Saccharomyces boulardii colon delivery system

Abstract

The invention discloses a saccharomyces boulardii colon delivery system which comprises the following steps: the survival rate in gastric juice is enhanced, and the residence time in the colon is increased, so that the treatment effect of the saccharomyces boulardii is enhanced. Saccharomyces boulardii is taken as a research object, in order to improve the bioavailability of the Saccharomyces boulardii in gastrointestinal tracts and prolong the adhesion time of bacteria in intestinal tracts, a layer-by-layer electrostatic self-assembly method is adopted, PDAD (poly dimethyl diallyl ammonium chloride), PSS (sodium polystyrene sulfonate), CS (chitosan) and SA (sodium alginate) are taken as biological membranes to coat the Saccharomyces boulardii, and the coated Saccharomyces boulardii is prepared. Then a series of characterization is carried out, and the effect of the delivery system is verified through in-vitro artificial gastric juice tolerance tests and mouse intestinal colonization.

Description

Technical Field

[0001] The invention discloses a Saccharomyces boulardii colon delivery system. Background Art

[0002] Saccharomyces boulardii ( Saccharomyces cerevisiae boulardii) is a unicellular fungus belonging to the genus Saccharomyces, a strain of the subspecies of Saccharomyces cerevisiae, and is the only yeast among probiotics. Saccharomyces boulardii can promote the proliferation and maturation of intestinal epithelial cells, making the villi longer and the crypts deeper, thereby improving the digestion and absorption capacity of nutrients; promoting the production of immunoglobulins (IgA) in the intestinal mucosa, building the first line of defense against pathogens. Saccharomyces boulardii strains secrete a 54kDa protease in animals. This enzyme can not only degrade Clostridium difficile ( C. difficile) secreted toxins A and B, and can also inhibit the binding of toxins to receptors on the brush border. Thus, the effects of enterotoxins and cytotoxins of Clostridium are reduced. The same effects are observed for toxins of molds and E. coli, and the harmful effects are reduced. The oligosaccharide structure on the surface of the Bladinidia yeast can adsorb pathogenic bacteria with flagella, such as E. coli, Salmonella, and the like. Bladinidia can significantly inhibit the excessive secretion of water and electrolytes (including chloride ions) caused by cholera toxin during infection with Vibrio cholerae. It has been found that a 120 kDa protease secreted by Bladinidia can affect the endothelial cells of the small intestine and large intestine, inhibit the activity of adenylate cyclase, and thus reduce the secretion of cAMP products and chloride from intestinal cells. During infection with E. coli, the phosphorylation of myoglobin light chains leads to the degradation of the tight junctions (mechanical barrier) between intestinal mucosal cells. Bladinidia can prevent this phosphorylation and reduce the permeability of intestinal mucosal cells, thereby reducing the transfer of pathogenic bacteria. Bladinidia can secrete polyamines (spermine and spermidine) in the mouse ileum. Polyamines can stimulate the maturation and metabolism of small intestinal cells. This helps the recovery of patients with diarrhea. IL-8 is a pro-inflammatory cytokine secreted during intestinal infection with E. coli. Bladinidia can reduce the secretion of IL-8 during E. coli infection; it has a protective effect in inflammatory bowel disease. Bladinidia can play a partial anti-infection role by regulating the phenotype, function, and migration of dendritic cells, inhibiting the response to bacterial antigens (LPS). Recent studies have shown that human bone marrow primary cells CD1c+CD11c+CD123-DC (mDC) cultured in supernatant culture medium containing Bladinidia significantly reduced the expression of microbial antigen lipopolysaccharide (LPS)-induced systemic stimulators CD40, CD80, and dendritic cell activation marker receptor CCR7. At the same time, the expression level of the anti-inflammatory factor IL-10 increased, while the key pro-inflammatory cytokines TNF-α and IL-6 significantly decreased. Ultimately, Bladinidia supernatant inhibited the proliferation of T cells through a mixed lymphocyte reaction. The nutritional effect on intestinal cells is manifested by increasing the levels of disaccharidases such as lactase, sucrose, maltase, fructase, and aminopeptidase in the small intestinal mucosa of humans and mice. More disaccharides can be broken down into monosaccharides and absorbed by intestinal cells into the blood circulation system. This is beneficial for the treatment of diarrhea, as the activity of enzymes is reduced during diarrhea, resulting in the inability to degrade and absorb carbohydrates. Bladinidia can induce the secretion of IgA in the small intestine of humans and mice, thereby improving the immunity of animals and building the first line of defense against pathogens.

[0003] In clinical practice, Bladinidia can improve allergic diarrhea and antibiotic diarrhea, but the number of yeast that reaches the intestinal tract to play a role is not enough due to the difficulty of yeast to survive in the highly acidic environment of gastric juice, and the treatment effect is greatly reduced. SUMMARY

[0004] The present application aims to provide a Saccharomyces boulardii colon delivery system to solve the problems in the background.

[0005] The present application provides the following technical solution: a Saccharomyces boulardii colon delivery system, characterized by comprising the following steps: First, prepare a four-layer material coated Saccharomyces boulardii colon delivery system, and characterize it by Zeta potential and transmission electron microscopy (TEM). In order to demonstrate the effect of different coating layers on the Saccharomyces boulardii delivery system, FDA staining and dilution spread plate method are used to determine the viability and viable bacterial count of the Saccharomyces boulardii colon delivery system. Second, analyze the in vivo and in vitro tolerance of the Saccharomyces boulardii colon delivery system. In vitro, perform artificial simulated gastric fluid (SGF) experiments and detect viable bacterial count. Use a small animal live imaging instrument to evaluate the retention of LY-SC-PPCS in the gastrointestinal tract in vivo.

[0006] Preferably, the Saccharomyces boulardii colon delivery system comprises Saccharomyces boulardii culture, Saccharomyces boulardii encapsulation, and Saccharomyces boulardii delivery system characterization.

[0007] Preferably, the Saccharomyces boulardii culture is as follows: take Saccharomyces boulardii glycerol bacteria from a -80℃ refrigerator, allow it to slightly thaw, take an appropriate amount to a shake flask containing 50 mL of culture medium in a clean bench, incubate overnight in a 30℃, 220 rpm shaker, and take it out when the OD value at 600 nm is about 3-5. After centrifugation, transfer it to a shake flask containing 50 mL of high-sugar YPD medium and ferment for 52 hours (calculate the amount to be taken out based on the OD value so that the initial OD value in the shake flask is 0.5. After fermentation, detect the OD value at 600 nm. According to the OD value result, take an appropriate amount of bacterial solution, wash it 2-3 times with 0.9% physiological saline, centrifuge and discard the supernatant, resuspend it with 0.9% physiological saline to make the bacterial concentration of the bacterial solution 10 9 CFU / mL for intragastric administration to mice.

[0008] Preferably, the encapsulation of the B. subtilis is by dissolving PDAD, PSS, SA in 0.15 M NaCl to form a solution of 2 mg / mL (PDAD, PSS) and 3 mg / mL SA respectively, dissolving CS in 1 % acetic acid and adjusting the pH to 5 with 1 % NaOH to form a solution of 2 mg / mL (pH 5.0) CS, dispersing B. subtilis in NaCl (0.15 M) solution and washing with NaCl (0.15 M) solution three times, then resuspending B. subtilis in PDAD and PSS solution sequentially for 15 min to get two-layered coated B. subtilis, followed by resuspending B. subtilis in CS solution and SA solution sequentially for 20 min to get four-layered coated B. subtilis. The washing step is always followed after each resuspension incubation.

[0009] Preferably, the characterization of the B. subtilis delivery system includes Zeta potential measurement, transmission electron microscopy (TEM), viability assay and in vitro and in vivo tolerance analysis of the B. subtilis delivery system.

[0010] Preferably, the Zeta potential measurement is by diluting and redispersing the different layer-encapsulated B. subtilis delivery system in deionized water, then determining the potential on the surface of the different layer-encapsulated B. subtilis delivery system by measuring electrophoretic light scattering using Zetasizer Nano-Sizer 90 (Malvern Instruments, Malvern, UK).

[0011] Preferably, the transmission electron microscopy (TEM) is by using low-speed centrifugation (3 min, 2000 x g) to precipitate the yeast cells, and immediately fixing at 4 °C in 2.5 % (v / v) glutaraldehyde for 12-24 h, then fixing the sample with 1 % osmium acid solution for 1-2 h, carefully removing the osmium acid waste liquid, rinsing with 0.1 M phosphate buffer PB (pH 7.4) for 3 times, 15 min each time, dehydrating the tissue sample in 30 %, 50 %, 70 %, 80 %, 95 %, 100 % ethanol sequentially, 10 min each time, and dehydrating twice in 100 % acetone, 20 min each time, after the infiltration embedding treatment, using an ultramicrotome to cut the resin block of the embedded sample into 70-90 nm ultrathin sections, then using a copper mesh to slice, staining the sections on the mesh with uranyl acetate and lead citrate sequentially, observing the morphology under a transmission electron microscope after drying, and collecting images for analysis.

[0012] Preferably, the viability assay includes FDA staining method to determine the bacterial viability and plate counting method to quantify the number of viable bacteria.

[0013] Preferably, the FDA staining method for determining bacterial viability is to study the viability of yeast cells by FDA assay, in which FDA is hydrolyzed by esterase in metabolically active cells to green fluorescent fluorescein, since FDA is insoluble in water, therefore, FDA stock solution (10 mg / mL) is prepared by dissolving FDA in acetone, 2 μL of stock solution is mixed with 0.5 mL of yeast suspension (phosphate buffer: 50 mM, pH 6.5), after 30 min of shaking incubation, the cells are washed with 0.15 M NaCl aqueous solution for three times, and then characterized by confocal laser scanning microscopy.

[0014] Preferably, the plate counting method for quantifying the number of viable bacteria is to determine the number of viable bacteria after different layers of encapsulation by plate counting, the appropriate dilution multiple is selected, and the bacterial solution is serially diluted with sterile water, 20 μL of bacteria from different encapsulation layers is coated on YPD solid agar plates, which are cultured in a 30 °C microbiological incubator for 48 h, and then the number of colonies on each plate is counted, and the CFU count is determined by the number of colonies, the dilution ratio and the volume of plate dilution.

[0015] Preferably, the in vitro and in vivo tolerance analysis of the Saccharomyces boulardii delivery system includes determination of the number of viable bacteria after treatment with artificial gastric juice, observation of bacterial morphology, and evaluation of gastrointestinal retention in vivo.

[0016] Preferably, the determination of the number of viable bacteria after treatment with artificial gastric juice is to quantitatively analyze the viability of Saccharomyces boulardii and Saccharomyces boulardii delivery system using dilution and coating plate counting method, and the density of 1 × 10 6 CFU. The cells of Saccharomyces boulardii and Saccharomyces boulardii delivery system are collected and suspended in 5 mL of SGF, which is prepared according to the Chinese Pharmacopoeia, and then incubated in a shaking water bath at 220 rpm at 37 °C, samples are collected at 0 h, 0.5 h, 1 h, 1.5 h and 2 h, centrifuged and washed, and the number of bacteria is determined by serial dilution of the sample with sterile water, coating the diluted sample on YPD solid medium, and counting the number of colonies after incubation in a microbiological incubator (30 °C) for 48 h.

[0017] Preferably, the observation of bacterial morphology is to study the morphological changes of LY-SC and LY-SC-PPCS after exposure in SGF by transmission electron microscopy (TEM) imaging technology and biological scanning electron microscopy (SEM) imaging technology. Specifically, Saccharomyces boulardii and Saccharomyces boulardii delivery system are suspended in SGF and incubated for 4 h, the SGF is removed by centrifugation, washed 3 times, fixed in 2.5% glutaraldehyde, and then photographed by transmission electron microscopy and biological scanning electron microscopy after a series of treatments such as gradient dehydration.

[0018] Preferably, the evaluation of the in vivo gastrointestinal retention is for further determining the survival of B. longum and B. longum delivery system in the digestive tract, female C57 BL / 6 mice are orally administered with B. longum and B. longum delivery system (1x10 8 CFU), at 4 h and 8 h, the mice and their gastrointestinal tracts are imaged by an in vivo IVIS imaging system (PerkinElmer, Lumina III) for automatic exposure model, the bioluminescence signal of the region of interest is quantified by IVIS Living Image 4.2 software, and is expressed by average radiation rate, unit: photon per second per cm 2 per sphere (p / s / cm 2 / sr).

[0019] Preferably, the statistical analysis is that the number of repeated tests / animals (n) used in each group represents biological repeated tests, all well plate combined studies are independently performed for 3 times, all dilutions for CFU analysis are plated in technical parallel, samples for ELISA are run in technical duplicate, all statistical and data distribution analysis are performed by Prism (GraphPad), all experimental results are expressed as mean ± standard deviation (SD), and statistical significance is expressed as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0020] The present application has the beneficial effects of: enhancing the survival rate in gastric juice and increasing the residence time in the colon, so as to enhance the treatment effect of B. longum. Taking B. longum as the research object, in order to improve the bioavailability of B. longum in the gastrointestinal tract and prolong the adhesion time of the bacteria in the intestinal tract. The B. longum is coated with a biological membrane by using the method of layer-by-layer electrostatic self-assembly, and polydimethyl diallyl ammonium chloride (PDAD), sodium polystyrene sulfonate (PSS), chitosan (CS) and sodium alginate (SA) are used as the biological membrane. Then a series of characterizations are performed, and the effect of the delivery system is verified through in vitro artificial gastric juice tolerance test and mouse intestinal colonization. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 (A) Zeta potential of different layers during encapsulation Figure 2 (B) Representative TEM images of B. longum delivery and B. longum delivery system. Scale bar; 1 μm Figure 3 (A) Representative images of LY-SC inverted fluorescence microscope after FDA staining of different encapsulation layers Figure 4For (B) Fluorescent quantification. Scale bar: 50 pm. (C). Quantification of viable LY-SC encapsulated in different layers; Figures 5-6 For (A-B) Representative SEM and TEM images of LY-SC and LY-SC-PPCS after 2 hours of exposure to SGF at 37 °C. Scale bar: A top: 3 pm bottom: 1 pm; B top: 5 pm bottom: 1 pm; Figure 7 For (C) Quantification of viability of LY-SC and LY-SC-PPCS exposed to simulated gastric fluid (SGF) for different times; Figure 8 For (A) Representative inverted fluorescence microscopy images of LY-SC with different encapsulation layers after FDA staining; Figure 9 For (B) Quantitative evaluation of bioluminescent signal of B. brayana and B. brayana delivery system in mice for 4 hours and 8 hours; DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the protection scope of the present application.

[0023] Please refer to Figures 1-9 The technical solutions provided by the present application are as follows: the B. brayana colon delivery system of the present application comprises the following two steps: First, a four-layer material coated B. brayana colon delivery system is prepared, and Zeta potential and transmission electron microscopy (TEM) are used to characterize the B. brayana colon delivery system. In order to prove the influence of different coating layers on the B. brayana delivery system, FDA staining and dilution coating plate method are used to determine the viability and viable bacterial count of the B. brayana colon delivery system.

[0024] Second, the in vitro and in vivo tolerance of the B. brayana colon delivery system is analyzed. In vitro, the artificial simulated gastric fluid (SGF) experiment is carried out, and the viable bacterial count is detected. The retention of LY-SC-PPCS in the gastrointestinal tract in vivo is evaluated by a small animal live imaging instrument.

[0025] Specifically, the B. brayana colon delivery system comprises B. brayana culture, B. brayana encapsulation, B. brayana delivery system characterization, bacterial viability determination, in vitro and in vivo tolerance analysis of B. brayana delivery system, and statistics.

[0026] 1. Culture of Saccharomyces boulardii: Take out the glycerol stock of Saccharomyces boulardii from the -80℃ freezer, wait for it to thaw slightly, and take an appropriate amount into a shake flask containing 50 mL of culture medium in a clean bench. Culture it in a shaker at 30℃ and 220 rpm overnight. When the OD value at 600nm is about 3-5, take it out, centrifuge it, and transfer it to a shake flask containing 50 mL of high-glucose YPD medium for fermentation for 52 hours (calculate the amount of takeout based on the OD value so that the initial OD value in the shake flask is 0.5). After fermentation is complete, measure the OD value at 600nm, take out an appropriate amount of bacterial solution based on the OD value, wash it with 0.9% saline 2-3 times, centrifuge and discard the supernatant, and resuspend it with 0.9% saline to a bacterial concentration of 10 9 CFU / mL, used for oral administration to mice. 2. Encapsulation of Saccharomyces boulardii: Prior to encapsulation, PDAD, PSS, and SA were dissolved in 0.15 M NaCl to form solutions with final concentrations of 2 mg / mL (PDAD, PSS) and 3 mg / mL SA, respectively. CS was dissolved in 1% acetic acid and the pH was adjusted to 5 with 1% NaOH to form a 2 mg / mL (pH 5.0) CS solution. First, Saccharomyces boulardii was dispersed in a 0.15 M NaCl solution and washed three times with a 0.15 M NaCl solution. The Saccharomyces boulardii was then resuspended in PDAD and PSS solutions, followed by incubation for 15 minutes to obtain a double-layer coating. Subsequently, the Saccharomyces boulardii was resuspended in CS and SA solutions, followed by incubation for 20 minutes to obtain a quadruple-layer coating. Washing steps were always performed after each resuspension and incubation step.

[0027] 3 Characterization of the Saccharomyces boulardii Delivery System 3.1 Zeta Potential Measurement: The Saccharomyces boulardii delivery systems with different numbers of encapsulated layers were diluted and redispersed in deionized water, and the potential on the surface of the Saccharomyces boulardii delivery systems with different encapsulated layers was determined by measuring electrophoretic light scattering using a ZetaSizer Nano-Sizer 90 (Malvern Instruments, Malvern, UK).

[0028] 3.2, Transmission electron microscopy (TEM): Yeast cells were pelleted by low-speed centrifugation (3 min, 2000 x g) and immediately fixed in 2.5% (v / v) glutaraldehyde at 4 °C for 12-24 h. Then the samples were fixed with 1% osmium tetroxide solution for 1-2 h. The osmium waste liquid was carefully removed, and the tissue samples were rinsed with 0.1 M phosphate buffer PB (pH 7.4) for 3 times, 15 min each time. The tissue samples were dehydrated in 30%, 50%, 70%, 80%, 95%, 100% ethanol, respectively, for 10 min each time, and finally dehydrated in 100% acetone for 20 min twice. After the infiltration embedding treatment, the resin blocks of the embedded samples were cut into 70-90 nm ultrathin sections by an ultramicrotome, and then the sections were collected on copper grids. The sections on the grid were stained with uranyl acetate and lead citrate, respectively, and then observed under a transmission electron microscope after drying, and images were collected for analysis.

[0029] 3.3, Viability assay: 3.31, FDA staining method for determining the viability of bacteria: The viability of yeast cells was studied by FDA assay, in which FDA was hydrolyzed to green fluorescent fluorescein by esterase in metabolically active cells. Since FDA is not soluble in water, FDA stock solution (10 mg / mL) was prepared by dissolving FDA in acetone. 2 μL of stock solution was mixed with 0.5 mL of yeast suspension (phosphate buffer: 50 mM, pH 6.5). After 30 min of incubation with shaking, the cells were washed with 0.15 M NaCl aqueous solution for three times, and then characterized by confocal laser scanning microscopy.

[0030] 3.32, Plate counting method for quantifying the number of viable bacteria: In order to quantitatively determine the number of viable bacteria after different layer encapsulation, plate counting was used. The appropriate dilution multiple was selected to continuously dilute the bacterial solution with sterile water, and 20 μL of bacteria from different encapsulation layers were spread on YPD solid agar plates, which were then cultured in a 30 °C microbial incubator for 48 h, and then the number of colonies on each plate was counted. The CFU count was determined by the number of colonies, the dilution ratio, and the volume of plate dilution.

[0031] 3.4, In vitro and in vivo tolerance analysis of B. subtilis delivery system: 3.41, Determination of viable bacteria after treatment with artificial gastric juice: The viability of B. subtilis and B. subtilis delivery system was quantitatively analyzed by dilution and spread plate counting method. Briefly, 1 x 10 6CFU density. C. bradei and C. bradei delivery system cells were collected and suspended in 5 mL of SGF, prepared according to the Chinese Pharmacopoeia. Then incubated in a shaking water bath at 37 °C with 220 rpm. Samples were collected at 0 h, 0.5 h, 1 h, 1.5 h, and 2 h, centrifuged, washed, and serially diluted by combining the samples with sterile water. Diluted samples were plated on YPD solid medium, and the number of colonies was determined by counting the colonies after incubation in a microbial incubator (30 °C) for 48 h.

[0032] 3.42, Morphology observation: Transmission electron microscopy (TEM) imaging technology and biological scanning microscope (SEM) imaging technology were applied to study the morphological changes of LY-SC and LY-SC-PPCS after exposure in SGF. Specifically, C. bradei and C. bradei delivery system were suspended in SGF and incubated for 4 h, then centrifuged to remove SGF, washed 3 times, and then placed in 2.5 % glutaraldehyde for fixation, followed by a series of gradient dehydration treatments, and then taken by transmission electron microscopy and biological scanning electron microscopy.

[0033] 3.43, Evaluation of in vivo gastrointestinal retention: To further determine the survival of C. bradei and C. bradei delivery system in the digestive tract, female C57 BL / 6 mice were orally administered C. bradei and C. bradei delivery system (1 x 10 8 CFU), and at 4 h and 8 h, the mice and their gastrointestinal tracts were imaged by the in vivo IVIS imaging system (PerkinElmer, Lumina III) for automatic exposure model imaging. The bioluminescence signal of the region of interest was quantified by IVIS Living Image 4.2 software, and was expressed by the average radiation rate, with units of photons per second per cm 2 per steradian (p / s / cm 2 / sr).

[0034] 3.5, Statistical analysis: The number of repeated experiments / animals (n) used in each group is shown in the figure legend, representing biological replicates. All well-plate combined studies were performed independently 3 times. All dilutions for CFU analysis were plated in technical replicates. Samples for ELISA were run in technical duplicates. All statistical and data distribution analyses were performed using Prism (GraphPad). All experimental results are expressed as mean ± standard deviation (SD). Statistical significance is represented as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0035] 3.6, Experimental results and analysis: 3.61 Characterization of Saccharomyces boulardii and Saccharomyces boulardii Delivery Systems: High colonic accumulation is a key indicator of the potential effectiveness of oral Saccharomyces boulardii delivery systems. Oral administration is a preferred route of administration due to its favorable patient compliance and cost-effectiveness. However, due to the complex and harsh gastrointestinal environment, probiotics are easily inactivated by strong acid and digestive enzymes, significantly impacting their effectiveness in the colon. To address this issue, in this study, live Saccharomyces boulardii was encapsulated in four different materials: PDAD, PSS, chitosan, and sodium alginate. This was achieved using electrostatic LbL assembly technology. PDAD and PSS were chosen as the first two layers to prevent direct contact of chitosan with the Saccharomyces boulardii cell wall, potentially affecting bacterial activity due to its antimicrobial properties. The chitosan was then coated with sodium alginate, and the two cross-linked to form a networked, eggshell-like structure with excellent stability. Furthermore, the negative charge of sodium alginate facilitates its accumulation at sites of inflammation. The presence of negatively charged phosphate groups on the yeast cell wall contributes to its overall negative surface charge. Based on the report that yeast cell surface is negatively charged, the LbL process starts with positively charged PDAD. The successful deposition of the material was confirmed by measuring the zeta potential. Zeta potential is used to characterize the kinetic potential, which reflects the charge on the bacterial surface. Figure 1 As shown, the zeta potential of the S. boulardii delivery system increased from -16.4 mV to 36.07 mV due to successful coating with the cationic polymer PDAD, and decreased to -33.75 mV due to effective coating with the anionic polymer PSS. Further addition of chitosan shifted the zeta potential of the S. boulardii delivery system back to 38.5 mV. Finally, further coating with sodium alginate again resulted in a negative zeta potential (-36 mV). As previously reported, the alternating negative and positive potentials of each polyelectrolyte layer deposition confirmed LbL self-assembly on the bacterial surface. Similarly, to further visualize the successful deposition and morphology of the materials, transmission electron microscopy (TEM) imaging was used to observe the morphological changes of the bacteria. Figure 2 As shown, the cell wall surface of natural yeast cells appears smooth and featureless. However, the surface of the Saccharomyces boulardii delivery system coated with four layers of material is significantly rougher and thicker, containing a large amount of granular material, indicating that the material has been successfully deposited on the surface of the bacteria.

[0036] 3.62. Viability test of bacteria after encapsulation procedure: After verifying that the Saccharomyces boulardii delivery system was successfully encapsulated, FDA staining was further used to study the viability of Saccharomyces boulardii. FDA is a fat-soluble dye that can quickly enter the cell through the cell membrane outside the cell. Inside the cell, FDA is hydrolyzed by intracellular esterases into fluorescein, which is a strong fluorescent dye that accumulates inside the cell. The amount of intracellular fluorescein accumulation is related to the viability of the cell and the activity of active esterases: the higher the fluorescence intensity, the stronger the ability of the intracellular active esterase to hydrolyze FDA, and the higher the cell viability. The viability test showed that the encapsulation of the four-layer material is compatible with the delivery of live Saccharomyces boulardii. By observing and capturing images under an inverted fluorescence microscope ( Figure 3 ), fluorescence quantitative analysis was performed using Image J software ( Figure 4 ). The viability of natural yeast is referenced to 1, and its fluorescence intensity decreases as the number of encapsulated layers increases. The calculated fluorescence intensity of the Saccharomyces boulardii delivery system S is 0.69, a decrease of 0.31. The decreased fluorescence intensity indicates a decrease in bacterial viability, but we believe that this is a sufficiently high survival rate for bacterial encapsulation. To quantify the number of viable bacteria remaining after encapsulation, dilution plates were used for counting. Figure 5 As shown in the figure, after four encapsulation steps, the number of viable cells increased from 2×10 6 reduced to 1.3×10 6 , a 35% decrease. This result is consistent with the FDA staining fluorescence intensity analysis, indicating that the initial survival rate after the encapsulation process is approximately 70%, further demonstrating the biosafety and compatibility of the encapsulation material.

[0037] 3.63. Analysis of in vitro tolerance of bacteria after encapsulation: As an oral Saccharomyces boulardii delivery system, the ability to accumulate at high levels in the colon is an important indicator for evaluating its potential effectiveness. Oral administration is considered a preferred route of administration due to its good patient compliance and economic benefits. However, due to the complex and harsh gastrointestinal environment, probiotics are easily inactivated in the presence of strong acid and digestive enzymes, which greatly affects the effectiveness of engineered bacteria in the colon. To solve this problem, we encapsulated probiotics in safe and non-toxic materials to resist the invasion of gastric acid. In order to verify its anti-gastric acid effect, we first conducted an in vitro simulated artificial gastric fluid (SGF) experiment. Transmission electron microscopy (TEM) and biological scanning electron microscopy (SEM) imaging techniques were used to study the morphological changes of bacteria after exposure to digestive tract solutions. Figure 5 and 6After 2h of SGF incubation, the morphology of B. subtilis delivery system remained completely intact, while the uncoated B. subtilis exhibited a damaged cell wall, indicating the excellent protective effect of the four-layer material coating. According to the literature, the protective effect of the material on the bacteria can be related to the formation of an insoluble sodium alginate skin at the end of sodium alginate. In order to further quantify the survival rate of bacteria in SGF, B. subtilis and B. subtilis delivery system were first incubated in SGF solution. And every 0.5 h dilution spread plate count. As shown in Figure 7 Figure 2, the number of viable B. subtilis decreased by 100-fold / ml within 2h of exposure to SGF, while the number of viable B. subtilis delivery system decreased by only 10-fold / ml. Further indicating the protective effect of the material's encapsulation on probiotics in the gastrointestinal environment. The protective effect of the material on the bacteria can be related to the formation of an insoluble sodium alginate skin at the end of sodium alginate.

[0038] 3.64, In vivo gastrointestinal retention of B. subtilis delivery system: Inspired by the high stability of B. subtilis delivery system in SGF, in order to track the in vivo distribution of B. subtilis delivery system in mice, we used a small animal live imaging system to visualize its distribution in mice. Live imaging technology can qualitatively and quantitatively study biological processes at the tissue, cell and molecular levels in the living state without damaging the animal. Fluorescent proteins can be used to label tumor cells (to observe tumor development), and fluorescent dyes or quantum dots can be used to label polypeptides, antibodies or drugs to observe their distribution in the body and their targeting of tumors. This technology plays an important role in the fields of life sciences and pharmaceutical research.

[0039] The B. subtilis delivery of the present application was labeled with mCherry fluorescent protein, and the luminescent signal was monitored to study the distribution and retention of B. subtilis and B. subtilis delivery system in the gastrointestinal tract. Two groups of mice were gavaged with 1x10 8 CFU of B. subtilis or B. subtilis delivery system, respectively, and the luminescent signal from the probiotics was monitored by in vivo imaging system (IVIS) at 4h and 8h. Figures 8-9 As shown in Figure 3, after 4h of gavage, the luminescent signal from the B. subtilis delivery system was 20 times higher than that from the B. subtilis; and after 8h, the luminescent signal from the B. subtilis delivery system was still 4 times higher than that from the B. subtilis delivery system, and the direct imaging results of the gastrointestinal tract also confirmed this result. This is likely due to the good mucosal adhesion of the two polysaccharides, which prolongs the residence time in the intestine. This further reveals that after being encapsulated by the material, the retention time of B. subtilis delivery in the gastrointestinal tract is prolonged, and the survival rate is greatly improved.

[0040] Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified, or some of the technical features can be replaced by equivalent features, by those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Saccharomyces boulardii colon delivery system, characterized in that: The method includes the following steps: In the first step, a four-layer coated Saccharomyces boulardii colon delivery system was prepared and characterized by zeta potential and transmission electron microscopy (TEM). To demonstrate the effects of different coatings on the Saccharomyces boulardii colon delivery system, FDA staining and dilution plate methods were used to determine the viability and number of surviving bacteria in the Saccharomyces boulardii colon delivery system. The second step was to analyze the in vivo and in vitro tolerance of the Saccharomyces boulardii colon delivery system. Simulated gastric fluid (SGF) experiments were conducted in vitro, and the number of viable bacteria was detected. In vivo gastrointestinal retention of LY-SC-PPCS was evaluated using a small animal in vivo imaging device. The Saccharomyces boulardii colon delivery system includes culturing Saccharomyces boulardii, encapsulating Saccharomyces boulardii, and characterizing the Saccharomyces boulardii delivery system; The statistical analysis was performed using the number of replicates / animals (n) used in each group, representing biological replicates. All plate binding studies were performed independently three times. All dilutions used for CFU analysis were plated in technical replicates. Samples used for ELISA were run in technical duplicates. All statistical and data distribution analyses were performed using Prism (GraphPad). All experimental results are expressed as mean ± standard deviation (SD). Statistical significance is indicated as *P < 0.05, **P < 0.01, *P < 0.001, and *P < 0.0001.

2. The boulardii colon delivery system according to claim 1, characterized in that The boulardii yeast culture was performed by taking out the boulardii yeast glycerol bacteria from a -80°C refrigerator, slightly thawing it, taking an appropriate amount into a shake flask containing 50 mL of culture medium in a clean bench, and culturing it overnight in a shaker at 30°C and 220 rpm. When the OD value at 600 nm was about 3-5, the culture was taken out, centrifuged, and transferred to a shake flask containing 50 mL of high-glucose YPD medium and fermented for 52 hours (the amount taken out was calculated based on the OD value so that the initial OD value in the shake flask was 0.

5. After fermentation was completed, the OD value was detected at 600 nm. According to the OD value result, an appropriate amount of bacterial solution was taken out, washed 2-3 times with 0.9% saline, centrifuged and discarded the supernatant, and resuspended with 0.9% saline to a bacterial concentration of 10 9 CFU / mL, used for oral administration to mice.

3. The Saccharomyces boulardii colon delivery system according to claim 1, characterized in that: The encapsulation of the Saccharomyces boulardii is as follows: before encapsulation, PDAD, PSS, and SA are dissolved in 0.15 M NaCl to form solutions with final concentrations of 2 mg / mL (PDAD, PSS) and 3 mg / mL SA, respectively; CS is dissolved in 1% acetic acid and the pH is adjusted to 5 with 1% NaOH to form a 2 mg / mL (pH 5.0) CS solution; first, the Saccharomyces boulardii is dispersed in a NaCl (0.15 M) solution and washed three times with a NaCl (0.15 M) solution; then, the Saccharomyces boulardii is resuspended in PDAD and PSS solutions in sequence and incubated for 15 minutes to obtain a two-layer coated Saccharomyces boulardii; then, the Saccharomyces boulardii is resuspended in a CS solution and an SA solution in sequence and incubated for 20 minutes to obtain a four-layer coated Saccharomyces boulardii; and washing steps are always performed after each resuspension and incubation step.

4. The Saccharomyces boulardii colon delivery system according to claim 1, characterized in that: The characterization of the Saccharomyces boulardii delivery system included zeta potential measurement, transmission electron microscopy (TEM), bacterial viability assay, and in vitro and in vivo tolerance analysis of the Saccharomyces boulardii delivery system.

5. The Saccharomyces boulardii colon delivery system according to claim 4, characterized in that: The zeta potential was measured by diluting and redispersing the Saccharomyces boulardii delivery systems with different numbers of encapsulated layers in deionized water, and then measuring the electrophoretic light scattering using a ZetaSizer Nano-Sizer 90 (Malvern Instruments, Malvern, UK) to determine the potential on the surface of the Saccharomyces boulardii delivery systems with different encapsulated layers.

6. The Saccharomyces boulardii colon delivery system according to claim 4, characterized in that: The transmission electron microscopy (TEM) method is to use low-speed centrifugation (3 min, 2000 × g) to precipitate yeast cells, and immediately fix them in 2.5% (v / v) glutaraldehyde at 4°C for 12-24 h, then fix the samples with 1% osmium acid solution for 1-2 h, carefully remove the osmium acid waste solution, rinse three times with 0.1M phosphate buffer PB (pH 7.4), each for 15 min, dehydrate the tissue samples in 30%, 50%, 70%, 80%, 95%, and 100% ethanol in sequence, each with an interval of 10 min, and finally dehydrate them twice with 100% acetone, each for 20 min. After infiltration and embedding, the resin block of the embedded sample is cut into 70-90 nm ultrathin sections using an ultrathin microtome, and then the sections are scooped out with a copper mesh. The sections on the grid are stained with uranyl acetate and lead citrate in sequence. After drying, the morphology is observed under a transmission electron microscope, and image analysis is collected.

7. The Saccharomyces boulardii colon delivery system according to claim 4, characterized in that: The bacterial viability assay includes an FDA staining method for determining bacterial viability and a plate count method for quantifying viable bacterial counts. The FDA staining method for determining bacterial viability studies yeast cell viability using the FDA assay, wherein FDA is hydrolyzed by esterases in metabolically active cells into green fluorescent fluorescein. Since FDA is insoluble in water, FDA is dissolved in acetone to prepare an FDA stock solution (10 mg / mL). 2 μL of the stock solution is mixed with 0.5 mL of yeast suspension (phosphate buffer: 50 mM, pH 6.5). After shaking and incubating for 30 minutes, the cells are washed three times with a 0.15 M NaCl aqueous solution and then characterized by confocal laser scanning microscopy. The plate count method for quantifying viable bacterial counts is to quantify the number of viable bacteria after different layers of encapsulation. The plate count method is used to determine the number of viable bacteria after different layers of encapsulation. The plate count method is used to determine the number of viable bacteria. The bacterial solution is serially diluted with sterile water at appropriate dilution multiples. 20 μL of bacteria from different encapsulated layers are spread on YPD solid agar plates and incubated in a 30°C microbial incubator for 48 hours. The number of colonies on each plate is then counted, and the CFU count is determined based on the number of colonies, dilution ratio, and plate dilution volume.

8. The Saccharomyces boulardii colon delivery system according to claim 4, characterized in that: The in vitro and in vivo tolerance analysis of the Saccharomyces boulardii delivery system includes determination of viable bacterial count after treatment with artificial gastric juice, observation of bacterial morphology, and evaluation of in vivo gastrointestinal retention.

9. The Saccharomyces boulardii colon delivery system according to claim 8, characterized in that: The viable bacterial count after artificial gastric juice treatment was determined by using the dilution plate count method to quantitatively analyze the activity of Saccharomyces boulardii and the Saccharomyces boulardii delivery system. 6 The density of CFU was calculated by collecting cells of Saccharomyces boulardii and the Saccharomyces boulardii delivery system and suspending them in 5 mL of SGF, which was prepared according to the Chinese Pharmacopoeia. The cells were then incubated at 37°C in a shaking water bath at 220 rpm. The samples were collected at 0 h, 0.5 h, 1 h, 1.5 h, and 2 h, washed by centrifugation, and serially diluted by combining the samples with sterile water. The diluted samples were spread on YPD solid medium, and the bacterial count was determined by counting the colonies after 48 h of incubation in a microbial incubator (30°C).

10. The Saccharomyces boulardii colon delivery system according to claim 8, characterized in that: The bacterial morphology observation is to use transmission electron microscopy (TEM) imaging technology and biological scanning microscopy (SEM) imaging technology to study the morphological changes of LY-SC and LY-SC-PPCS after exposure to SGF. Specifically, Saccharomyces boulardii and the Saccharomyces boulardii delivery system were suspended in SGF and incubated for 4 hours, and then the SGF was removed by centrifugation. After washing three times, they were fixed with 2.5% glutaraldehyde and subjected to a series of treatments such as gradient dehydration, and then photographed by transmission electron microscopy and biological scanning electron microscopy. The in vivo gastrointestinal retention evaluation is to further determine the survival of Saccharomyces boulardii and the Saccharomyces boulardii delivery system in the digestive tract. Female C57 BL / 6 mice were orally administered Saccharomyces boulardii and the Saccharomyces boulardii delivery system (1×10 8 CFU), mice and their gastrointestinal tract were imaged using an in vivo IVIS imaging system (PerkinElmer, Lumina III) at 4 and 8 h of exposure. The bioluminescent signals of the regions of interest were quantified using IVIS Living Image 4.2 software and expressed as the average radiance in photons per second per cm 2 per steradian (p / s / cm 2 / sr).

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