 A metal-polyphenol nanoparticle solution with a concentration of 1.2 was diluted to 1 mL with PBS.

[0027] The microorganisms mentioned in step (2) are preferably at least one of Gram-negative bacteria, Gram-positive bacteria and yeast.

[0028] The preferred Gram-positive bacterium is *Lactobacillus plantarum* FS4722.

[0029] The preferred Gram-negative bacterium is Escherichia coli Nissle 1917.

[0030] The yeast is preferably brewer's yeast.

[0031] The metal ion mentioned in step (2) is preferably Cu. 2+ Zn 2+ and Fe 3+ At least one of the following; more preferably Fe 3+ .

[0032] The metal-polyphenol nanoparticles mentioned in step (2) are preferably copper-polyphenol nanoparticles.

[0033] The mixing method described in step (2) is preferably vortex.

[0034] The solid-liquid separation method described in step (2) is preferably centrifugation; more preferably, centrifugation at 1500-2000 g for 1-3 min.

[0035] The washing solution used in step (2) is preferably PBS buffer.

[0036] The number of cleaning cycles in step (2) is preferably 2 to 3.

[0037] A microorganism coated with a metal-polyphenol secondary coordination nanocoating was prepared by the above method. The thickness of the nanocoating is 80-100 nm. It has a higher ability to withstand environmental stress or can form a biofilm more quickly.

[0038] The above preparation method is applied in the preparation of microecological regulation systems.

[0039] The above-mentioned microorganisms coated with metal-polyphenol secondary coordination nanocoatings are used in the preparation of probiotic formulations.

[0040] The microorganisms mentioned are at least one of Lactobacillus plantarum FS4722 and Escherichia coli Nissle 1917.

[0041] The PBS used in this invention is preferably 0.01M PBS with pH 7.4.

[0042] The present invention has the following advantages and effects compared with the prior art:

[0043] (1) This invention provides a nanocoating system formed by secondary coordination of metal-polyphenol nanoparticles and metal ions. This system can rapidly assemble into a continuous coating with a thickness of about 100 nm on the surface of polystyrene microspheres within 5 min. Compared with the limitations of traditional metal-polyphenol coordination networks, which typically only form a thickness of about 10 nm and require multiple coordination cycles to obtain a thicker coating, this invention achieves rapid and efficient construction of thick coatings through secondary coordination, significantly simplifying the preparation steps and improving the preparation efficiency.

[0044] (2) The metal-polyphenol secondary coordination nanocoating constructed in this invention has good versatility and can be applied to microbial cells of different types, shapes, and sizes, including Gram-positive bacteria *Lactobacillus plantarum* FS4722, Gram-negative bacteria *Escherichia coli* Nissle 1917 (EcN), and *Saccharomyces cerevisiae*. This coating achieves uniform embedding of different bacterial cell surfaces without affecting the growth and normal metabolic processes of microorganisms, indicating that the coating system provided by this invention has good biocompatibility and safety, and is suitable for a variety of microorganisms.

[0045] (3) The nano-coating constructed in this invention exhibits selective effects in the regulation of biological functions. Experimental results show that the coating has no significant effect on the biofilm formation of Gram-positive probiotic FS4722, but promotes the biofilm formation of Gram-negative probiotic EcN. This phenomenon reveals the differential interaction between the coating structure and the surface characteristics of different groups of microorganisms, providing a new technical path for the targeted regulation of biofilms of specific probiotics based on exogenous interface engineering. This invention not only helps to improve the biofilm capacity of specific probiotics, but also provides important reference value for microbial engineering, probiotic formulation optimization, and the design of microecological intervention strategies. Attached Figure Description

[0046] Figure 1 shows the construction and characterization results of three types of nanoparticles; where a is the transmission electron microscope image and appearance image of the three types of nanoparticles, and b is the scanning electron microscope image of the three types of nanoparticles.

[0047] Figure 2 shows the results of constructing a secondary coordination nanocoating; where a is a scanning electron microscope image and appearance image of the coordination assembly of three nanoparticles and three metal ions on the surface of polystyrene microspheres, b is a particle size distribution map of polystyrene microspheres and nanocoated polystyrene microspheres detected by laser particle size analyzer, c is the particle size of polystyrene microspheres and nanocoated polystyrene microspheres, d is a transmission electron microscope image and elemental distribution map of nanocoated polystyrene microspheres, and e is a cytotoxicity analysis map of Cu-NPs.

[0048] Figure 3 shows the general applicability of the nanocoating on different types of microbial cells; where a is a scanning electron microscope image of three natural and nanocoated microbial cells, b is the growth curve of three natural and nanocoated microorganisms, ce is the particle size of three natural and nanocoated microorganisms, and f is a scanning electron microscope image of the nanocoated EcN dividing normally in LB medium.

[0049] Figure 4 shows transmission electron microscopy images of natural and nano-coated EcN.

[0050] Figure 5 shows the results of the effect of nano-coating on the formation of biofilm by probiotics; a is a comparison of the crystal violet staining results of biofilms of natural and nano-coated FS4722, b is a comparison of the crystal violet staining results of biofilms of natural and nano-coated EcN, c is the effect of nanoparticle addition on FS4722 biofilm, d is the effect of nanoparticle addition on EcN biofilm, e is a scanning electron microscope image of the effect of nanoparticle addition on the formation of nano-coating, and f is the effect of different materials on the formation of EcN biofilm.

[0051] Figure 6 shows the results of biofilm formation ability of nano-coated EcN in simulated intestinal mucus; a is a laser confocal microscope image of biofilm formation by natural and nano-coated EcN in simulated mucus, b is a scanning electron microscope image of biofilm formation by natural and nano-coated EcN in simulated mucus, and c is a cytotoxicity analysis of natural and nano-coated EcN.

[0052] Figure 7 shows the results of the in vitro environmental resistance of the EcN nano-coating. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0054] This study first investigated the nanoparticle system formed by metal ions and organic ligands using polystyrene microspheres as a carrier, obtaining a scheme for constructing a continuous coating with a thickness of approximately 100 nm through secondary coordination. Next, experiments were conducted using microorganisms as a carrier. The results showed that this method can form a thick and stable coating on the surface of microorganisms, not only improving structural integrity but also providing stronger interfacial protection. Furthermore, this method is applicable to different types of probiotics and exhibits differentiated effects on the biofilm behavior of Gram-positive and Gram-negative probiotics. This provides a new technical pathway for enhancing probiotic activity and achieving controllable biofilm regulation, contributing to the development of probiotic engineering and microecological regulation technologies.

[0055] Example 1: Preparation of metal-polyphenol nanoparticles

[0056] In this embodiment, three biocompatible metal ions (Cu) were first selected. 2+ Zn 2+ Fe 3+ Metal-polyphenol nanoparticles were prepared using tannic acid (TA) and polyvinylpyrrolidone (PVP). PVP was added to ensure uniform dispersion of the nanoparticles in solution and to improve their biocompatibility. The specific steps are as follows:

[0057] 150 mg of PVP (K30) was dissolved in 30 mL of MOPS buffer (100 mM, pH 8.0), and then different metal ions (40.335 mg Cu) were added. 2+ 89.247 mg Zn 2+ Or 81.087 mg Fe 3+ The copper ions originated from copper chloride dihydrate, the zinc ions from zinc chloride, and the iron ions from ferric chloride hexahydrate. 255.18 mg of tannic acid (TA) was added under stirring at 600 rpm, resulting in a metal ion to TA molar ratio of 2:1. The pH of the mixture was adjusted to 8.0 with 1M NaOH solution. After reacting for another 5 min at room temperature, the final product was separated by centrifugation (10000 g, 10 min). The product was washed three times with deionized water to obtain three types of nanoparticles containing different metal ions: Cu-NPs, Zn-NPs, and Fe-NPs.

[0058] Transmission electron microscopy and surface images of the three nanoparticles are shown in Figure 1a, and scanning electron microscopy images are shown in Figure 1b. The results in Figure 1 show that the three nanoparticles have similar spherical morphology and particle size, approximately 30 nm.

[0059] Example 2: Preparation of metal-polyphenol secondary coordination nanocoating

[0060] Because the TA surface is rich in phenolic hydroxyl groups, the uncoordinated phenolic hydroxyl groups in the nanoparticles may further coordinate with metal ions to form a nanocoating. Typically, metal-polyphenol coordination systems can only form a thin layer of about 10 nm after a single coordination assembly. Therefore, we speculate that using nanoparticles instead of phenolic ligands to assemble with metal ions may lead to the formation of thicker films. Three types of nanoparticles were used as ligands, and further coordinated with metal ions (Cu... 2+ Zn 2+ Fe 3+ Nano-coatings were prepared by secondary coordination assembly on the surface of polystyrene (PS) microspheres through arrangement and combination. The specific steps are as follows:

[0061] (1) Assembly of three types of nanoparticles on PS surface: 50 μL of polystyrene microsphere (PS) solution (solid content 2.5%, Jiangsu Zhichuan Technology Co., Ltd.) was taken and washed twice with water, and then resuspended in 400 μL of water. Next, 50 μL of metal ion solution with a concentration of 4 mg / mL (Cu) was added to each. 2+ Zn 2+ Fe 3+ The nanoparticles were then briefly vortexed for dispersion. Immediately afterwards, 50 μL of freshly prepared nanoparticle solutions (the three types of nanoparticles were diluted with water to OD) were added separately. 600 The concentration was approximately 1.2 μL, and the dispersion was vortexed for 20 s. The pH was adjusted by adding 500 μL of PBS buffer (1×, pH 7.4), and the suspension was vortexed for 5 min to promote collisions between PS and nanoparticles. Finally, the mixture was washed three times with deionized water (2000 g, 2 min) to remove free nanoparticles. It is important to note that during assembly, metal ions were added first, followed by nanoparticles. This is because reversing the order of addition would prevent the formation of a continuous coating.

[0062] As can be seen from the scanning electron microscope image (Figure 2a), only Cu-NPs and Fe... 3+ The combination formed a uniform and complete coating. The nanoparticle structure on the coating surface is similar to that in Figure 1b, and is uniformly and continuously distributed on the PS surface. A laser particle size analyzer was used to analyze the PS@Fe... 3+ Particle size analysis was performed on Cu-NPs. Figures 2b and 2c show that the PS particle size is approximately 1.82 μm. The PS@Fe after secondary coordination encapsulation... 3+ The Cu-NPs particle size is approximately 1.91 μm, therefore the calculated thickness of the nanocoating is approximately 90 nm. Transmission electron microscopy images also show a coating thickness close to 90 nm, and Cu elements are uniformly distributed on the PS surface, further confirming that Cu-NPs form a continuous nanocoating on the PS surface (see d in Figure 2).

[0063] (2) Cytotoxicity analysis of Cu-NPs: RAW264.7 mouse mononuclear macrophage leukemia cells (1×10⁻⁶) were used. 4 (1 × 10⁶ cells / well) were seeded into 96-well plates and incubated at 37°C in growth medium (DMEM medium containing 10% v / v fetal bovine serum and 1% penicillin antibiotics) for 24 h. Then, 200 μL of fresh medium containing different concentrations (0, 1, 5, 10, 25, 50 μg / mL) of Cu-NPs (prepared according to Example 1) was added to the medium, and incubation continued for 24 h. Afterward, 10 μL of CCK-8 solution was added to each well and incubated for 1 h. Finally, the OD value of the medium at 450 nm was recorded using a multi-mode microplate reader. Human colorectal adenocarcinoma cells Caco-2 (1 × 10⁶ cells / well) were seeded into each well. 4 Cu-NPs were seeded into 96-well plates and incubated at 37°C for 24 h in DMEM medium (containing 20% ​​v / v fetal bovine serum, 1% antibiotics, and 1% non-essential amino acids). The medium was then replaced with 200 μL of fresh medium containing different concentrations (0, 10, 50, 100, 150, 200 μg / mL) of Cu-NPs, and the OD value at 450 nm was recorded according to the experimental method described above. The results (see Figure 2e) indicate that the cytotoxicity of Cu-NPs was negligible.

[0064] Example 3: Application of nanocoatings on microbial cells

[0065] To explore the versatility of nanocoatings on microbial cells, three representative microorganisms were selected: *Lactobacillus plantarum* FS4722 (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 22750, disclosed in Chinese patent "CN202210167880.6 - A strain of *Lactobacillus plantarum* and its application in lowering uric acid, weight loss, and anti-inflammation"), *Escherichia coli* Nissle 1917 (abbreviated as EcN, purchased from Biobw), and *Saccharomyces cerevisiae* (CEN.PK2-1C). The specific steps are as follows:

[0066] (1) Assembly of nanocoatings on cell surface: FS4722, EcN, and Saccharomyces cerevisiae were cultured overnight in their respective culture media (MRS, LB, and YPD media, respectively; FS4722 and EcN were cultured at 37°C, and Saccharomyces cerevisiae at 30°C). 1 mL of bacterial culture (OD) was collected. 600 Centrifuge at 2000 g for 5 min (approximately 3), and wash cells three times with PBS (1×, pH 7.4). Resuspend the washed cells in 400 μL of water, then add 50 μL of Fe...3+ The solution (4 mg / mL) was added to the cell suspension, and the mixture was gently vortexed for about 20 s. Then, 50 μL of the freshly prepared diluted Cu-NPs solution (with an OD of 4 mg / mL) prepared according to Example 2 was added. 600 The pH was adjusted to approximately 1.2, and the mixture was vortexed for about 20 s. 500 μL of PBS was added to the mixture to adjust the pH, and the mixture was vortexed for 5 min at room temperature. Finally, the embedded cells were collected after washing three times with PBS (2000 g, 2 min). The embedded microorganisms were then inoculated into their respective culture media and cultured continuously. OD was measured at regular intervals. 600 Draw the growth curve.

[0067] (2) Characterization and activity of nano-coated microorganisms: Scanning electron microscopy revealed that the nano-coating successfully formed on the surface of three different types of microbial cells (see Figure 3a), indicating that the application of the coating has universality without limiting cell surface properties, size, and shape. The growth curves of the microorganisms showed that the coating had a slight inhibitory effect on the early stage of cell growth, but eventually reached a growth trend close to that of the natural cells (see Figure 3b). Particle size analysis using a laser particle size analyzer showed that the particle sizes of the natural Saccharomyces cerevisiae, FS4722, and EcN were 3.05, 1.21, and 0.94 μm, respectively, while the particle sizes of the cells after nano-coating were 3.15, 1.31, and 1.03 μm, respectively. The changes in particle size data indicate that the coating thickness was approximately 100 nm on the surface of different microbial cells. In addition, the growth status of the nano-coated EcN in LB medium was observed using scanning electron microscopy, and the results showed that the coating did not affect the normal division of bacteria (see Figure 3f). In addition, transmission electron microscopy also revealed that the nanoparticles formed a uniform and continuous coating on the EcN surface (see Figure 4).

[0068] Example 4: The effect of nanocoating on biofilm formation by probiotics

[0069] Existing research indicates that copper ions and copper-containing nanoparticles can inhibit bacterial biofilm formation at certain concentrations. However, in our experiments, we found that trace amounts of Cu-NPs can promote the formation of probiotic biofilms through oxidative stress.

[0070] (1) First, the biofilm formation ability of nano-coated probiotics compared with that of natural probiotics was tested. 1×10⁶ micrograms were inoculated into each well of a 96-well plate. 6CFU nanocoated Gram-positive bacteria FS4722 and Gram-negative bacteria EcN (prepared according to Example 3) were cultured statically at 37°C to form biofilms; simultaneously, untreated natural strains FS4722 and EcN served as controls. After 24 h, the culture medium was discarded, and the biofilms were washed with PBS (1×, pH 7.4). The dried biofilms were stained with 0.1% crystal violet for 15 min. The crystal violet staining solution was discarded, and the biofilms were washed three times with PBS and then dried. The crystal violet-stained biofilms were dissolved in 95% ethanol and transferred to new 96-well plates, and the absorbance was measured at 570 nm using a microplate reader. The results (see Figures 5a and b) showed that Gram-positive bacteria had a stronger ability to form biofilms (deeper crystal violet staining), but the nanocoating had no significant effect on the biofilm formation of Gram-positive bacteria FS4722, while it significantly promoted the biofilm formation of Gram-negative bacteria EcN.

[0071] (2) Further investigation was conducted on the effect of Cu-NPs on the biofilm formation of Gram-positive bacteria FS4722 and Gram-negative bacteria EcN. 1 mL of the probiotics cultured overnight was taken and adjusted to OD. 600 The concentration was approximately 3. After washing, the cell pellet was supplemented with 0, 25, 50, 75, 100, 125, 150, and 200 μL of OD200, respectively. 600 The Cu-NPs concentration was approximately 1.2, and the volume was increased to 1 mL with PBS; then the resulting mixture was diluted to 1 × 10⁻⁶. 6 CFUs were inoculated into 96-well plates and incubated statically at 37°C for 24 h. Crystal violet staining was performed using the same procedure as described above, and results were collected using a microplate reader. Furthermore, the effect of different amounts of Cu-NPs on coating formation was investigated; 1 mL of probiotics cultured overnight was taken and adjusted to OD0.05. 600 The concentration was approximately 3. After washing, the cell pellet was resuspended in 400 μL of PBS, and then 50 μL of Fe was added. 3+ The solution (4 mg / mL) was gently vortexed for about 20 s, and then 0, 25, 50, and 75 μL of OD solution were added respectively. 600Cu-NPs of approximately 1.2 were added to a 1 mL volume with PBS, followed by electron microscopy. The results (Figure 5c) showed that for Gram-positive bacteria FS4722, a nanoparticle addition of 25 μL significantly promoted biofilm formation. However, at this level, the nanoparticles were insufficient to form a continuous and complete nanocoating on the probiotics (see Figure 5e), and biofilm formation began to be inhibited at a nanoparticle addition of 150 μL. Figure 5e also shows that a nanoparticle addition of 50 μL was the optimal embedding condition, at which point a complete coating formed on the surface of all probiotics. At an addition of 75 μL, a large number of additional nanoparticles accumulated on the bacterial surface. The results (Figure 5d) showed that for Gram-negative bacteria EcN, a nanoparticle addition of 50 μL significantly promoted biofilm formation, and biofilm formation began to be inhibited at a nanoparticle addition of 200 μL. This may be because Gram-positive bacteria lack a physical outer membrane barrier, making them more sensitive to the effects of nanoparticles. This further confirms that the nanocoating selectively promotes biofilm behavior.

[0072] (3) In addition, the effects of other embedding materials on EcN biofilms were also investigated, including:

[0073] ① ECN@EL-100 group: anionic polymer EUDRAGIT L 100-55 (abbreviated as EL 100-55, Shanghai Evonik Industries): 1 mL of overnight cultured ECN (OD 600 After adjusting to approximately 3, wash three times with PBS (1×, pH 7.4), then add 200 μL of Ca. 2+ (5 mg / mL, anhydrous calcium chloride) Vortex for 1 min, then add 200 μL EL 100-55 (1 mg / mL) and vortex for 1 min. Finally, add 600 μL PBS and adjust the pH to 5.0. Vortex for 5 min and wash three times with PBS (pH 5.0).

[0074] ②ECN@Fe 3+ -TA: Single-layer metal-phenol coordination network Fe 3+ -TA: Take 1 mL of 1×10 8 Add 10 μL TA (40 mg / mL) to the CFU EcN bacterial culture, followed by 10 μL Fe. 3+ (10 mg / mL), and finally add 880 μL PBS (1×, pH 7.4), vortex for 60 s and wash three times with PBS (pH 7.4);

[0075] ③ECN+Fe 3+Prepared according to step (1) of Example 3, but without adding Cu-NPs, containing only 50 μL Fe. 3+ EcN (4 mg / mL).

[0076] The results (f in Figure 5) indicate that the monolayer metal-phenol network also has a certain promoting effect on EcN biofilms, but the nano-coating has the best effect. Furthermore, Fe was used to form the nano-coating. 3+ The fact that it does not affect biofilm formation indicates that an appropriate amount of Cu-NPs is the main factor promoting the formation of biofilms in Gram-negative bacteria EcN.

[0077] Example 5: The ability of EcN nanocoating to form a biofilm in simulated intestinal mucus

[0078] The following steps were taken to investigate the biofilm formation ability of Gram-negative bacteria EcN treated with nanocoating in a simulated intestinal mucus environment:

[0079] (1) Construction of EcN expressing red fluorescent protein (mCherry): The plasmid pBBR1MCS2-Tac-mCherry (kanamycin resistant, Hunan Fenghui Biotechnology Co., Ltd.) was transformed into Escherichia coli Nissle 1917 to obtain EcN strain expressing red fluorescent protein. The EcN strain was used in all subsequent experiments.

[0080] (2) Cultivation of biofilms in simulated mucus: Coated EcN cells were obtained according to step (1) of Example 3. Simulated mucus was obtained by dissolving 30 mg / mL mucin (derived from porcine stomach, purchased from Sigma-Aldrich) in PBS (1×, pH 7.4) containing 0.05% agar. The simulated mucus contained 50 μg / mL kanamycin. Following step (1) of Example 4, the ability of natural and nano-coated EcN cells to form biofilms after culturing in simulated mucus for 12, 24, and 48 h was tested. The structure of the biofilms was examined by scanning electron microscopy and laser confocal microscopy. The results (Figures 6a and 6b) showed that after culturing in mucus for 12 h, there was no significant difference in the total bacterial count in the biofilms of natural and nano-coated EcN cells. However, nano-coated EcN cells tended to aggregate more readily, which was beneficial for subsequent biofilm formation. Furthermore, with the extension of culturing time to 24 and 48 h, nano-coated EcN cells formed more biofilms and a greater bacterial count. This indicates that the biofilm-promoting effect of nanoparticles can still be maintained in a simulated intestinal mucus system.

[0081] (3) Cytotoxicity of EcN nanocoating: Caco-2 cells and human colon cancer cells HT29 (1×10⁻⁶) were subjected to cytotoxicity. 4 / well) were seeded into 96-well plates and incubated in growth medium at 37 °C for 24 h. Natural EcN and nano-coated EcN (1×10) were seeded separately into wells. 7 CFU / mL solution was added to each well and co-cultured for 24 h. Then, 10 μL of CCK-8 solution was added to each well and incubated for 1 h. Finally, the OD value of the culture medium at 450 nm was recorded using a multi-mode microplate reader. The results (Figure 6c) indicate that the cytotoxicity of the EcN nanocoating is negligible.

[0082] Example 6: In vitro environmental resistance of EcN nanocoating

[0083] In addition to biofilm formation ability, the effects of other environmental conditions on the nano-coated EcN were investigated, including UV irradiation, lyophilization, antibiotics, and hydrogen peroxide (H2O2). The specific steps were as follows: equal amounts of natural and nano-coated EcN (1×10⁻⁶) were... 8 The samples were treated with UV germicidal lamp (λ = 254 nm, 20 W) for 40 min, lyophilized for 12 h, and in ciprofloxacin (10 μg / mL) and H2O2 (8 mM) solutions at 37 °C for 1 h. After treatment under different conditions, bacteria were collected from each sample and washed twice. Finally, the bacteria were serially diluted and plated on LB agar plates, and colonies were counted after overnight incubation. The results in Figure 7 show that the nanocoating in this study had the strongest protective effect on probiotics under the above-mentioned adverse in vitro environmental conditions.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing microorganisms coated with a metal-polyphenol secondary coordination nanocoating, characterized in that... The process includes the following steps: (1) dissolving polyvinylpyrrolidone in MOPS buffer, then adding biocompatible metal ions, followed by polyphenols, adjusting the pH of the system to 7.8-8.2, self-assembling at room temperature to form metal-polyphenol nanoparticles, separating the solid and liquid, washing the obtained solid, and obtaining metal-polyphenol nanoparticles; (2) adding biocompatible metal ions to microorganisms, mixing evenly, adding the metal-polyphenol nanoparticles obtained in step (1), mixing evenly again, obtaining an assembly system, coordinating assembly, separating the solid and liquid, washing the obtained solid, and obtaining microorganisms coated with a metal-polyphenol secondary coordination nanocoating; the metal ions mentioned in step (1) are Cu 2+ The polyvinylpyrrolidone mentioned in step (1) is polyvinylpyrrolidone K30; the polyphenol mentioned in step (1) is tannic acid; the metal ion mentioned in step (1) and the polyphenol are mixed in a molar ratio of 2:0.9-1.1; the metal ion mentioned in step (2) is Fe 3+ The microorganisms mentioned in step (2) are at least one of Gram-negative bacteria, Gram-positive bacteria, and yeast; the assembly system mentioned in step (2) is obtained through the following steps: take a concentration of OD 600 Centrifuge 1 mL of microbial culture medium with a concentration of 2.8–3.2, collect the bacterial cells, wash with PBS, and resuspend the cells in PBS. Add 0.19–0.21 mg of metal ions to the resulting bacterial suspension, mix well, and then add 40–60 μL of OD200 solution. 600 A metal-polyphenol nanoparticle solution with a concentration of 1.1–1.3 was diluted to 1 mL with PBS.

2. The method for preparing microorganisms coated with a metal-polyphenol secondary coordination nanocoating according to claim 1, characterized in that: The Cu 2+ Derived from copper chloride or its hydrate; the Fe 3+ The bacteria are derived from ferric chloride or its hydrate; the Gram-positive bacteria are *Lactobacillus plantarum* FS4722; the Gram-negative bacteria are *Escherichia coli* Nissle 1917; and the yeast is *Saccharomyces cerevisiae*.

3. The method for preparing microorganisms coated with a metal-polyphenol secondary coordination nanocoating according to claim 1, characterized in that: The MOPS buffer mentioned in step (1) is a MOPS buffer with pH 7.8-8.2 and 90-110 mM; the pH mentioned in step (1) is 8.

0.

4. The method for preparing microorganisms coated with a metal-polyphenol secondary coordination nanocoating according to claim 1, characterized in that: The self-assembly time in step (1) is 3 to 10 minutes; the solid-liquid separation method in step (1) is centrifugation; the cleaning solution in step (1) is deionized water or purified water; the mixing method in step (2) is vortexing; the solid-liquid separation method in step (2) is centrifugation; the cleaning solution in step (2) is PBS buffer.

5. A microorganism coated with a metal-polyphenol secondary coordination nanocoating, characterized in that: It is obtained by the preparation method according to any one of claims 1 to 4.

6. The application of the preparation method according to any one of claims 1 to 4 in the preparation of a microecological regulation system.

7. The application of the microorganisms coated with the metal-polyphenol secondary coordination nanocoating as described in claim 5 in the preparation of probiotic formulations.

8. The application according to claim 7, characterized in that: The microorganisms mentioned are at least one of Lactobacillus plantarum FS4722 and Escherichia coli Nissle 1917.

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