A method for preparing a biomineralized-coated bacteria, the biomineralized-coated bacteria and applications thereof

Abstract

The application provides a preparation method of a biomineralization coated bacterium, the biomineralization coated bacterium and application thereof, and relates to the technical field of microbial medicine preparation.The preparation method of the biomineralization coated bacterium comprises the following steps: mixing bacteria and a polyvinylpyrrolidone solution to obtain a bacterium suspension; mixing a CaCl2 solution with the bacterium suspension, and then adding an Na2CO3 aqueous solution; stirring for 0.5-1.5 hours, centrifuging, collecting the precipitate and obtaining the biomineralization bacterium. The method is simple and convenient, and is used for assisting biological interface mineralization through electrostatic interaction to deposit a super-resistant and self-adapting bacterium protection coating. The method is universal for coating various species including obligate and facultative anaerobes. The preparation procedure and the formed coating have negligible influence on the activity of the bacterium, and the bacterium is free from the double invasion of gastric acid and bile acid, so that the activity and quantity of the bacterium reaching the intestinal tract are ensured.

Description

Technical Field

[0001] This invention relates to the field of microbial drug preparation technology, and in particular to a method for preparing biomineralized coated bacteria, biomineralized coated bacteria and their applications. Background Technology

[0002] The gut microbiome, composed of a vast array of microorganisms, has proven crucial for maintaining host health. In-depth research has shown that disruption of the gut microbiome balance leads to impaired gut homeostasis, subsequently triggering a variety of diseases. Supplementation with beneficial microorganisms is often used as an attractive strategy for disease prevention and treatment because the introduced species can reduce pathogen colonization and maintain a healthy microbial composition. With its excellent patient compliance, oral delivery of probiotics to the gut microbiome has become the most attractive method of supplementation due to its non-invasive nature. A key step in the effectiveness of microbial biotherapy is ensuring the probiotics are viable during manufacturing and maintain sufficient numbers after oral delivery to the gut. Therefore, there is an urgent need for protective methods to prepare oral live biological therapeutics.

[0003] Currently, the formation of dry powders, enteric coatings, and pellets, along with the addition of whey, glycerol, trehalose, pepsin, and other protective agents, has been utilized to reduce environmental threats to bacterial therapies. The use of alginate, chitosan, cellulose, metallophenolic networks, and metal-organic frameworks to individually encapsulate entire coatings has been explored as an effective tool for physically protecting bacteria from external attacks. For example, coating with additional self-assembled lipid membranes endows bacteria with the ability to resist gastrointestinal-related threats. However, oral biotherapies prepared by existing methods face unavoidable difficulties throughout the entire process from bacterial culture to intestinal colonization, resulting in extremely low usability.

[0004] During the manufacturing process, initial exposure to oxygen can be toxic to the enzymes and nucleic acids of anaerobic bacteria, which represent the vast majority of beneficial microorganisms. Secondly, to ensure safety and reduce potential contamination risks, therapeutic products are frequently sterilized, leading to cellular stress, altered cell structure, and even bacterial cell death. Finally, after oral ingestion, gastrointestinal-related luminal damage, particularly from gastric and bile acids, inactivates the ingested microorganisms, resulting in an insufficient number of viable cells upon arrival in the intestines. Therefore, a method is urgently needed to protect the microorganisms throughout the entire process from manufacturing to colonization in the intestines. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing biomineralized coated bacteria, the biomineralized coated bacteria, and their application in the preparation of super-resistant and adaptive oral intestinal microbial drugs, thereby protecting the bacteria from the dual attack of gastric acid and bile acid and ensuring the activity and quantity of the bacteria when they reach the intestine.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing biomineralized coated bacteria, comprising the following steps: mixing bacteria with a polyvinylpyrrolidone solution to obtain a bacterial suspension; mixing a CaCl2 solution with the bacterial suspension, and then adding a Na2CO3 aqueous solution; stirring for 0.5–1.5 h, centrifuging, and collecting the precipitate to obtain biomineralized bacteria.

[0008] Preferably, the ratio of the bacteria to the polyvinylpyrrolidone solution is 1×10⁻⁶. 7 ~1×10 8 CFUs: 1-2 mL.

[0009] Preferably, the concentration of the polyvinylpyrrolidone solution is 1 to 3 mg / mL.

[0010] Preferably, the CaCl2 concentration is 0.30–0.36 M, and the ratio of bacteria to CaCl2 solution is 1 × 10⁻⁶. 7 ~1×10 8 CFUs: 0.1-0.3 mL.

[0011] Preferably, the CaCl2 solution is mixed with the bacterial suspension and then stirred for 10–30 minutes.

[0012] Preferably, the concentration of the Na2CO3 aqueous solution is 0.30-0.36M, and the amount of Na2CO3 aqueous solution used is equal in volume to the amount of CaCl2 used.

[0013] Preferably, the centrifugation speed is 8000-12000 rpm and the centrifugation time is 0.5-1.5 min.

[0014] Preferably, after centrifugation to separate the biomineralizing bacteria, the bacteria are washed 2 to 4 times with PBS solution.

[0015] The present invention also provides a method for preparing the biomineralized coated bacteria.

[0016] The present invention also provides the application of the aforementioned biomineralized coated bacteria in the preparation of super-resistant and adaptive intestinal oral microbial drugs.

[0017] This invention provides a method for preparing biomineralized coated bacteria. A simple electrostatic interaction-assisted biointerface mineralization method is used to deposit a highly resistant and adaptive bacterial protective coating. This method is applicable to a variety of species, including obligate and facultative anaerobes, and the preparation procedure and the resulting coating have negligible impact on bacterial viability. Due to its physical barrier effect, the resulting single-cell coating endows individually packaged anaerobic bacteria with resistance to damage associated with manufacturing and product sterilization, including oxygen exposure, ultraviolet irradiation, and 75% ethanol. After oral administration, a spontaneous metathesis reaction occurs between the mineral coating and gastric acid, enabling rapid neutralization of the low pH in the stomach and demineralization-triggered release of coated bacteria. Simultaneously, the calcium ions generated during decomposition induce micellar aggregation of bile acids, thereby protecting the released bacteria from gastric and bile acid attack and preventing subsequent intestinal colonization. Under the coating of the biomineral coating, the probiotic Bacteroides fragilis strain exhibited impressive therapeutic effects in a DSS-induced mouse colitis model. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of biomineralization at the interface, where a) mineral coating is prepared on the surface of bacteria; b) the coated bacteria's resistance to environmental attacks; c) the neutralization of gastric acid, adaptive release of the coated bacteria, and calcium ion-triggered bile acid aggregation, through a dual decomposition reaction of the biomineral coating in the gastrointestinal tract after oral ingestion.

[0019] Figure 2 Characterization images of the biomineral coatings include: a) representative TEM images of Bacteroides fragilis BF839 native bacteria and Bacteroides fragilis BF839-coated bacteria, scale bar: 5 μm; b) typical SEM images and EDS elemental mappings of Bacteroides fragilis BF839-coated bacteria, scale bar: 1 μm; c) FTIR spectra of Bacteroides fragilis BF839 native bacteria and Bacteroides fragilis BF839-coated bacteria; d) Zeta potentials of Bacteroides fragilis BF839 after different treatments; e) flow cytometry histograms of calcein-labeled coated BF839; f) confocal images of Bacteroides fragilis BF839-coated bacteria, with the green channel representing the calcein-labeled biomineral coating, scale bar: 8 μm.

[0020] Figure 3 Cell compatibility and versatility of biomineral coatings are shown, where a) growth curves of cells cultured in GAM broth at 37°C; b) cell viability; c) representative TEM images of native and coated EcNs, scale bar: 2 μm; d) Zeta potentials of native and coated EcNs; e) typical TEM images of native and coated Bifs, scale bar: 1 μm; f) Zeta potentials of native and coated Bifs. Error bars represent standard deviations. n = 3 independent experiments. Data are expressed as mean ± SD.

[0021] Figure 4 To assess resistance to manufacturing-related threads, the following were used: a) air exposure for 30 days; c) UV exposure for 30 minutes; error bars represent standard deviations. n = 3 independent experiments, and data are expressed as mean ± SD; b) representative TEM images of native BF839 exposed to air; d) representative TEM images of coated BF839 exposed to UV radiation, scale bar: 2 μm; e) uncoated coated BF839 and native BF839 exposed to UV radiation for 30 minutes.

[0022] Figure 5 Adaptive plots of biomineral coatings, including: a) digital photographs of native and coated bacteria with SGF; b) bacterial number dependence; c) flow cytometry histograms of bacteria; d) confocal image of calcein-labeled coated BF839, with the green channel indicating the calcein-labeled mineral coating, scale bar: 8 μm; e) number of surviving bacteria, with error bars representing standard deviations for n = 3 independent experiments, and data expressed as mean ± SD; f) TEM images of native and coated BF839 after SGF incubation, scale bar: 2 μm.

[0023] Figure 6 For in vivo resistance and adaptation, where a) confocal images of EcN, with green and red channels representing calcein-labeled mineral coatings and EcN expressing mCherry, scale bar: 10 μm; b) IVIS images of the gastrointestinal tract, scale bar representing radiometric intensity (p / s / cm). 2 c) Bacterial retention in the stomach; d) Bacterial retention in the gut. Error bars represent standard deviation. n = 3 independent experiments. Data are expressed as mean ± SD.

[0024] Figure 7 The images show the therapeutic effects of the coated bacteria, including: a) double-blind histopathological analysis; b) changes in mouse body weight during infection and treatment; c) mean length; d) digital photographs of the intestine 5 days after treatment, scale bar: 1 cm; e) serum tumor necrosis factor-α levels 5 days after treatment; f) mean number of MPO-positive cells counted in the colon; g) representative images of MPO staining; h) representative images of H&E staining in the colon, with red and blue arrows representing epithelial damage and inflammation, respectively, scale bar: 150 μm. Error bars represent standard deviations. n = 3 independent experiments. Data are expressed as mean ± SD.

[0025] Figure 8 Representative SEM image of Bacteroides fragilis BF839 native bacteria, scale bar: 1μm;

[0026] Figure 9Plates containing native Bacteroides fragilis BF839 bacteria, Bacteroides fragilis BF839-coated bacteria, and uncoated Bacteroides fragilis BF839-coated bacteria.

[0027] Figure 10 For the treatment of exposure to 75% (v / v) ethanol for 10 min, a) the number of surviving bacteria after exposure to 75% (v / v) ethanol for 10 min at 37°C with equal amounts of native Bacteroides fragilis BF839 and Bacteroides fragilis BF839-coated bacteria; significance was assessed using one-way ANOVA, and p-values ​​are given, ***p<0.001, ****p<0.0001; b) representative TEM images of native Bacteroides fragilis BF839 and Bacteroides fragilis BF839-coated bacteria to 75% (v / v) ethanol, scale bar: 2 μm;

[0028] Figure 11 For the diagram of exposure to bile acid solution, a) with 0.33M Ca 2+ Bile acid solution (0.3 mg / mL) after mixing and standing for 5 minutes. -1 (a) Digital photographs of the corresponding solutions; (b) Equal amounts of Bacteroides fragilis BF839 protozoa exposed to bile acid solution (0.3 mg / mL) -1 ) or with 0.33M Ca 2+ The number of bacteria surviving in the corresponding solutions after mixing at 37°C for 1 hour was assessed using the Student's t-test, and p-values ​​were given, *p<0.05;

[0029] Figure 12 Representative confocal images of native EcNs carrying pBBR1MCS2-Tac-mCherry in the stomach at 0, 1 and 5 h after oral administration. The red channel shows EcN expression of mCherry. Scale bar: 10 μm.

[0030] Figure 13 For representative confocal images, a) native EcN carrying pBBR1MCS2-Tac-mCherry; b) representative confocal images of EcN coated in the intestine 0, 1 and 5 hours after oral administration; green and red channels indicate EcN with calcein-labeled coating, and red channels indicate EcN expressing mCherry. Scale bar: 10 μm.

[0031] Figure 14 a) Representative IVIS images of the gastrointestinal tract harvested 1 hour after drug administration; b) Representative IVIS images of the gastrointestinal tract harvested 5 hours after drug administration. Scale bars represent radiometric values ​​(p / s / cm). 2 1×10⁻⁶ sr) was administered to mice via oral tube feeding. 7The natural EcN or coated EcN of CFU were obtained by using EcN expressing LuxCDABE and EcN coated with mineral layers labeled with calcein, respectively. Detailed Implementation

[0032] This invention provides a method for preparing biomineralized coated bacteria, comprising the following steps: mixing bacteria with a polyvinylpyrrolidone solution to obtain a bacterial suspension; mixing a CaCl2 solution with the bacterial suspension, and then adding a Na2CO3 aqueous solution; stirring for 0.5–1.5 h, centrifuging, and collecting the precipitate to obtain biomineralized bacteria.

[0033] In the preparation method described in this invention, bacteria are mixed with a polyvinylpyrrolidone solution to obtain a bacterial suspension, wherein the preferred ratio of bacteria to polyvinylpyrrolidone solution is 1×10⁻⁶. 7 ~1×10 8 CFUs: 1–2 mL, more preferably 1 × 10⁻⁶ 8 CFUs: 1-2 mL, preferably 1×10⁻⁶. 8 CFUs: 1.5 mL; the concentration of the polyvinylpyrrolidone solution is preferably 1-3 mg / mL, more preferably 1.5-2.5 mg / mL, and even more preferably 1.8-2.2 mg / mL; the present invention uses polyvinylpyrrolidone surfactant as a stabilizer, which binds to the surface of negative bacteria through electrostatic interaction.

[0034] In the preparation method described in this invention, CaCl2 solution is mixed with bacterial suspension and stirred, and then Na2CO3 aqueous solution is added; the concentration of CaCl2 is preferably 0.30-0.36M, more preferably 0.31-0.35M, and even more preferably 0.32-0.34M; the ratio of bacteria to CaCl2 solution is preferably 1×10⁻⁶. 7 ~1×10 8 CFUs: 0.1–0.3 mL, more preferably 1 × 10⁻⁶ mL. 8 CFUs: 0.1–0.3 mL, preferably 1 × 10⁻⁶ mL. 8 CFUs: 0.2 mL; the stirring time is preferably 10–30 min, more preferably 15–25 min, and even more preferably 18–22 min; the concentration of the Na2CO3 aqueous solution is preferably 0.30–0.36 M, more preferably 0.31–0.35 M, and even more preferably 0.32–0.34 M; the amount of Na2CO3 aqueous solution is preferably equal in volume to that of CaCl2; calcium ions are adsorbed onto the PVP-stabilized bacterial cell surface by complexing with the introduced pyrrolidone group, and the absorbed calcium ions guide the heterogeneous nucleation and deposition of calcium carbonate (CaCO3) on the bacterial cell surface to form a biomineral coating.

[0035] In the preparation method described in this invention, after adding Na2CO3 aqueous solution, the mixture is stirred, centrifuged, and the precipitate is collected to obtain biomineralizing bacteria. The stirring time is preferably 0.5-1.5 h, more preferably 0.6-1.4 h, and even more preferably 0.8-1.2 h. The centrifugation speed is preferably 8000-12000 rpm, more preferably 9000-11000 rpm, and even more preferably 9500-11500 rpm. The centrifugation time is preferably 0.5-1.5 min, more preferably 0.6-1.4 min, and even more preferably 0.8-1.2 min.

[0036] In the preparation method described in this invention, after centrifuging to separate the biomineralizing bacteria, the bacteria are washed with PBS solution, preferably 2 to 4 times, and more preferably 3 times.

[0037] The present invention also provides a method for preparing the biomineralized coated bacteria.

[0038] The present invention also provides the application of the aforementioned biomineralized coated bacteria in the preparation of super-resistant and adaptive intestinal oral microbial drugs.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Bacteroides fragilis BF839 was purchased from the China Culture Collection Center for Microbial Cultures (GMCC, China). Phosphate-buffered saline (1×PBS) was provided by Sigma-Aldrich (USA). Na2CO3, CaCl2 and polyvinylpyrrolidone were purchased from Adamas-beta (Shanghai, China).

[0042] 1×10 8 CFUs of Bacteroides fragilis BF839 were mixed with 1.5 mL of a 2 mg / mL polyvinylpyrrolidone solution to obtain a bacterial suspension. 0.2 mL of a 0.33 M CaCl2 solution was mixed with the bacterial suspension and stirred for 20 min. Then, 0.2 mL of a 0.33 M Na2CO3 aqueous solution was added, and the mixture was stirred for 1 h. The mixture was centrifuged at 10,000 rpm for 1 min, and the precipitate was collected to obtain biomineralized bacteria. The bacteria were washed three times with PBS.

[0043] Example 2

[0044] 1×10 7CFUs of Bacteroides fragilis BF839 were mixed with 1 mL of 1 mg / mL polyvinylpyrrolidone solution to obtain a bacterial suspension. 0.1 mL of 0.30 M CaCl2 solution was mixed with the bacterial suspension and stirred for 10 min. Then, 0.1 mL of 0.30 M Na2CO3 aqueous solution was added, and the mixture was stirred for 0.5 h. The mixture was centrifuged at 8000 rpm for 1.5 min, and the precipitate was collected to obtain biomineralized bacteria. The bacteria were washed twice with PBS.

[0045] Example 3

[0046] 1×10 8 CFUs of Bacteroides fragilis BF839 were mixed with 2 mL of a 3 mg / mL polyvinylpyrrolidone solution to obtain a bacterial suspension. 0.3 mL of a 0.36 M CaCl2 solution was mixed with the bacterial suspension and stirred for 30 min. Then, 0.3 mL of a 0.36 M Na2CO3 aqueous solution was added, and the mixture was stirred for 1.5 h. The mixture was centrifuged at 12000 rpm for 0.5 min, and the precipitate was collected to obtain biomineralized bacteria. The bacteria were washed four times with PBS.

[0047] Example 4

[0048] 1×10 7 CFUs of Bacteroides fragilis BF839 were mixed with 2 mL of 1.5 mg / mL polyvinylpyrrolidone solution to obtain a bacterial suspension. 0.2 mL of 0.32 M CaCl2 solution was mixed with the bacterial suspension and stirred for 15 min. Then, 0.2 mL of 0.32 M Na2CO3 aqueous solution was added, and the mixture was stirred for 0.8 h. The mixture was centrifuged at 9000 rpm for 0.8 min, and the precipitate was collected to obtain biomineralized bacteria. The bacteria were washed three times with PBS.

[0049] Example 5

[0050] 1×10 8 CFUs of Bacteroides fragilis BF839 were mixed with 1 mL of 2.5 mg / mL polyvinylpyrrolidone solution to obtain a bacterial suspension. 0.2 mL of 0.34 M CaCl2 solution was mixed with the bacterial suspension and stirred for 25 min. Then, 0.2 mL of 0.34 M Na2CO3 aqueous solution was added, and the mixture was stirred for 1.2 h. The mixture was centrifuged at 11000 rpm for 1.2 min, and the precipitate was collected to obtain biomineralized bacteria. The bacteria were washed three times with PBS.

[0051] Experimental Example

[0052] Calcein was purchased from Adamas-beta (Shanghai, China); plasmids pBBR1MCS2-Tac-mCherry (anti-kanamycin) and pMD18-luxCDABE (anti-ampicillin) were purchased from domestic suppliers; simulated gastric juice (SGF, pH 1.2) was prepared by dissolving 3.2 g pepsin and 2.0 g NaCl in 1 L deionized water, then 7 mL concentrated HCl solution was added, and the pH was adjusted to 1.2 with dilute HCl solution. The resulting buffer solution was then filtered through a 0.22 μm membrane; bacteria were grown in GAM medium at 37 °C with appropriate antibiotics added.

[0053] Female ICR mice (6–8 weeks, 17–20 g) and female C57BL / 6 mice (6–8 weeks, 17–20 g) were provided by J.J. Laboratory Animal Center (Shanghai, China). All mice were housed and fed under 12 / 12h dark / light cycles and specific pathogen-free (SPF) conditions at an ambient temperature of 25°C and a humidity of 55%. All animal procedures complied with the guidelines for laboratory animal care from the Shanghai Medical College, and the animal protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

[0054] Characterization and observation experiment of coated bacteria

[0055] The morphology of the original Bacteroides fragilis BF839 bacteria and the Bacteroides fragilis BF839-coated bacteria obtained in Example 1 was observed by transmission electron microscopy (TEM, HITACH, Japan). One drop of the original Bacteroides fragilis BF839 bacterial sample solution and the Bacteroides fragilis BF839-coated bacterial sample solution obtained in Example 1 were deposited on a carbon-coated copper grid, respectively, and then washed twice with ddH2O for 5 min each time. Before observation, the samples were air-dried at room temperature.

[0056] Scanning electron microscopy (SEM, ZEISS 1550VP FESEM) was used to observe the morphology of the original Bacteroides fragilis BF839 bacteria and the Bacteroides fragilis BF839-coated bacteria obtained in Example 1. The original Bacteroides fragilis BF839 bacteria and the Bacteroides fragilis BF839-coated bacteria obtained in Example 1 were collected by centrifugation (4000g, 5min), washed three times with PBS, and fixed in 3% glutaraldehyde solution for 1h at room temperature. The fixed bacteria were washed twice with PBS and dehydrated continuously for 15min in 30%, 50%, 70%, 80%, 90%, and 100% ethanol. They were then air-dried before observation.

[0057] Fourier transform infrared spectroscopy (FTIR, Bruker Tensor 27 spectrometer, USA) was used to examine the surface infrared signals of *Bacteroides fragilis* BF839 native bacteria and *Bacteroides fragilis* BF839-coated bacteria obtained in Example 1. Samples of *Bacteroides fragilis* BF839 native bacteria and *Bacteroides fragilis* BF839-coated bacteria obtained in Example 1 were collected by centrifugation (4000g, 5min) and washed twice with ddH2O, then freeze-dried for 12h. Infrared spectra of samples and background were analyzed using the KBr disc technique at 450–4000 cm⁻¹. -1 Recorded within the range, with a resolution of 4cm. -1 For each FTIR spectrum, 64 scans were collected and averaged for each sample and background under specific conditions.

[0058] 1×10 8 CFUs of Bacteroides fragilis BF839 were dispersed in deionized water containing 10 mM PVP. 0.2 mL of CaCl2 (0.33 M) was added to the bacterial suspension at room temperature, and after stirring for 20 min, 0.2 mL of a 20:1 Na2CO3:calcein mixture was added to the mixture. After stirring for 1 h, the biomineralized bacteria were separated by centrifugation (10000 rpm, 1 min) and washed three times with PBS. The labeled Bacteroides fragilis BF839 coated with the PBS was diluted to 10⁻⁶. 5 CFUs were dropped onto agar gel on a glass slide and covered with a glass microscope slide.

[0059] The prepared samples were observed to ensure uniform CaCO3 coating using a laser scanning confocal microscope (LSCM, Leica TCS SP8, German). Uniformly coated bacteria were selected as the final sample. The zeta potential of the biomineralized bacterial samples was examined using dynamic light scattering (DLS, Malvern Zetasizer nano ZS, UK). Flow cytometry (Beckman CytoFlex, USA) was used to measure the calcein-labeled biomineralized bacterial samples, with *Bacteroides fragilis* BF839 as a control group. The gastrointestinal tract of mice was imaged using an in vivo imaging system (IVIS, INIS Lumina II, Caliper).

[0060] Transmission electron microscopy (TEM) revealed that Bacteroides fragilis BF839-coated bacteria exhibited a rougher surface with higher contrast compared to native Bacteroides fragilis BF839 cells. Figure 2 (a) ; Typical scanning electron microscopy (SEM) images show individual BF839 cells completely encapsulated after mineralization at the biointerface. Figure 2 b and Figure 8); Energy-dispersive X-ray spectroscopy (EDS) analysis of the elemental spectrum of *Bacteroides fragilis* BF839-coated bacteria showed that calcium signals were uniformly distributed on the *Bacteroides fragilis* BF839-coated bacteria. Figure 2 b); The existence of the mineralized layer was further verified by Fourier transform infrared (FTIR) spectroscopy, in which the Bacteroides fragilis BF839-coated bacteria only had the main absorption region of CaCO3 ( Figure 2 c); The zeta potential of Bacteroides fragilis BF839 after interaction with PVP, as measured by dynamic light scattering (DLS), showed a significant increase from -37.8 ± 1.7 mV to 25.5 ± 2.7 mV, further increasing to near positive values ​​under calcium ion absorbance. Figure 2 d); After surface mineralization, the zeta potential decreased to -13.4±0.8 mV, indicating successful formation of the coating structure; Flow cytometry histograms of labeled Bacteroides fragilis BF839-coated bacteria showed that the fluorescence intensity of BF839 and BF839 mixed with calcein significantly increased ( Figure 2 e); Quantitative analysis of the labeled BF839 showed a coating efficiency of 88.2%. Laser scanning confocal microscopy (LSCM) imaging combined with bright fluorescence signals ( Figure 2 f) indicates that a CaCO3 coating was generated on each BF839 cell.

[0061] Growth curve determination of Bacteroides fragilis BF839-coated bacteria

[0062] Centrifuge (4000g, 5min) the Bacteroides fragilis BF839-coated bacteria obtained in Example 1, collect the bacterial pellet, and wash with PBS. The original Bacteroides fragilis BF839 bacteria, the Bacteroides fragilis BF839-coated bacteria, and the Bacteroides fragilis BF839-coated bacteria with the coating removed after soaking in simulated gastric acid for 30 minutes were diluted to a GAM optical density (OD) value of 0.15, and incubated with gentle shaking at 37°C. The OD values ​​of the cultures were recorded at 600nm in 96-well plates at 0.5h intervals for 12h using a microplate reader (BioTek, USA).

[0063] By recording OD 600nm The optical density values ​​at a certain point were used to evaluate the growth curve of *Bacteroides fragilis* BF839-coated bacteria in Glufu anaerobic medium (GAM), such as... Figure 3As shown in Figure a, the results indicate that the proliferation of coated BF839 bacteria was completely inhibited, in stark contrast to the original Bacteroides fragilis BF839 bacteria. This inhibition of the coated BF839 bacteria is attributed to the presence of a robust CaCO3 layer that inhibits bacterial division and material exchange, thus promoting growth. After desalting the coated BF839 bacteria with hydrochloric acid (pH 4.0), their growth curves were found to be comparable to those of the original BF839 bacteria, indicating little difference in cell viability between the original and uncoated BF839 bacteria. Plate counts showed that the growth of coated BF839 bacteria was almost completely inhibited in GAM medium, while the viability of the original and uncoated BF839 bacteria remained consistent, further clarifying that the preparation procedure and mineral coating formation had a limited negative impact on bacterial activity. Figure 3 b and Figure 9 ).

[0064] In vitro gastric acid neutralization assay of Bacteroides fragilis BF839-coated bacteria

[0065] An in vitro study of gastric acid neutralization was conducted by measuring the pH of simulated gastric acid (SGF) (pH 1.2) using a pH meter (Mettler Toledo, Columbus, OH, USA). An equal amount of *Bacteroides fragilis* BF839-coated bacteria (1 × 10⁻⁶) was added to 20 mL of simulated gastric acid (SGF) every 1 minute. 8 After CFU, the pH change was recorded at room temperature.

[0066] like Figure 5 As shown in Figure a, the immediate generation of bubbles and completion of the reaction within minutes after mixing Bacteroides fragilis BF839-coated bacteria with simulated gastric acid (SGF, pH 1.2) indicates the decomposition of the CaCO3 coating. Figure 5 As shown in b, the spontaneous reaction between the CaCO3 coating and the acid caused a continuous increase in pH value, as the bacterial count increased from 23 × 10⁻⁶. 8 (CFU) increased to 35×10 8 (CFU) colony formation and a sharp increase in pH from 2 to 5.5 confirmed the ability of Bacteroides fragilis BF839-coated bacteria to effectively neutralize acidic solutions. After incubation with 1.2 mL of LSGF for 5 min, BF839 (1 × 10⁻⁶) coated with a mineral layer labeled with calcein (CFU) formed. 8 Flow cytometry histograms of CFU showed a significant decrease in fluorescence intensity. Figure 5c). As the SGF volume increased to 2.4 mL, the mineral layer completely decoated as the fluorescence intensity of the coated bacteria decreased to a level similar to that of the native Bacteroides fragilis BF839. LSCM further showed acid-induced removal of the CaCO3 layer, exhibiting a significantly weakened green fluorescence signal after SGF treatment, which subsequently disappeared completely upon increasing the volume of the acidic solution used. Figure 5 d); Plate counts showed that the number of bacteria coated with Bacteroides fragilis BF839 was almost the same as that of the original Bacteroides fragilis BF839 bacteria. Figure 5 e) Quantitative counting confirmed that acid-mediated CaCO3 layer decomposition completely uncoated Bacteroides fragilis BF839-coated bacteria, and the viability of the released bacteria remained unaffected after SGF damage.

[0067] TEM imaging showed that the mineral coating around BF839 was dissolved by acid, and both the surface and internal components of the bacteria remained in their normal shape. Figure 5 f) After the Bacteroides fragilis BF839 protozoan is subjected to acidic damage, the bacterial cell structure shrinks or even breaks down. It was found that calcium ions produced by the metathesis reaction can induce bile acid aggregation, which can reduce its toxicity to the released BF839, thereby increasing its survival rate after entering the duodenum. Figure 11 (a and b).

[0068] Determination of resistance to environmental damage by Bacteroides fragilis BF839-coated bacteria

[0069] Equal amounts of *Bacteroides fragilis* BF839-coated bacteria and *Bacteroides fragilis* BF839 native bacteria were stored in the air at 4°C for 30 days, or exposed to 100 μW / cm². 2 The samples were exposed to ultraviolet radiation for 30 minutes. Simultaneously, equal amounts of *Bacteroides fragilis* BF839-coated bacteria and *Bacteroides fragilis* BF839 native bacteria were resuspended in 1 mL of *Bacteroides fragilis* activation broth, and 75% ethanol was added. The samples were then gently incubated at 37°C with shaking. After 30 days of air exposure or 30 minutes of ultraviolet irradiation, 50 μL of each sample was taken, washed with PBS, serially diluted with PBS, and plated on agar plates. After incubation at 37°C overnight, colonies were counted. Samples were then observed using TEM after 30 days of air exposure or 30 minutes of ultraviolet irradiation.

[0070] like Figure 4 As shown in Figure a, over 75% of the uncoated Bacteroides fragilis BF839-coated bacteria remained viable after being stored in the air at 4°C for 30 days. Under the same experimental conditions, the number of surviving native Bacteroides fragilis BF839 bacteria was negligible (less than 6 orders of magnitude).

[0071] TEM observations of bacterial morphology confirmed that oxygen-induced cell membrane vesicle production and corresponding bubbling cell death occurred in the original bacteria Bacteroides fragilis BF839 ( Figure 4 b) In addition, it is avoided in Bacteroides fragilis BF839-coated bacteria. The resulting increased tolerance to oxygen toxicity can be explained by the isolation effect of the biomineral layer on the cell surface, which hinders the diffusion of oxygen molecules.

[0072] like Figure 4 As shown in c, after 30 minutes of ultraviolet irradiation (100 μW / cm²), 2 Under the same treatment, the surviving count of uncoated Bacteroides fragilis BF839-coated bacteria did not decrease significantly. In contrast, under the same treatment, the original Bacteroides fragilis BF839 bacteria were almost completely killed, indicating that the CaCO3 coating can shield the bacterial cells from the harmful effects of ultraviolet radiation. TEM observation of the original Bacteroides fragilis BF839 bacteria showed that the bacterial cells contained a large number of aggregated structures (…). Figure 4 d) reflects the inactivation of intracellular proteins and enzymes. The absence of structural failure observed in irradiated Bacteroides fragilis BF839-coated bacteria further supports the ability of the mineral layer to protect the encapsulated bacteria from UV sterilization. Figure 4 e).

[0073] In the presence of a CaCO3 coating, the quantitative survival rate of uncoated Bacteroides fragilis BF839-coated bacteria after direct incubation in 75% ethanol for 10 min was nearly 6 orders of magnitude higher than that of native Bacteroides fragilis BF839 bacteria. Figure 10 a).

[0074] TEM images demonstrate that exposure to 75% ethanol damages or even dissolves the bacterial envelope, leading to the inactivation of the original Bacteroides fragilis BF839 bacteria. However, due to the protective effect of the mineral coating, the uncoated Bacteroides fragilis BF839 bacteria retain their original envelope structure and smooth cell surface. Figure 10 b) indicates that bacterial biomineralization greatly enhances their resistance to manufacturing-related environmental attacks.

[0075] Retention experiment of Bacteroides fragilis BF839-coated bacteria in the gastrointestinal tract

[0076] Female ICR mice (6–8 weeks old, 17–20 g) were divided into two groups and administered 1×10⁻⁶ mg orally via tube feeding to each group. 7CFU-coated Bacteroides fragilis BF839 and native Bacteroides fragilis BF839. mCherry expressing EcN was coated with a calcein-labeled mineral layer. Mice were euthanized at 0, 1, and 5 hours post-administration. Gastrointestinal contents, including the stomach and intestines, were serially diluted with PBS, and 50 μL of each dilution was plated onto solid GAM agar plates and incubated at 37°C for 12 h before counting. After oral administration of native ECNs containing the pMD18-luxCDABE plasmid or ECNs coated with calcein, Bacteroides fragilis BF839-coated bacteria showed decreased calcein signaling from 0 to 5 h, but increased mCherry signaling in the gastric contents, indicating demineralization and that the released bacteria were protected from the neutralized gastric pH. Figure 13 a and supplement Figure 12 Conversely, the mCherry signal of the native *Bacteroides fragilis* BF839 was observed to gradually disappear due to damage from gastric acid. Furthermore, compared to the native *Bacteroides fragilis* BF839, more mCherry-tagged bacteria were observed in the contents extracted from the intestines of the *Bacteroides fragilis* BF839-coated bacterial group at 1 and 5 hours after tube feeding, indicating an improved survival rate of the *Bacteroides fragilis* BF839-coated bacteria (Supplement). Figure 13 (a and b).

[0077] Gastrointestinal tract samples were collected at 1 h and 5 h post-ingestion for in vivo imaging system (IVIS) measurements. Representative IVIS images captured at predetermined time points showed a signal mismatch between luxCDABE luminescence and calcein fluorescence, indicating successful demineralization and survival of Bacteroides fragilis BF839-coated bacteria in the gastrointestinal tract. Figure 6 b and Figure 14 In the Bacteroides fragilis BF839 protobacterial group, the luxCDABE luminescence signal emitted by the sampling bundle was negligible, which in turn reflects the protective effect of the mineral coating.

[0078] like Figure 6 As shown in Figure c, the survival rates of Bacteroides fragilis BF839-coated bacteria in the stomach were significantly improved at 1 h and 5 h after administration, being 2-fold and almost 6-fold higher than those of the native Bacteroides fragilis BF839 group, respectively. Similarly, compared to the native Bacteroides fragilis BF839 group, the survival rates of Bacteroides fragilis BF839-coated bacteria in the intestine were almost 5-fold and more than 4.5-fold higher, respectively. Figure 6 d). In summary, the dual-decomposition reaction-mediated gastric acid neutralization and bacterial release demonstrate the resistance and adaptability of the mineral coating, which effectively protects the coated bacteria from gastrointestinal stressors during oral administration.

[0079] like Figure 3As shown in cf, TEM imaging, together with zeta potential measurements, claims that facultative anaerobic Escherichia coli Nissle 1917 (EcN) and obligate anaerobic Bifidobacterium (Bif) can be similarly coated, highlighting the broad applicability of this method to different strains of biomineralization.

[0080] DSS-induced mouse colitis model experiment

[0081] Six- to eight-week-old female C57BL / 6 mice were given 3% DSS salt (molecular weight 36,000-50,000 kDa; Sangon, China) in sterile drinking water for 7 days.

[0082] Mice were randomly divided into three groups and orally administered either native Bacteroides fragilis BF839 bacteria or Bacteroides fragilis BF839-coated bacteria (1×10⁻⁶). 7 CFUs / mouse / day or PBS, for 5 days. Untreated healthy mice were used as controls.

[0083] Mice were weighed daily and then euthanized by CO2 asphyxiation. 1 mL of blood was collected from each mouse and stored in a 1.5 mL Eppendorf tube, then incubated at 37°C for 30 min. The collected blood was centrifuged at 4000 g for 5 min to separate serum from each mouse. The concentration of tumor necrosis factor-α was then determined using an ELISA kit (MultiSciences Biotech, China). The colon was collected for length measurement and finally fixed in 4% formalin for blinded histopathological analysis.

[0084] Colon samples were fixed in 4% formalin and processed according to the prescribed paraffin embedding procedure. Paraffin-embedded samples were sectioned at 4 μm and then stained with hematoxylin and eosin (H&E) and MPO. Samples were scanned using a 3D HISTECHPannoramic 250 (3DHISTECH, Hungary). MPO activity was examined by MPO staining and the number of brown MPO-positive cells was counted.

[0085] All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using Prism 8.0 (GraphPad, USA). One-way ANOVA was used to analyze the results, and the minimum significance test was performed between the three groups. Unpaired Student's t-tests were used for comparisons between the two groups. A p-value < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) was considered statistically significant.

[0086] Mice were fed 3% DSS in drinking water for one week to induce colitis, followed by daily oral administration of topical BF839 for 5 days. Figure 7 a). For example Figure 7 As shown in b, the mice's weight was recorded daily, and the results indicated that DSS feeding led to a sustained decrease in body weight compared to healthy mice. DSS mice treated with *Bacteroides fragilis* BF839-coated bacteria recovered body weight more effectively than the *Bacteroides fragilis* BF839 native bacteria and PBS treatment groups. Mean intestinal length harvested and measured on day 5 post-treatment indicated that DSS could induce a reduction in intestinal length, and that the use of *Bacteroides fragilis* BF839 native bacteria exerted a moderately beneficial effect. Figure 7 (c and d). The average length of the intestine sampled from mice coated with Bacteroides fragilis BF839 was similar to that of healthy mice, but significantly longer than that of mice with native Bacteroides fragilis BF839.

[0087] Compared with native Bacteroides fragilis BF839 and PBS treatment, the Bacteroides fragilis BF839-coated bacterial treatment group significantly reduced the inflammatory response, as evidenced by lower serum levels of tumor necrosis factor-(TNF-α), one of the key inflammatory cytokines. Figure 7 e). Similar levels of tumor necrosis factor-α were perceived in mice coated with Bacteroides fragilis BF839 compared to healthy mice.

[0088] Furthermore, the mean number of MPO-positive cells in colon tissue sections from mice treated with Bacteroides fragilis BF839 was significantly lower than that in the Bacteroides fragilis BF839 group compared to the native bacteria and PBS group. Figure 7 f and g). Typical images of Bacteroides fragilis BF839-coated bacterial colon stained with hematoxylin and eosin (H&E) show undetectable histological damage, including mucosal epithelial cell shedding and lamina propria leakage and intestinal gland disappearance, similar to healthy mice. Figure 7 h). The Bacteroides fragilis BF839-coated bacteria demonstrated effective therapeutic efficacy, and the enhanced efficacy could be explained by the increased oral bioavailability of the coated bacteria due to the mineral coating, which improves intestinal length extension, inflammatory response reduction, and limited histological damage.

[0089] As can be seen from the above embodiments, the present invention provides a method for preparing biomineralized coated bacteria, biomineralized coated bacteria, and their application in the preparation of highly resistant and adaptive oral microbial drugs for the gut, which can protect bacterial biopharmaceuticals from external damage from manufacturing to reaching the gut. Electrostatic interactions assist in the mineralization of the bio-interface to deposit a highly resistant and adaptive bacterial protective coating, and the preparation process and the resulting coating have negligible impact on bacterial viability. Due to its physical barrier effect, the resulting single-cell coating endows individually packaged anaerobic bacteria with resistance to damage associated with manufacturing and product sterilization, including oxygen exposure, ultraviolet irradiation, and 75% ethanol. After oral administration, a spontaneous metathesis reaction occurs between the mineral coating and gastric acid, enabling rapid neutralization of the low pH in the stomach and demineralization-triggered release of coated bacteria. Simultaneously, the calcium ions generated during decomposition induce micelle aggregation of bile acids, thereby protecting the released bacteria from gastric acid and bile acid attack, thus preventing subsequent intestinal colonization. With the help of biomineral coatings, the probiotic Bacteroides fragilis strain showed excellent therapeutic effects in a DSS-induced mouse colitis model. Biomineralization can be used to prepare multifunctional bacterial biopharmaceuticals for various biomedical applications.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing biomineralized coated bacteria, characterized in that, Includes the following steps: Bacteroides, Escherichia, or Bifidobacterium bacteria were mixed with a polyvinylpyrrolidone solution to obtain a bacterial suspension. CaCl2 solution was mixed with the bacterial suspension, and then Na2CO3 aqueous solution was added. The mixture was stirred for 0.5 to 1.5 hours, centrifuged, and the precipitate was collected to obtain biomineralized bacteria.

2. The method for preparing biomineralized coated bacteria as described in claim 1, characterized in that, The ratio of bacteria to polyvinylpyrrolidone solution was 1×10⁻⁶. 7 ~1×10 8 CFUs: 1~2mL.

3. The method for preparing biomineralized coated bacteria as described in claim 2, characterized in that, The concentration of the polyvinylpyrrolidone solution is 1~3 mg / mL.

4. The method for preparing biomineralized coated bacteria as described in claim 1, characterized in that, The CaCl2 concentration is 0.30~0.36M, and the ratio of bacteria to CaCl2 solution is 1×10⁻⁶. 7 ~1×10 8 CFUs: 0.1~0.3mL.

5. The method for preparing biomineralized coated bacteria as described in claim 4, characterized in that, After mixing the CaCl2 solution with the bacterial suspension, stir for 10-30 minutes.

6. The method for preparing biomineralized coated bacteria as described in claim 1, characterized in that, The concentration of the Na2CO3 aqueous solution is 0.30~0.36M, and the amount of Na2CO3 aqueous solution used is equal in volume to the amount of CaCl2 used.

7. The method for preparing biomineralized coated bacteria as described in claim 1, characterized in that, The centrifugation speed is 8000~12000 rpm, and the centrifugation time is 0.5~1.5 min.

8. The method for preparing biomineralized coated bacteria as described in claim 1, characterized in that, After centrifugation to separate the biomineralizing bacteria, the bacteria are washed 2-4 times with PBS solution.

9. Biomineralized coated bacteria prepared by the method according to any one of claims 1 to 8.

10. The use of the biomineralized coated bacteria according to claim 9 in the preparation of oral microbial drugs, characterized in that, The drug has properties of resisting gastric acid, bile acid, ultraviolet radiation and / or ethanol damage; and when the bacteria in the biomineralized coated bacteria are Bacteroides or Bifidobacterium, the drug also has properties of resisting oxidative toxicity.

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