A liposome-encapsulated probiotic with improved environmental tolerance, and methods of making and using the same

Abstract

The application discloses a kind of liposome reinforced coating probiotics with improved environmental tolerance and its preparation method and application.The method comprises the following steps: culturing bacteria to 10 8 CFU / mL~10 9 CFU / mL, centrifugal collection bacterial body is washed and stored; phospholipid, cholesterol and pluronic polymer are dissolved in organic solvent, and the solvent is evaporated to obtain a lipid film; the bacterial body is resuspended in CaCl2 solution and reacted for 10~30 min, the obtained bacterial solution is transferred to the lipid film, and the reaction is continued for 10~30 min to obtain bacteria with liposome reinforced coating.The method can improve the extreme environmental tolerance of probiotics during processing, storage and delivery, and can efficiently resist freeze-drying damage and damage to the digestive tract, thereby solving the problems of high damage during processing and storage of probiotics and low oral delivery efficiency.

Description

Technical Field

[0001] This invention relates to the field of probiotic products technology, specifically to a probiotic with an improved environmental tolerance and a lipid film-reinforced coating, its preparation method, and its application. Background Technology

[0002] Probiotics are a class of live microorganisms used as food supplements to improve gut microbiota balance and provide health benefits to the host. Currently, probiotics have become a research hotspot in the fields of food nutrition and biomedicine, and are widely used in the prevention and adjunctive treatment of gastrointestinal diseases. However, orally administered probiotics are easily affected by extreme environmental factors such as gastric acid, bile salts, and digestive enzymes when passing through the human digestive tract, leading to a significant reduction in activity and making it difficult for them to effectively reach the intestines and colonize. Studies have shown that only when the concentration of live bacteria in the colon reaches 10... 6 ~10 7 Probiotics can only effectively exert their physiological functions when the concentration of CFU / g of intestinal contents is reached. Therefore, constructing a delivery system that can protect probiotics from extreme environmental stresses in the digestive tract is of great significance for the development of probiotic preparations.

[0003] To achieve efficient delivery of probiotics, researchers have developed various microencapsulation technologies, which involve encapsulating probiotics in microcapsules using suitable carrier materials to protect their activity and control release. Traditional microencapsulation methods, such as spray drying, extrusion, freeze drying, and emulsification, all have certain limitations, restricting their application efficiency and scope. Spray drying is suitable for industrial production, but the heat and dehydration stress during the preparation process can easily lead to a significant decrease in probiotic activity, which is difficult to completely avoid even with optimized temperature parameters. Freeze drying can better maintain the stability of the carrier structure, but the ice crystals formed may damage the integrity of the probiotic cell membrane, causing irreversible damage and reducing the survival rate of live bacteria. Extrusion has mild preparation conditions and has less impact on probiotic activity, but its product has a large particle size and poor uniformity, which is not conducive to the uniform distribution and colonization of probiotics in the intestine. Emulsification produces microcapsules with a wide particle size range (25 μm to 2 mm), which can meet different application needs, but their morphology is difficult to control precisely and they are prone to agglomeration.

[0004] Single-cell nanoencapsulation is a novel encapsulation strategy that constructs artificial nanoshells, typically less than 100 nm thick, on the surface of a single living cell, endowing cells with new functional properties. This technology is usually based on non-covalent interactions such as electrostatic interactions and hydrophilic-hydrophobic interactions, or utilizes covalent bonding reactions between probiotic surface functional groups and modifying materials to modify the bacterial surface. Through mechanisms such as constructing physical barriers, regulating the microenvironment, and achieving targeted delivery, single-cell encapsulation technology can significantly improve the stability and physiological function of probiotics in vivo and in vitro. The surface of probiotics is rich in various functional groups (such as free thiols, amino groups, hydroxyl groups, and carboxyl groups), providing abundant modification sites for chemical coupling and encapsulation; the negative charge on their surface also facilitates supramolecular electrostatic interactions. These characteristics lay a solid foundation for the surface modification of probiotics.

[0005] To address the shortcomings of existing probiotic delivery systems in terms of tolerability and controllability, this invention proposes a lipid membrane-reinforced coating probiotic with high tolerance to extreme environments, its preparation method, and its application, aiming to provide new ideas and technical support for the research and development of probiotic preparations. Summary of the Invention

[0006] In view of the defects or deficiencies in the existing technology, and in order to maximize the protection of probiotics from damage by extreme environments during processing, storage and delivery, the primary objective of this invention is to provide a method for bacterial surface coating. This method can improve the tolerance of probiotics to extreme environments during processing, storage and delivery. This method can achieve efficient resistance of bacteria, especially probiotics, to freeze-drying damage and digestive tract damage, and can solve the problems of large damage during probiotic processing and storage and low oral delivery efficiency.

[0007] Another object of the present invention is to provide coated probiotics prepared by the above method.

[0008] Another object of the present invention is to provide the application of the coated probiotics prepared by the above method.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A method for coating a bacterial surface includes the following steps:

[0011] S1: Culture the bacteria to 10... 8 CFU / mL ~10 9 CFU / mL, collect bacterial cells by centrifugation, wash and use for later use;

[0012] S2: Dissolve phospholipids, cholesterol, and Prönkel polymer in an organic solvent, mix well, and evaporate the solvent to obtain a lipid film;

[0013] S3: Resuspend the bacterial refill from step S1 in CaCl2 solution. After reacting for 10–30 min, the resulting bacterial solution is transferred to the lipid film obtained in S2, and the reaction continues for 10–30 min to obtain bacteria with a surface lipid film-reinforced coating.

[0014] Further, the bacteria mentioned in step S1 are probiotics; probiotics are defined by the Food and Agriculture Organization of the United Nations and the World Health Organization as "live microorganisms that, when applied in adequate amounts, can provide health benefits to the host"; further still, the bacteria mentioned in step S1 include at least one of lactobacillus, bifidobacterium, and Escherichia coli; even further still, the bacteria mentioned in step S1 are Bifidobacterium longum ATCC BAA-999 or Escherichia coli nissle 1917.

[0015] Furthermore, the washing described in step S1 is washing with PBS, and even further, washing with PBS 2-3 times.

[0016] Further, the phospholipids mentioned in step S2 include at least one of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA), dioleoyltrimethylammonium propane (DOTAP), distearylphosphatidylcholine (DSPC), and distearylphosphatidylethanolamine (DSPE); and even more specifically, at least one of dioleoylphosphatidylcholine (DOPC) and dioleoyltrimethylammonium propane (DOTAP).

[0017] Further, the Pluronic polymer mentioned in step S2 includes at least one of Pluronic® P65 (Poloxamer 185), Pluronic® F-68 (Poloxamer 188), Pluronic® P108 (Poloxamer 108), Pluronic® P123 (Poloxamer 403), and Pluronic® F-127 (Poloxamer 407); and even more specifically, Pluronic® F-127 (Poloxamer 407).

[0018] Further, the mass ratio of phospholipids, cholesterol and Prönnicke polymer in step S2 is (10-20):1:(70-100).

[0019] Furthermore, the phospholipids, cholesterol, and Pluronic polymer mentioned in step S2 are dioleoylphosphatidylcholine (DOPC), dioleoyltrimethylammonium propane (DOTAP), cholesterol, and Pluronic® F-127 (Poloxamer 407), with a mass ratio of 10:1:3:88.

[0020] Furthermore, in step S3, the concentration of the CaCl2 solution was 12.5 mM, and the concentration of bacteria after resuspending was 10. 8 CFU / mL ~10 9 CFU / mL.

[0021] Furthermore, in step S3, the ratio of bacterial cells to lipid film is 10:1. 8 CFU~10 9 CFU: 8–10 mg.

[0022] A lipid membrane-reinforced coating probiotic was prepared by the above method, and the lipid membrane-reinforced coating probiotic exhibits high tolerance to extreme environments.

[0023] The above-mentioned lipid membrane-reinforced coating probiotics are used in the preparation of intestinal flora regulation drugs.

[0024] Furthermore, the gut microbiota-regulating drugs described herein are applicable to the treatment of diseases including but not limited to: intestinal diseases such as ulcerative colitis, irritable bowel syndrome, and infectious enteritis; metabolic diseases such as obesity and diabetes; and gastrointestinal tumors such as colorectal cancer.

[0025] The application of the above-mentioned lipid membrane-reinforced coating probiotics in the preparation of drugs for treating enteritis.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] (1) Unlike traditional microencapsulation technologies (emulsion cross-linking, freeze-drying, spray drying, electrostatic spraying), this invention employs single-cell nanoencapsulation technology. This encapsulation strategy, which constructs artificial nanoshells typically <100nm thick on the surface of individual live bacteria, not only reduces production costs (eliminating the need for large specialized equipment like freeze dryers) but also avoids damage to probiotic activity caused by freeze-drying and spray drying processes. Furthermore, compared to the rapid transport of large-sized delivery carriers in the intestines, nanoscale encapsulation carriers can help overcome flow effects, thereby prolonging the intestinal retention time of probiotics.

[0028] (2) The lipid membrane reinforced coating probiotics prepared in this invention can significantly improve the survival rate of probiotics during processing, storage and digestive tract delivery under the protection of the coating, thereby significantly improving the stability and physiological efficacy of probiotics in vivo and in vitro.

[0029] (3) The lipid membrane reinforced coating probiotics of the present invention can survive and remain better in the digestive tract environment to exert physiological effects and bring health benefits to the host (such as: treatment of ulcerative colitis). At the same time, the simple preparation method can realize large-scale production and storage. Attached Figure Description

[0030] Figure 1 These are TEM and SEM images of Escherichia coli with a lipid membrane-reinforced coating.

[0031] Figure 2 The images show the DLS test results of E. coli with different formulations of lipid film-reinforced coatings.

[0032] Figure 3 This is a graph showing the evaluation results of the tolerance of E. coli with different formulations of lipid membrane-reinforced coatings to damage in the in vitro digestive tract environment.

[0033] Figure 4 This is a graph showing the evaluation results of the tolerance of Bifidobacteria with different formulations of lipid membrane-reinforced coatings to damage in the in vitro digestive tract environment.

[0034] Figure 5 This is a graph showing the evaluation results of the tolerance of Bifidobacteria with different formulations of lipid film-reinforced coatings to freeze-drying damage.

[0035] Figure 6 This is a graph showing the evaluation results of the survival and retention of Bifidobacteria with different formulations of lipid membrane-reinforced coating in the mouse intestine.

[0036] Figure 7 This is a graph showing the evaluation results of the therapeutic effect of orally administered lipid membrane-reinforced Bifidobacterium on ulcerative colitis in mice. Detailed Implementation

[0037] To enable those skilled in the art to better understand and implement the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments are not intended to limit the present invention.

[0038] Example 1: Preparation of DOPC lipid film-reinforced Escherichia coli (ELP)

[0039] The method for preparing DOPC lipid membrane-reinforced Escherichia coli (ELP) includes the following steps:

[0040] (1) Prepare a lipid component mixed solution with a total concentration of 2 mg / mL containing DOPC (10 mg / mL of mother liquor, dissolved in chloroform) and cholesterol using methanol, wherein the mass ratio of DOPC to cholesterol is 10:1;

[0041] (2) Place 500 μL of lipid component mixture solution and 500 μL of Pluronic® F-127 (16 mg / mL) dissolved in methanol in a sample vial and dry by rotary evaporation (37°C, 120 rpm) to form a lipid film;

[0042] (3) Take OD 600nm =0.8 Escherichia coli Nissle 1917 (approximately 6 × 10⁸) 8 Centrifuge 0.5 mL of CFU, wash twice with PBS, resuspend in 1 mL of CaCl2 solution (12.5 mM) and react for 10 min. Then transfer all of it to the sample bottle described in step (2), fully hydrate the membrane and continue to react for 10 min to obtain lipid membrane-reinforced coated Escherichia coli, denoted as ELP.

[0043] Example 2: Preparation of DOPC-DOTAP lipid membrane reinforced coating of Escherichia coli (ELPD)

[0044] The preparation method of DOPC-DOTAP lipid membrane-reinforced Escherichia coli (ELPD) includes the following steps:

[0045] (1) Prepare a lipid component mixed solution with a total concentration of 2 mg / mL containing DOPC (10 mg / mL of mother liquor, dissolved in chloroform) and cholesterol using methanol, wherein the mass ratio of DOPC to cholesterol is 10:1;

[0046] (2) 500 μL of DOPC lipid fraction mixed solution, 500 μL of Pluronic® F-127 (16 mg / mL) dissolved in methanol and 28 μL of DOTAP (10 mg / mL, dissolved in chloroform) were placed in a sample vial and dried by rotary evaporation (37°C, 120 rpm) to form a lipid film.

[0047] (3) Take OD 600nm =0.8 Escherichia coli Nissle 1917 (approximately 6 × 10⁸) 8 Centrifuge 0.5 mL of CFU, wash twice with PBS, resuspend in 1 mL of CaCl2 solution (12.5 mM) and react for 10 min. Then transfer all of it to the sample bottle described in step (2), fully hydrate the membrane and continue to react for 10 min to obtain lipid membrane-reinforced coated Escherichia coli, denoted as ELPD.

[0048] Example 3: Preparation of DOPC-DOTAP lipid membrane reinforced coating Bifidobacterium (BLPD)

[0049] The preparation method of DOPC-DOTAP lipid membrane reinforced coating Bifidobacterium (BLPD) includes the following steps:

[0050] (1) Prepare a lipid component mixed solution with a total concentration of 2 mg / mL containing DOPC (10 mg / mL of mother liquor, dissolved in chloroform) and cholesterol using methanol, wherein the mass ratio of DOPC to cholesterol is 10:1;

[0051] (2) 500 μL of DOPC lipid fraction mixed solution, 500 μL of Pluronic® F-127 (16 mg / mL) dissolved in methanol and 28 μL of DOTAP (10 mg / mL, dissolved in chloroform) were placed in a sample vial and dried by rotary evaporation (37°C, 120 rpm) to form a lipid film.

[0052] (3) Take OD 600nm =1 Bifidobacterium longum ATCC BAA-999 (approximately 1×10⁻⁶) 8 Centrifuge 1 mL of CFU, wash twice with PBS, then resuspend in 1 mL of CaCl2 solution (12.5 mM) and react for 10 min. Then transfer all of it to the sample bottle described in step (2), fully hydrate the membrane and continue to react for 10 min to obtain lipid membrane-reinforced coated Escherichia coli, denoted as ELPD.

[0053] Comparative Example 1: Preparation of Uncoated Escherichia coli (EcN)

[0054] A method for preparing uncoated Escherichia coli (EcN) includes the following steps:

[0055] (1) Place 1 mL of methanol in a sample vial and dry it by rotary evaporation (37°C, 120 rpm).

[0056] (2) Take OD 600nm =0.8 Escherichia coli Nissle 1917 (approximately 6 × 10⁸) 8 Centrifuge 0.5 mL of CFU, wash twice with PBS, resuspend in 1 mL of CaCl2 solution (12.5 mM) and react for 10 min. Then transfer all of it to the sample bottle described in step (1) and react for 10 min to obtain uncoated Escherichia coli, denoted as EcN.

[0057] Comparative Example 2: Preparation of DOPC lipid film-coated Escherichia coli (EL)

[0058] A method for preparing DOPC lipid film-coated Escherichia coli (EL) includes the following steps:

[0059] (1) Prepare a lipid component mixed solution with a total concentration of 2 mg / mL containing DOPC (10 mg / mL of mother liquor, dissolved in chloroform) and cholesterol using methanol, wherein the mass ratio of DOPC to cholesterol is 10:1;

[0060] (2) Place 500 μL of lipid component mixture and 500 μL of methanol in a sample vial and dry by rotary evaporation (37℃, 120 rpm) to form a lipid film;

[0061] (3) Take OD 600nm =0.8 Escherichia coli Nissle 1917 (approximately 6 × 10⁸) 8 Centrifuge 0.5 mL of CFU, wash twice with PBS, resuspend in 1 mL of CaCl2 solution (12.5 mM) and react for 10 min. Then transfer all of it to the sample bottle described in step (2), fully hydrate the membrane and continue to react for 10 min to obtain DOPC lipid membrane coated Escherichia coli, denoted as EL.

[0062] Comparative Example 3. Preparation of Uncoated Bifidobacterium (Bif)

[0063] (1) Place 1 mL of methanol in a sample vial and dry it by rotary evaporation (37°C, 120 rpm).

[0064] (2) Take OD 600nm =1 Bifidobacterium longum ATCC BAA-999 (approximately 1×10⁻⁶) 8 Centrifuge 1 mL of CFU, wash twice with PBS, resuspend in 1 mL of CaCl2 solution (12.5 mM) and react for 10 min. Then transfer all of it to the sample bottle described in step (1) and react for 10 min to obtain uncoated Bifidobacterium, denoted as Bif.

[0065] Comparative Example 4. Preparation of DOPC lipid film-coated Bifidobacterium (BL)

[0066] (1) Prepare a lipid component mixed solution with a total concentration of 2 mg / mL containing DOPC (10 mg / mL of mother liquor, dissolved in chloroform) and cholesterol using methanol, wherein the mass ratio of DOPC to cholesterol is 10:1;

[0067] (2) Place 500 μL of lipid component mixture and 500 μL of methanol in a sample vial and dry by rotary evaporation (37℃, 120 rpm) to form a lipid film;

[0068] (3) Take OD 600nm=1 Bifidobacterium longum ATCC BAA-999 (approximately 1×10⁻⁶) 8 Centrifuge 1 mL of CFU, wash twice with PBS, resuspend in 1 mL of CaCl2 solution (12.5 mM) and react for 10 min. Then transfer all of it to the sample bottle described in b, fully hydrate the membrane and continue to react for 10 min to obtain DOPC lipid membrane coated Bifidobacterium, denoted as BL.

[0069] Example 4: Coated / uncoated *E. coli* obtained from Examples 1, 2, Comparative Example 1, and Comparative Example 2 were used for TEM, DLS, and in vitro digestive tract environment tolerance testing.

[0070] (1) Morphological characterization of Escherichia coli with different formulations of lipid film-reinforced coating

[0071] The morphological characteristics of EcN, EL, and ELP were observed using scanning electron microscopy (SEM), and the microstructure of *E. coli* was observed using transmission electron microscopy (TEM) after uranium acetate staining. The results are as follows: Figure 1 As shown in the TEM images, DOPC lipids clearly form a distinct coating on the bacterial surface, while the combination of Pluronic® F-127 and DOPC lipids further forms a uniform and continuous shell. SEM images show that the composite shell formed by DOPC lipids and Pluronic® F-127 provides a smoother surface for ECF, in stark contrast to unencapsulated bacteria, demonstrating the successful construction of the coating on the bacterial surface.

[0072] (2) Particle size and zeta potential tests of Escherichia coli with different formulations of lipid film-reinforced coating

[0073] The particle size and zeta potential of EcN, EL, and ELP were measured using a Malvern particle size analyzer (DLS), and the results are as follows: Figure 2 As shown, compared to EcN, the potential of ELF increased from -30.65 mV to 23.52 mV, and the average particle size increased from 1213.33 nm to 2696.67 nm. This further confirms the formation of the coating.

[0074] (3) Evaluation of the tolerance of Escherichia coli with different formulations of lipid film-reinforced coating to damage in vitro digestive tract environment

[0075] The survival of bacteria coated in simulated gastric juice (SGF), simulated intestinal juice (SIF), and bile salts was recorded using plate colony counting. Equal volumes of EcN, EL, ELP, and ELPD were placed in 1 mL of SGF solution (pH 2.0, containing 0.9% NaCl and 3.2 g / L pepsin) and incubated with shaking at 37 °C. At predetermined time points (0 min, 15 min, and 30 min), 100 μL of each sample was used for dilution and plating, incubated at 37 °C for 24 h, and then the colony count was performed to assess bacterial survival. Figure 3 As shown in Figure A, almost all coated bacterial groups showed higher viable bacterial counts after treatment with SGF compared to the uncoated group (EcN). ELPD exhibited the best tolerance to SGF, with a 19200-fold increase in viable bacterial count after 15 min of incubation compared to EcN, a 25.6-fold increase compared to ELP (without cationic lipid components), and an 839.16-fold increase compared to EL (without cationic lipid components and no Prönnicke-reinforced lipid coating). Similarly, its survival rate was 70% in bile salt solution (0.3 mg / mL) and 86% in intestinal fluid (pH 6.8, containing 6.8 g / L KH2PO4 and 10 g / L trypsin), significantly higher than that of unmodified bacteria (bile salt incubation survival rate: 19%, intestinal fluid incubation survival rate: 21%). Figure 3 (As shown in B and C in the figure). The above results indicate that the Pronnick material-reinforced lipid coating plays a key role in improving the digestive damage resistance of probiotics encapsulated in lipid membranes, and the lipid membrane-reinforced coating can greatly enhance the tolerance of E. coli to the extreme environment of the digestive tract.

[0076] Example 5 uses the coated / uncoated Bifidobacterium obtained in Example 3 and Comparative Example 3 for in vitro digestive tract environment tolerance testing, tolerance to freeze-drying damage evaluation, survival and retention in mouse intestines evaluation, and therapeutic effect on mouse ulcerative colitis.

[0077] (1) Evaluation of the tolerance of lipid membrane-reinforced Bifidobacterium to in vitro digestive tract environmental damage.

[0078] The survival of coated Bifidobacteria in simulated gastric juice (SGF) was recorded using plate colony counting. Equal volumes of Bif and BLPD were placed in 1 mL of SGF solution (pH 2.0, containing 0.9% NaCl and 3.2 g / L pepsin), and incubated with shaking at 37 °C. At predetermined time points (0 min and 10 min), 100 μL of each sample was used for dilution and plating, incubated at 37 °C for 48 h, and then the colony count was performed to assess bacterial survival. Figure 4The results showed that, similar to those of the E. coli test, the number of BLFD viable cells increased by 14,396 times compared to uncoated Bif. This indicates that lipid membrane-reinforced coating encapsulation is a universally applicable method for improving the tolerance of probiotics to gastric acid.

[0079] (2) Evaluation results of the tolerance of Bifidobacterium with lipid membrane reinforced coating to freeze-drying damage;

[0080] Bacterial damage during the freeze-drying-rehydration process was recorded using plate colony counting. Equal volumes of Bif and BLPD were freeze-dried for 24 h and then stored at 4°C. At predetermined time points (0 d, 7 d, 14 d, and 42 d), 1 mL of 0.9% NaCl was added for rehydration. After serial dilution, 100 μL of each sample was used for plating and incubated at 37°C for 48 h. After incubation, the number of colonies was counted to assess bacterial survival. Figure 5 The results showed that BLFD was more resistant to mechanical stress during the freeze-drying process than uncoated Bif. After 42 days of freeze-drying and rehydration, the survival rate of BLFD was 42 times higher than that of Bif.

[0081] (3) Evaluation results of the survival and retention of lipid membrane-reinforced Bifidobacterium in the mouse intestine;

[0082] a. Stability evaluation of lipid membrane-reinforced Bifidobacterium in mouse intestine

[0083] The coated bacterial BLPD was prepared using an engineered *Bifidobacterium longum* ATCC BAA-999 strain (chloramphenicol resistant, 6 ng / mL) containing the pKO403-LacZ-cm plasmid, preserved in the laboratory. The engineered *Bifidobacterium longum* ATCC BAA-999 strain containing the pKO403-LacZ-cm plasmid was obtained by electroporation. Logarithmically growing *Bifidobacterium longum* ATCC BAA-999 cells were collected, washed with electroporation buffer, and resuspended. 150 μL of the resuspended bacterial solution was mixed with plasmid pKO403-LacZ-cm (400 ng / mL) (pKO403-LacZ-cm is commercially available) in an electroporation cuvette for electroporation (2500 V, 2 pulses, pulse duration 4.1–4.5 ms). After electroporation, the bacterial cells were revived on antibiotic-free MRS medium and plated on selective plates containing chloramphenicol (6 ng / mL). Positive clones were screened under anaerobic conditions. Single clones were picked and further amplified in chloramphenicol-containing MRS medium to obtain engineered Bifidobacterium strains stably carrying the pKO403-LacZ-cm plasmid. Eighteen 6–8 week old BALB / c mice were randomly divided into two groups of nine each, and administered equal amounts (1 × 10⁻⁶ ng / mL) of the plasmid to each group via gavage. 8Chloramphenicol-resistant Bif suspension or BLPD suspension (CFU / mouse) was administered. Three mice from each group were sacrificed at 4 h, 24 h, and 72 h after gavage. Cecal and colonic tissues and their contents were collected, homogenized, serially diluted, and spread on sterile MRS plates. After incubation at 37 ℃ for 48 h, colony counting was performed.

[0084] The results are as follows Figure 6 As shown in Figure A, the number of viable bacteria in the intestines of the BLPD group was significantly higher than that of the Bif group at all time points. Four hours after gavage, the number of viable bacteria in the cecum and colon of BLPD was 1.38 times and 4.81 times that of the Bif group, respectively; with increasing time, the difference further widened, and the trend became more pronounced at 24 h and 72 h. This indicates that BLPD maintains high tolerance and stability in the complex digestive tract environment.

[0085] b. Distribution and retention of Bifidobacteria with lipid membrane-reinforced coating in mice

[0086] 1 μM DIR fluorescent dye was mixed with Bif and BLPD bacterial suspensions, respectively, and incubated at 37°C for 15 min. After washing three times with PBS, free DIR was removed by centrifugation to obtain DIR-labeled Bif and BLPD. Six 6–8 week old BALB / c mice were randomly divided into two groups of three each, and administered equal amounts (1 × 10⁻⁶ DIR dye) by gavage to each group. 8 DIR-labeled Bif suspension or DIR-labeled BLPD suspension (CFU / mouse) were administered via gavage. Four hours after gavage, the fluorescence distribution in the two groups of mice was observed and compared using a small animal in vivo imaging system.

[0087] The results are as follows Figure 6 As shown in B and C, the fluorescence intensity in the abdominal region of mice in the BLPD gavage group was significantly higher than that in the Bif group, approximately 1.9 times higher, indicating that BLPD has a superior retention capacity in vivo.

[0088] (4) Evaluation results of the therapeutic effect of oral administration of Bifidobacterium with lipid membrane reinforcement coating on ulcerative colitis in mice.

[0089] Twenty male BALB / c mice aged 6–8 weeks, weighing 22±2 g, were randomly divided into four groups of five mice each after one week of acclimatization: healthy control group (Ctrl), colitis model group (PBS), uncoated bacteria group (Bif), lipid-coated bacteria group (BL), and reinforced coated bacteria group (BLPD).

[0090] During the experiment, healthy control mice drank ordinary drinking water throughout the entire process; mice in the other groups were fed drinking water containing 3.5% sodium dextran sulfate (DSS, molecular weight 36,000–50,000) for 7 days to establish an ulcerative colitis model. After modeling, mice in each group were administered the corresponding interventions by gavage: healthy control and model groups were administered 100 μL of PBS by gavage, and the uncoated bacterial group was administered 100 μL of Bif suspension (1×10⁻⁶). 8 CFU / animal), lipid-coated bacterial group administered 100 μL BL suspension (1×10) by gavage. 8 CFU / animal), reinforced coating bacterial group was administered 100 μL BLPD suspension (1×10) by gavage. 8 CFU / animal), once daily for 5 consecutive days.

[0091] After the intervention, the mice were euthanized, and colon tissue was collected for histopathological staining and myeloperoxidase (MPO) immunohistochemical staining. Results are as follows: Figure 7 As shown in the HE staining results: In the healthy control group (Ctrl) mice, the colonic tissue structure was intact, the mucosa and basal layer were tightly connected, the crypt morphology and depth were normal, and the goblet cells were neatly arranged; In the model group (PBS) mice, the colonic tissue structure was severely damaged, the crypt structure was blurred, a large number of goblet cells were missing, and obvious inflammatory cell infiltration was visible; In the uncoated bacterial group (Bif) mice, the colonic crypt structure was significantly damaged, the tissue was loose overall, and the pathological damage was not significantly improved compared with the model group. This may be attributed to the extreme environment of the upper gastrointestinal tract, resulting in insufficient concentration of live bacteria reaching the colon, making it difficult to achieve the therapeutic effect. Due to the encapsulation of the lipid coating, BL can improve the tolerance of bacteria to the extreme environment of the upper gastrointestinal tract to a certain extent, but due to the instability of the lipid membrane, its protective effect is limited. Therefore, the colonic tissue structure of the lipid-coated group (BL) mice was slightly improved compared with the Bif group, but there was still obvious pathological damage overall. In contrast, the colonic mucosa, submucosa and muscular layer of the BLPD group mice were intact, the connections between the layers were tight, the inflammatory cell infiltration was significantly reduced, the number of goblet cells increased, and the intestinal morphology was significantly improved, which was closest to the healthy control group. MPO is a heme protein found in neutrophils. When stimulated by external inflammation, it can lead to neutrophil aggregation and the release of MPO. Therefore, MPO level is a recognized biomarker for characterizing the severity of inflammation and can be used to assess neutrophil infiltration. Immunohistochemical staining results for MPO showed that a large number of brown MPO-positive cells were observed in the colon tissue of the model group mice, indicating severe neutrophil infiltration. The number of positive cells in the Bif and BL groups was slightly reduced compared to the model group. The BLPD group mice showed the fewest MPO-positive cells in their colon tissue, with a significantly decreased MPO expression level.

[0092] The above results further confirm that encapsulating probiotics with a lipid-reinforced coating can significantly enhance the stability of the strains in the intestinal environment, ensuring their effective arrival in the intestine and performance of physiological functions, thereby alleviating colitis-related pathological damage. Among these, the BLPD treatment group was the most effective in reducing the inflammatory response in colitis mice.

[0093] 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 coating a bacterial surface, characterized in that, Includes the following steps: S1: Culture the bacteria to 10... 8 CFU / mL ~10 9 CFU / mL, collect bacterial cells by centrifugation, wash and use for later use; S2: Dissolve phospholipids, cholesterol, and Prönkel polymer in an organic solvent, mix well, and evaporate the solvent to obtain a lipid film; S3: The bacterial cells from step S1 are resuspended in CaCl2 solution and reacted for 10-30 min. The resulting bacterial solution is then transferred to the lipid film obtained in S2, and the reaction is continued for 10-30 min to obtain bacteria with a surface lipid film-reinforced coating.

2. The method according to claim 1, characterized in that, The bacteria mentioned in step S1 are probiotics.

3. The method according to claim 2, characterized in that, The bacteria mentioned in step S1 include at least one of lactobacillus, bifidobacteria, and Escherichia coli.

4. The method according to claim 1, characterized in that, The phospholipids mentioned in step S2 include at least one of dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, dioleoylphosphatidic acid, dioleoyltrimethylammonium propane, distearylphosphatidylcholine, and distearylphosphatidylethanolamine; The Plonic polymer mentioned in step S2 includes at least one of poloxamer 185, poloxamer 188, poloxamer 108, poloxamer 403, and poloxamer 407; The mass ratio of phospholipids, cholesterol and Pranic polymer in step S2 is (10-20):1:(70-100).

5. The method according to claim 4, characterized in that, The phospholipids mentioned in step S2 include at least one of dioleoylphosphatidylcholine and dioleoyltrimethylammonium propane; The Plunk polymer mentioned in step S2 includes poloxamer 407.

6. The method according to claim 5, characterized in that, The phospholipids, cholesterol, and Pronek polymer mentioned in step S2 are dioleoylphosphatidylcholine, dioleoyltrimethylammonium propane, cholesterol, and poloxamer 407. The mass ratio of dioleoylphosphatidylcholine, cholesterol, dioleoyltrimethylammonium propane, and poloxamer 407 is 10:1:3:

88.

7. The method according to claim 1, characterized in that, In step S3, the concentration of the CaCl2 solution was 12.5 mM, and the concentration of bacteria after resuspending was 10. 8 CFU / mL ~10 9 CFU / mL; In step S3, the ratio of bacterial cells to lipid membrane is 10:

1. 8 CFU~10 9 CFU: 8–10 mg.

8. A lipid membrane-reinforced coating probiotic, prepared by the method described in any one of claims 1 to 7.

9. The use of the lipid membrane-reinforced coating probiotics according to claim 8 in the preparation of intestinal flora regulation drugs.

10. The use of the lipid membrane-reinforced coating probiotic of claim 8 in the preparation of a medicament for treating enteritis.

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