Colon-targeting probiotic microspheres based on pure biofilm self-assembly and application thereof

Abstract

The application discloses a kind of colon-targeted probiotic microspheres based on pure biofilm self-assembly and application thereof, and belongs to the field of microorganism and food bioengineering technology.The pure biofilm self-assembly microspheres prepared by the application present a dense and porous three-dimensional sponge-like network structure.While having excellent hardness, it can resist the erosion of gastric acid and small intestinal juice, thereby effectively protecting the embedded probiotics from maintaining activity before reaching the colon.In the stomach and small intestine stage, the microsphere structure remains intact, avoiding the premature release of probiotics.After entering the colon, the microsphere structure disintegrates, achieving targeted release of probiotics, with a release rate of 98.1% and a survival rate of 91.2%.In addition, the microspheres are formed by self-assembly of pure biofilm without additional synthetic scaffold materials, have excellent biocompatibility, and no additional safety risks.Therefore, the microspheres are suitable as oral delivery carriers for probiotics and can be applied in the fields of food and medicine.

Description

Technical Field

[0001] This invention relates to a colon-targeting probiotic microsphere based on pure biomembrane self-assembly and its application, belonging to the field of microbial and food bioengineering technology. Background Technology

[0002] Currently, targeted delivery of probiotics mainly relies on physical encapsulation with macromolecular polymers such as sodium alginate, which is a common and traditional method. However, this traditional delivery system often only considers the stable encapsulation of the strain in practical applications, neglecting issues such as the passive inhibition of strain activity, insufficient microsphere release precision, and colonization failure. Current physical encapsulation technologies are highly dependent on exogenous chemical or physical wall materials, which causes the encapsulated probiotics to be in a state similar to "dormancy" or "passive defense" for a long time. In this state, the biological activity of probiotics, which naturally secrete beneficial metabolites (such as extracellular polymeric substances like EPS), is significantly inhibited, preventing them from fully exerting their expected probiotic functions. At the same time, traditional microsphere carriers based on polymer materials are prone to non-specific swelling or premature degradation when subjected to the highly acidic environment of gastric juice and the complex effects of bile salts in intestinal juice. This phenomenon directly leads to leakage of probiotics before reaching the target site (such as the colon), making it difficult for the system to achieve precise, site-specific release based on the specific enzyme environment of the colon, thereby reducing the targeting and efficiency of delivery. When exogenous wall materials degrade at the target site in the intestine, the released probiotics are often in a free state. Due to the lack of spatial network protection of their own biofilm, the free strains are unable to resist the physical clearance of intestinal peristalsis, and their specific adhesion to intestinal mucin is weak, making it impossible to form long-term effective intestinal colonization, which affects the potential of probiotics to exert their functions for a long time.

[0003] In summary, these limitations collectively restrict the effectiveness and application prospects of traditional physical embedding delivery systems. Summary of the Invention

[0004] The primary objective of this invention is to provide a probiotic microsphere (SBM) based on pure biomembrane self-assembly and its preparation process. The probiotic microsphere process of this invention breaks through the limitation of traditional physical encapsulation of probiotics, which is highly dependent on exogenous wall materials. SBMs completely remove the exogenous framework, not only preserving almost all the biological activity of probiotics, but also being able to withstand extreme environments and target adhesion to the intestinal mucosa under the protection of a large amount of extracellular polymer (EPS) matrix induced in situ.

[0005] Another objective of this invention is to provide a sacrificial template method for preparing the pure biomembrane microspheres. This method is highly universal and adjustable, supporting the introduction of prebiotics to construct a "synbiotic" microenvironment. Simultaneously, it provides oral formulations or food compositions containing these microspheres to achieve the dual effects of "high-survival-rate delivery" and "endogenous targeted colonization" of probiotics. Furthermore, during in-situ induced self-assembly, by adding substrates or precursors, the strains can utilize their own biosynthetic pathways to synthesize products such as ceramides.

[0006] This invention is not a simple physical mixture of probiotics and exogenous prebiotics (such as inulin). During in situ induction, the dense EPS three-dimensional network secreted by probiotics achieves molecular-level in situ embedding of prebiotic molecules. After this structure disintegrates in the colonic environment, it provides the released bacteria with a zero-distance, dedicated carbon source niche, resulting in a significantly faster proliferation rate and better gut microbiota regulation than the traditional "free bacteria + free prebiotic" combination, achieving an unexpected synergistic effect.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a method for preparing probiotic microspheres based on the self-assembly of pure biomembranes, the method comprising: S1, the probiotic suspension is mixed with a mixed solution containing sodium alginate and prebiotics to prepare a primary solution; S2, the primary solution of S1 is dropped into the curing liquid and cured to obtain primary gel beads; S3: The primary gel beads of S2 are transferred into a culture medium containing 10-30 g / L carbon source at a solid-liquid ratio of 1:5-20. Under microaerobic or anaerobic conditions, fermentation is carried out for 24-72 h to activate the quorum sensing system, encapsulate prebiotics in situ, and collect the culture medium. S4, filter and wash the culture medium from S3 to collect the gel beads; place the gel beads in a chelating agent solution and stir to chelate and remove Ca. 2+ Then, centrifugation and washing with water were performed to obtain probiotic microspheres based on the self-assembly of pure biofilms.

[0008] In one embodiment of the present invention, in S1, the prebiotic is selected from one or more of inulin, galactooligosaccharides, fructooligosaccharides, and lactulose.

[0009] In one embodiment of the present invention, in S1, the probiotics are selected from one or more of the genera *Lactobacillus plantarum*, *Lactobacillus*, *Lactobacillus casei*, *Bifidobacterium*, and *Bacillus*.

[0010] In one embodiment of the present invention, the probiotic is selected from Lactobacillus plantarum (Lactobacillus plantarum). Lactiplantibacillus plantarum Lactobacillus rhamnosus ( Lactobacillus rhamnosus Lactobacillus casei ( Lacticaseibacillus casei ), Lactobacillus acidophilus ( Lactobacillus acidophilus ) or Bacillus coagulans ( Bacillus coagulans One or more of the following.

[0011] In one embodiment of the present invention, in S1, the concentration of the bacterial suspension is 10. 8 ~10 10 CFU / mL.

[0012] In one embodiment of the present invention, in S1, the concentration of the alginate is 1.0% to 3.0% (w / v), and the concentration of the prebiotic is 0.5% to 2.0% (w / v).

[0013] In one embodiment of the present invention, in S1, the volume ratio of the bacterial suspension to the mixed solution is 1:2~10.

[0014] In one embodiment of the present invention, in S2, the curing agent is a 0.05~0.2 M divalent calcium ion crosslinking liquid; preferably, it is a 0.05~0.2 M calcium chloride.

[0015] In one embodiment of the present invention, in S2, curing takes 15 to 60 minutes.

[0016] In one embodiment of the present invention, in S3, the carbon source includes one or more of sucrose, glucose, glycerol, lactose, and galactose.

[0017] In one embodiment of the present invention, in S3, a precursor substrate is further added to the culture medium, and the probiotics use the precursor substrate and synthesize active substances through their own biosynthetic pathway.

[0018] In one embodiment of the present invention, the active substance precursor substrate includes L-serine and palmitic acid.

[0019] In one embodiment of the present invention, the active substance includes at least one of ceramide, sphingosine, phytosphingosine, sphingomyelin or glycosylceramide.

[0020] In one embodiment of the present invention, in S4, the chelating agent is sodium citrate.

[0021] A second objective of this invention is to provide probiotic microspheres based on self-assembly of pure biomembranes prepared according to the method.

[0022] A third objective of this invention is to provide a product containing the probiotic microspheres based on pure biofilm self-assembly.

[0023] In one embodiment of the present invention, the probiotic microspheres contain at least 10 strains. 8 ~10 10 CFU / mL.

[0024] In one embodiment of the present invention, the product includes food or medicine.

[0025] In one embodiment of the present invention, the product is in the form of lyophilized powder, granules, liquid preparation, or ointment.

[0026] Beneficial effects The pure biomembrane self-assembled microspheres of this invention possess a dense, porous three-dimensional sponge-like network structure. While exhibiting excellent rigidity, they are resistant to the erosion of gastric acid and small intestinal fluid, effectively protecting the encapsulated probiotics and maintaining their activity before reaching the colon. They retain structural integrity in the stomach and small intestine, preventing premature release. Upon entering the colon, under the influence of pH changes and the action of coliform extracts (or β-galactosidase), the structure disintegrates, and the probiotics are released in a targeted manner, achieving a release rate of 98.1% and a survival rate of 91.2%. Furthermore, the microspheres are formed by the self-assembly of a pure biomembrane, without additional synthetic framework materials, exhibiting good biocompatibility and no additional safety risks. They are suitable as oral delivery carriers for probiotics in the food and pharmaceutical fields. (1) Three-dimensional structural features: The pure biofilm self-assembled microspheres of the present invention break the technical prejudice that conventional embedding materials are solid or amorphous. After the sacrificial template is removed, they present a three-dimensional sponge-like network structure formed by dense filamentous / flocculent EPS and strains, and have the spatial arrangement features of "dense outer layer and porous interior".

[0027] (2) Chemical and mechanical properties: Fourier transform infrared spectroscopy (FTIR) confirmed that the alginate characteristic peaks in the structure of the pure biofilm self-assembled microspheres of this invention were significantly weakened or disappeared, and the structure was mainly composed of amide I / II bands (protein) secreted by the bacteria and polysaccharide matrix (1000-1200 cm⁻¹). -1 The microspheres exhibit an independent structure. The zeta potential on the surface of the microspheres is weakly negative (approximately -15 mV) dominated by lactic acid bacteria, and single-particle compression deformation tests show that they possess excellent compressive strength.

[0028] (3) Resistance to gastric and intestinal fluids and adhesion properties: The dense EPS layer on the outer layer of the pure biofilm self-assembled microspheres of this invention enables the internal bacteria to resist the erosion of simulated gastric and intestinal fluids. After undergoing two-step digestion treatment with simulated gastric and intestinal fluids, the viable bacteria count can still be maintained at 8.2 Log CFU / g. At the same time, the abundant protein and polysaccharide matrix on the surface of the microspheres, through hydrogen bonding and electrostatic interaction, makes its adhesion rate to intestinal mucin (68.7%) significantly better than that of free strains (12.4%).

[0029] (4) Extreme processing stability: Because the EPS three-dimensional network of the pure biofilm self-assembled microspheres of this invention provides strong antifreeze and anti-drying physical support during freezing and dehydration, the difference in viable bacterial survival rate before and after freeze-drying is minimal, and a solid powder with high viable bacterial loading can be obtained. This solid powder also exhibits excellent thermal stability and can withstand short-term high-temperature heat treatment (15-30 min) at 65℃ to 75℃ without structural disintegration. Attached Figure Description

[0030] Figure 1 The images are SEM (scanning electron microscope) comparisons, showing the morphological characteristics. Figure (A) shows a smooth calcium alginate film (traditional embedding), and Figure (B) shows a biofilm microsphere with surface wrinkles and an internal sponge-like porous network. Figure 2 The images show the physicochemical and structural confirmation spectra. The left image is an FTIR comparison (confirming the disappearance of the alginate peak and the enhancement of the EPS characteristic peak); the right image shows the film-forming genes (e.g., ...). luxS A comparison of RT-qPCR expression levels of [product name]; Figure 3 These are curves from an in vitro digestion model. The left figure shows the survival rate in simulated gastric and intestinal fluids; the right figure shows the cumulative release kinetics curve (explosive release trend) in a colonic environment. Figure 4 This is a histogram comparing adhesion rates, demonstrating the adhesion advantage of microspheres to intestinal mucin. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] Culture of *Lactobacillus plantarum*: The bacterial culture preserved in glycerol tubes was inoculated into liquid MRS medium and incubated statically at 37°C for 24 h to obtain activated bacterial culture. The fully activated bacterial culture was inoculated into 100 mL of liquid MRS medium at an inoculation rate of 10% (v / v) and incubated at 37°C until mid-log phase (fermentation time is 10-12 h). The culture was then diluted to 1×10⁻⁶. 9 Preparation of biomembrane self-assembled microspheres using CFU / mL.

[0033] The culture of Lactobacillus gasseri and Lactobacillus rhamnosus is the same as that of Lactobacillus plantarum.

[0034] Liquid MRS medium: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, ammonium citrate 2 g / L, dipotassium hydrogen citrate 2 g / L, Tween 80 1 ml / L, sodium acetate 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.05 g / L.

[0035] Modified MRS broth: Based on the basic formulation of liquid MRS medium, the carbon source glucose was replaced with 20 g / L sucrose, while the other components remained unchanged.

[0036] Example 1: A chimeric prebiotic (inulin) *Lactobacillus plantarum* ( Lactiplantibacillus plantarum Preparation of self-assembled microspheres of pure biomembrane This embodiment provides a method using *Lactobacillus plantarum* (… L. plantarum The preparation method of pure biomembrane self-assembled microspheres and their application in prebiotic encapsulation, including *Lactobacillus plantarum* (accession number GDMCC No: 65484), the specific steps are as follows: S1. Activation and precursor mixing: Take a suspension of *Lactobacillus plantarum* (1.0 × 10⁻⁶) during the late logarithmic growth phase. 9 The primary solution is obtained by thoroughly mixing sodium alginate and prebiotic (inulin) at a volume ratio of 1:5 with a mixed solution containing sodium alginate and prebiotic (inulin); the mixed solution is a 0.1 M PBS buffer system containing 2.0% (w / v, g / mL) sodium alginate and 1.0% (w / v, g / mL) inulin. S2. Primary Template Construction: The primary solution from S1 was uniformly dripped into 0.1 M CaCl2 crosslinking solution using an electrostatic high-voltage microencapsulation apparatus (voltage 5.0 kV, injection speed 10 mL / h, needle inner diameter 21G). After the addition was complete, the mixture was cured at room temperature for 30 min, and then washed to obtain calcium alginate-inulin-probiotic primary gel beads with uniform particle size (approximately 800 μm). S3, In-situ Induced Self-Assembly: The primary gel beads from S2 were transferred to a modified MRS broth containing 20 g / L sucrose at a ratio of 1:10 (w / v, g / mL), and anaerobically cultured at 37℃ and 50 rpm for 48 h. The culture medium was then collected. Within this confined space, the quorum sensing system (QS) was activated, and the bacteria secreted large amounts of extracellular polysaccharides and membrane proteins to encapsulate inulin molecules in situ.

[0037] S4. In-situ removal of the sacrificial template: The culture medium from S3 was filtered through an 800 μm filter, washed 2-3 times with PBS buffer, and the gel beads were collected. The gel beads were then placed in 0.05 M, pH 6.0 sodium citrate solution and stirred at 50 rpm for 30 min to chelate and remove Ca. 2+After centrifugation and washing with water, sodium alginate and Ca were completely removed. 2+ Pure biofilm microspheres (SBMs).

[0038] Example 2: Spatiotemporal evolution dynamics of the self-assembly process and verification of EPS secretion This embodiment aims to verify the synergistic contribution of "confined space" and "in-situ induction" to the construction of a three-dimensional support network for SBMs. Through comparative experiments, this embodiment demonstrates that only under the dual synergistic effect of specific template retardation conditions and the substrate concentration induced by the QS system can pure biomembrane microspheres existing independently of the exogenous framework be obtained, thus overcoming the inhibition of probiotic activity by wall materials in traditional polymer physical encapsulation techniques. Specifically: 1. Effect of sodium alginate addition on the formation of self-assembled microspheres from pure biofilms The specific implementation method is the same as in Example 1, except that the amount of sodium alginate added in S1 is adjusted to 1%, 2%, 3%, 4%, and 5%, respectively. The results are shown in Table 1. Table 1

[0039] Experimental results show that the concentration of sodium alginate does not exhibit a simple linear relationship with film-forming effect. When the concentration exceeds 3%, the dense gel network severely hinders mass transfer between the inside and outside, preventing the effective penetration of carbon sources from the external culture medium. Simultaneously, QS signaling molecules secreted by the internal cells are physically isolated, failing to reach the threshold for quorum sensing activation, ultimately suppressing the secretion of extracellular polysaccharides and proteins. However, at a concentration of 2.0%, an ideal "semi-permeable confined niche" is formed, maintaining the physical morphology of the early self-assembly stage while allowing for the effective accumulation of signaling molecules.

[0040] 2. Effect of the volume ratio of bacterial suspension to a mixed solution containing sodium alginate and prebiotics on the formation of self-assembled microspheres from pure biofilms. The specific implementation method is the same as in Example 1, except that the volume ratio of the bacterial suspension in S1 to the mixed solution containing sodium alginate and prebiotics was adjusted to 1:1, 1:2, 1:5, 1:10, and 1:12, respectively. The results are shown in Table 2. Table 2

[0041] Experimental results confirm that the volume ratio of bacterial suspension to mixed solution not only determines the initial biomass within the microspheres, but is also a key threshold condition for the successful construction and eventual complete removal of the "sacrificial template".

[0042] When the bacterial culture ratio is too high (e.g., 1:1 or 1:2), the high concentration of bacterial cells creates physical steric hindrance, blocking the reaction between sodium alginate and Ca. 2+The formation of the "egg-box structure" at the cross-linking sites leads to a significant reduction in the strength of the primary gel, making the gel prone to breakage in the S3 stage and causing failure of self-assembly and embedding.

[0043] When the bacterial culture ratio is too low (e.g., 1:10 or 1:12), the cross-linking density of sodium alginate is too high, resulting in extremely low internal porosity. This makes it difficult for sodium citrate in the S4 stage to effectively penetrate the microsphere core. Simultaneously, the initial implantation sites of the live bacteria are too sparse, causing their secreted EPS three-dimensional network to become tightly entangled with undegraded sodium alginate. Ultimately, this leads to incomplete removal, with some sodium alginate physically encapsulated within the biofilm network, forming a hybrid framework. This significantly reduces the intelligent response disintegration rate of the microspheres in the colonic environment and may even inhibit probiotic activity.

[0044] In summary, a volume ratio of 1:5 provides a crucial balance between microfluidics and crosslinking density. This balance maintains the structural integrity of the primary template in stage S3 while ensuring the formation of loosely suitable channels within the template, enabling complete Ca removal in stage S4. 2+ .

[0045] 3. Effect of prebiotic addition on the formation of self-assembled microspheres from pure biomembranes The specific implementation method is the same as in Example 1, except that the amount of prebiotics added in S1 is adjusted to 0.5%, 1%, 2%, 3%, and 5%, respectively. The results are shown in Table 3. Table 3

[0046] Experimental results show that the amount of prebiotics added is not necessarily better the higher it is, but rather there is a strict concentration window (0.5%~2.0%, preferably 1.0%). The establishment of this concentration mainly depends on the "cross-linking steric hindrance" of the primary gel template and the "osmotic pressure balance" of the microbial niche inside the microspheres.

[0047] When the prebiotic concentration exceeds 3%, it has a dual negative impact on encapsulation and the final biofilm performance. High concentrations of prebiotics increase solution viscosity, leading to uneven droplet formation and tailing in the microcapsule formation. Excessive prebiotics create steric hindrance, interfering with the reaction of sodium alginate and Ca. 2+ Cross-linking loosens the structure of the gel beads, reduces their strength, and makes them prone to breakage. Simultaneously, high concentrations of prebiotics generate high osmotic pressure during the self-assembly stage, leading to plasmolysis and growth arrest of probiotics, inhibiting secondary proliferation and quorum sensing, and significantly reducing the secretion of extracellular polysaccharides and membrane-forming proteins. Unmetabolized prebiotics occupy space, blocking the interweaving of the EPS network, causing the decalcified biomembrane microspheres to collapse and deform.

[0048] In summary, a concentration of 1.0% is the key balance point. It can achieve in-situ chimerism of prebiotics, providing carbon sources for probiotics to exert synergistic effects, while avoiding high osmotic stress and cross-linking interference, ensuring that the biomembrane structure is dense and intact after the exogenous skeleton is removed.

[0049] 4. Effect of fermentation time on the formation of self-assembled microspheres from pure biofilms To verify the dynamic formation process of biofilm during the in situ culture stage, samples were taken and analyzed at different time points (0 h, 12 h, 24 h, 48 h) in S3 of Example 1.

[0050] Polysaccharides were quantified using the phenol-sulfuric acid method, and proteins were quantified using the BCA method. The results are shown in Table 4. With the extension of the culture time in the confined space, the polysaccharide and protein contents inside the microspheres increased exponentially, proving that the three-dimensional biomembrane network was forming.

[0051] Table 4

[0052] 5. Effect of the ratio of primary gel to culture medium on the formation of self-assembled microspheres from pure biomembranes The specific implementation method is the same as in Example 1, except that the ratio of the primary gel to the culture medium in S3 is adjusted to 1:1, 1:2, 1:5, 1:10, 1:20, and 1:25, respectively. The results are shown in Table 5. Table 5

[0053] Experimental data strongly demonstrate that the ratio of primary gel to culture medium (solid-liquid ratio) regulates the total nutrient mass transfer and the local enrichment concentration of quorum sensing (QS) signaling molecules within the system: If the ratio is too small (e.g., 1:1), the total volume of the primary gel in the system is too large, while the absolute amount of total carbon source provided by the external culture medium is too low. Under the stress of overcrowding and starvation, the probiotics inside the gel will deplete their nutrient substrate in a very short time, their metabolic activity will rapidly decline, and they will enter the death phase prematurely. At the same time, the strains will lose their anabolic metabolic capacity due to starvation before they can produce sufficient concentrations of signaling molecules. The QS system cannot be activated, membrane-forming genes are silenced, and the content of extracellular polysaccharides and extracellular proteins decreases significantly, ultimately leading to the complete inability to form a biomembrane support network.

[0054] When the ratio is set to 1:10, the sufficient external culture medium ensures a continuous and stable transfer of carbon source to the gel interior, providing ample energy for cell proliferation and membrane formation. On the other hand, the calcium alginate physical framework restricts the diffusion of bacteria to the outside of the primary gel, forming a physical barrier. This allows QS signaling molecules to accumulate efficiently inside the microspheres, rapidly exceeding the activation threshold, initiating membrane-forming gene expression, and causing the bacteria to secrete large amounts of EPS and extracellular proteins, completing the self-assembly of a pure biomembrane.

[0055] When the culture medium ratio is too high (e.g., 1:20 or 1:25), the concentration gradient inside and outside the primary gel is too large, causing the QS signal molecules generated inside the microspheres to permeate and dilute into the culture medium, making it difficult for them to accumulate inside the microspheres. At the same time, some bacteria on the surface of the microspheres will migrate into the culture medium, resulting in a loose internal structure of the microspheres, which are very easy to disintegrate in the subsequent decalcification step.

[0056] 6. Effect of sucrose addition in culture medium on the formation of self-assembled microspheres from pure biofilms. The specific implementation method is the same as in Example 1, except that the amount of sucrose added in S3 is adjusted to 5 g / L, 8 g / L, 10 g / L, 20 g / L, 30 g / L, and 50 g / L, respectively. The results are shown in Table 6. Table 6

[0057] Example 3: A chimeric Lactobacillus gasseri containing prebiotics (fructooligosaccharides) and ceramides ( Lactobacillus gasseri Preparation of self-assembled microspheres of pure biomembrane This embodiment provides a method using Lactobacillus gasseri (… L. gasseri The preparation method of pure biomembrane self-assembled microspheres and their application in encapsulating prebiotics, including Lactobacillus gasseri BN9 (accession number GDMCC No: 64320), the specific steps are as follows: S1. Activation and Precursor Mixing: Take Lactobacillus gasseri from the late logarithmic growth stage (… L. gasseri ) suspension (1.0×10 9 The primary solution is obtained by thoroughly mixing sodium alginate and prebiotic (fructooligosaccharide) at a volume ratio of 1:10 with the sodium alginate and prebiotic (fructooligosaccharide); the primary solution is a 0.1 M PBS buffer system containing 3.0% (w / v, g / mL) sodium alginate and 0.5% (w / v, g / mL) fructooligosaccharide. S2. Primary Template Construction: The primary solution from S1 was uniformly dropped into 0.1 M CaCl2 crosslinking solution using an electrostatic high-voltage microencapsulation apparatus (voltage 5.0 kV, injection speed 10 mL / h, needle inner diameter 21G). After the addition was complete, the mixture was cured at room temperature for 30 min, and then washed to obtain calcium alginate-fructooligosaccharide-probiotic primary gel beads with uniform particle size (approximately 800 μm). S3, In-situ induced self-assembly: The primary gel beads of S2 were transferred at a ratio of 1:15 (w / v, g / mL) into a modified MRS broth containing 0.5 g / L L-serine and 0.1 g / L palmitic acid, and anaerobically cultured at 37℃ and 50 rpm for 48 h. The culture medium was then collected. S4. In-situ removal of the sacrificial template: The culture medium from S3 was filtered through an 800 μm filter, washed 2-3 times with PBS buffer, and the gel beads were collected. The gel beads were then placed in 0.05 M, pH 6.0 sodium citrate solution and gently stirred at 100 rpm for 30 min to chelate and remove Ca. 2+ After centrifugation and washing with water, pure biofilm microspheres (SBMs) were obtained.

[0058] The results showed that the oxidative degradation rate of the ceramide synthesized inside the BN9 biofilm microspheres obtained after decalcification was <5% under the condition of being placed in an open environment at room temperature for 30 days, which was significantly lower than that of free ceramide.

[0059] Example 4: A chimeric prebiotic (fructooligosaccharide) Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus Preparation of self-assembled microspheres of pure biomembrane This embodiment provides a Lactobacillus rhamnosus HN001 ( L. rhamnosus The method for self-assembling microspheres to encapsulate prebiotics involves the following steps: S1. Activation and precursor mixing: Take a suspension of *Lactobacillus rhamnosus* HN001 from the late logarithmic growth stage (1.0 × 10⁻⁶). 9 The solution is prepared by mixing sodium alginate and prebiotic (fructooligosaccharides) at a volume ratio of 1:5 to obtain a primary solution; the primary solution is a 0.1 M PBS buffer system containing 1.50% (w / v, g / mL) sodium alginate and 0.5% (w / v, g / mL) fructooligosaccharides. S2. Primary Template Construction: The primary solution from S1 was uniformly dropped into 0.1 M CaCl2 crosslinking solution using an electrostatic high-voltage microencapsulation apparatus (voltage 5.0 kV, injection speed 10 mL / h, needle inner diameter 21G). After the addition was complete, the mixture was cured at room temperature for 30 min, and then washed to obtain calcium alginate-fructooligosaccharide-probiotic primary gel beads with uniform particle size (approximately 800 μm). S3, In-situ induced self-assembly: The primary gel beads of S2 were transferred into modified MRS broth containing 2% sucrose at a ratio of 1:5 (w / v, g / mL) and anaerobic cultured at 37℃ and 50 rpm for 72 h. The culture medium was then collected. S4. In-situ removal of the sacrificial template: The culture medium from S3 was filtered through an 800 μm filter, washed 2–3 times with PBS buffer, and the gel beads were collected. The gel beads were then placed in 0.1 M EDTA solution and gently stirred at 75 rpm for 30 min to chelate and remove Ca. 2+ After centrifugation and washing with water, pure biofilm microspheres (SBMs) were obtained.

[0060] Comparative Example 1: Preparation of Traditional Calcium Alginate Physically Encapsulated Microspheres The difference between this comparative example and Example 1 is that sodium alginate was directly used for physical encapsulation, as detailed below: Figure 1 The scanned electron microscope (SEM) showed that the SBMs were regular spherical with a dense, wrinkled biofilm structure on their surface, while the traditional physically embedded microspheres had smooth surfaces, uniform structures, and no biofilm-like morphology. Further scanning electron microscopy confirmed that the SBMs had densely packed bacteria on their surface, forming a three-dimensional network biofilm matrix, while the traditional microspheres only showed a smooth calcium alginate matrix with scattered bacteria embedded within it.

[0061] Traditional physical embedding group: Lactobacillus plantarum in the late logarithmic growth stage ( L. plantarum ) suspension (1.0×10 9 The solution (CFU / mL) was mixed with 2.0% (w / v) sodium alginate at a volume ratio of 1:5 and then added dropwise to 0.1 M CaCl2 for solidification for 30 minutes. No in-situ culture induction or decalcification treatment was performed.

[0062] FTIR results ( Figure 2 This confirms that the structure of microspheres prepared by the traditional physical encapsulation method is entirely dependent on the exogenous chemical wall material of alginate (1600 cm). -1 Nearby COO - (It exhibits extremely strong stretching and vibration), and the internal strains are distributed in a scattered, dotted pattern.

[0063] membrane-forming genes luxS The RT-qPCR results showed that the traditional physical embedding group luxSThe gene expression level was approximately 20% of that in the pure biofilm microspheres of Example 1, indicating that the traditional encapsulation method did not activate the probiotic quorum sensing membrane formation process and did not form an endogenous biofilm network interwoven with extracellular polymers. In the final traditional calcium alginate physically encapsulated microspheres, the live bacteria amount was only at the initial encapsulation level, and in-situ proliferation and enrichment of probiotics were not achieved.

[0064] Example 5: Evaluation of extreme tolerance, colon targeting, and stability of pilot-scale samples (1) Gastrointestinal barrier tolerance test (INFOGEST 2.0 model) The samples were sequentially treated with simulated gastric fluid (SGF, pH 2.0, containing pepsin, 2 h) and simulated small intestinal fluid (SIF, containing porcine bile salts, 2 h), and the viable bacterial count was recorded. A control group was set up, which consisted of an untreated suspension of *Lactobacillus plantarum* from the same batch as in Example 1.

[0065] Table 7

[0066] The results showed that free probiotics could hardly tolerate the dual stress of acidic gastric juice and bile salts, and no viable bacteria were detected after two steps of digestion. The results of traditional calcium alginate physically encapsulated microspheres showed that the gel structure of calcium alginate was gradually destroyed under the influence of acidic environment and bile salts, with the final survival rate only maintaining 5.45 Log CFU / g. In contrast, the pure biofilm self-assembled microspheres prepared in Example 1 of this invention, relying on the physical barrier effect of the dense EPS three-dimensional network, could effectively isolate hydrogen ions and digestive enzymes in gastric juice. After two steps of digestion, they still maintained a viable bacterial count of 8.2 Log CFU / g, with a survival rate far higher than that of traditional encapsulation methods. This fully demonstrates that the microspheres have excellent gastrointestinal barrier tolerance and can ensure that a sufficient number of active probiotics can successfully reach the colon to exert their effects. Figure 3 ).

[0067] (2) Colonic smart response release kinetics After incubating the samples with SGF and SJF for 2 h each, they were then transferred to simulated colonic fluid (SCF) containing 0.1% coliform mixed enzyme system (pH 6.8) to monitor the release rate of microspheres.

[0068] Table 8

[0069] The results showed that the release rate of traditional calcium alginate-encapsulated microspheres in colonic fluid remained slow. This is because alginate, as an exogenous macromolecule, is difficult to be rapidly degraded by colonic microorganisms. Therefore, only half of the probiotics could be released slowly, failing to achieve targeted and concentrated replenishment. In contrast, the pure biofilm self-assembled microspheres prepared in Example 1 of this invention are mainly composed of extracellular polysaccharides secreted by the probiotics themselves. Under the action of specific polysaccharide-degrading enzymes secreted by colonic microorganisms, the EPS network is rapidly cleaved and degraded. The release rate significantly accelerated after 1 hour, and a burst release of 92.8% was achieved after 2 hours, with a final release rate of over 98%. This meets the requirements for colon-targeted delivery and can rapidly release sufficient active probiotics to exert physiological effects after reaching the colon. Figure 3 ).

[0070] (3) Extreme processing stability verification (thermal stability and vacuum freeze-drying stability) The structural maintenance ability of the SBMs prepared in Example 1 during thermodynamic and freeze-drying processes.

[0071] Table 9

[0072] (4) Intestinal mucosal colonization capacity - incubation of porcine gastric mucin in vitro.

[0073] (a) Mucin coating: Porcine gastric mucin (Type II, Sigma) was dissolved in 0.1 M, pH 7.4 PBS buffer to prepare a 10 mg / mL mucin solution. The mucin solution was added to 24-well cell culture plates and coated overnight at 4°C. The next day, the liquid in the wells was aspirated, and the plates were washed three times with sterile PBS buffer.

[0074] (b) Sample incubation: The free bacterial suspension (control group), the conventionally physically embedded microspheres prepared in Comparative Example 1, and the pure biofilm microspheres (SBMs) prepared in Example 1 were resuspended in PBS, and the initial viable count of each sample was adjusted to 1.0 × 10⁻⁶. 8 Then add it to the 24-well plate from step (a) and incubate at 37°C for 2 hours.

[0075] (c) Washing and Elution: After incubation, the supernatant was discarded, and the plate was washed three times with sterile PBS buffer. Then, 1 mL of PBS elution buffer containing 0.1% (v / v) Triton X-100 was added to each well, and the plate was vigorously shaken for 15 min at room temperature to allow for complete detachment of the bacteria and microspheres tightly bound to Mucin. The eluent collected from the microsphere group was homogenized to release the viable bacteria inside.

[0076] (d) Viable cell count and calculation: Collect the final eluent from each well, perform serial dilutions, and spread it onto MRS solid agar plates. After incubation at 37°C for 48 h, count the colonies (recorded as the number of adherent viable cells). N 1). The formula for calculating adhesion rate is: Adhesion rate (%) = ( N 1 / Initial total number of live bacteria added N 0) × 100%.

[0077] Test data shows ( Figure 4 Under the same conditions, the adhesion efficiency of free-state bacteria was relatively low (12.4%). Comparative Example 1 showed an even lower adhesion rate of 8.5%; this result is mainly attributed to the use of a hydrophilic, smooth calcium alginate surface, a surface characteristic that makes it extremely difficult for microorganisms to adhere and cling to the surface. In contrast, the adhesion rate of the pure biofilm microspheres prepared in Example 1 was significantly improved to 68.7%. This indicates that the microsphere surface is exposed with a large number of specific surface adhesion proteins (Mub) and related polysaccharides.

[0078] (5) Evaluation of microsphere encapsulation rate and viable bacteria enrichment load The number of viable bacteria and the encapsulation efficiency inside the microspheres were determined using the standard plate coating counting method. After step S2 (primary template construction), the initial encapsulation rates of the traditional physical encapsulation group (Comparative Example 1) and the primary gel beads of the present invention were both between 92% and 95%, with no significant difference.

[0079] However, after step S3 (in-situ induced self-assembly), the viable bacterial load of the traditional physically embedded group remained at approximately 2.5 × 10⁻⁶ due to the lack of carbon source supply and confined space for proliferation. 8 CFU / g wet weight; while the SBMs of this invention, due to the secondary large-scale proliferation of probiotics in the microecological niche using prebiotics and sucrose, have a final viable cell load of 1.8 × 10⁻⁶. 10 CFU / g wet weight. This indicates that the preparation method of the present invention not only achieves a high initial encapsulation rate, but also realizes a cascade amplification and enrichment of the number of live bacteria inside the microspheres by nearly two orders of magnitude through an in-situ self-assembly mechanism, significantly improving the probiotic delivery efficiency per unit mass of microspheres.

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing probiotic microspheres based on the self-assembly of pure biomembranes, characterized in that, The method is as follows: S1, a primary solution is prepared by mixing a probiotic suspension with a mixed solution containing sodium alginate and prebiotics at a volume ratio of 1:2 to 10; wherein the concentration of sodium alginate is 1.0% to 3.0% (w / v), and the concentration of prebiotics is 0.5% to 2.0% (w / v); the concentration of the bacterial suspension is 10... 8 ~10 10 CFU / mL; S2, the primary solution of S1 is dropped into the curing liquid and cured to obtain primary gel beads; S3: The primary gel beads of S2 are transferred into a culture medium containing 10-30 g / L carbon source at a solid-liquid ratio of 1:5-20. Under microaerobic or anaerobic conditions, fermentation is carried out for 24-72 h to activate the quorum sensing system, encapsulate prebiotics in situ, and collect the culture medium. S4, filter and wash the culture medium from S3, and collect the gel beads; place the gel beads in a chelating agent solution and stir to chelate and remove Ca. 2+ Then, centrifugation and washing with water were performed to obtain probiotic microspheres based on self-assembly of pure biofilm. The probiotic microspheres are unaffected by gastric and intestinal fluids, have a survival rate of >90%, are released into the colon with a targeted release, and have an adhesion rate of >60%. Among them, after S4 treatment, the obtained probiotic microspheres still maintain the complete microsphere morphology without using calcium alginate as the structural framework. Its structure is independently maintained by a three-dimensional sponge-like network formed by the interweaving of extracellular polymers secreted by probiotics and bacterial cells, with a dense outer layer and a porous inner layer. In S1, the prebiotic is selected from one or more of inulin, galactooligosaccharides, fructooligosaccharides and lactulose; The probiotics are selected from one or more of the genera *Lactobacillus plantarum*, *Lactobacillus*, *Lactobacillus*, *Bifidobacterium*, and *Bacillus*. In S2, the curing liquid is a 0.05~0.2 M divalent calcium ion crosslinking liquid.

2. The method according to claim 1, characterized in that, In S3, the carbon source includes one or more of sucrose, glucose, glycerol, lactose, and galactose.

3. The method according to claim 1, characterized in that, In S3, a precursor substrate is also added to the culture medium, and the probiotics use the precursor substrate and synthesize active substances through their own biosynthetic pathway.

4. A probiotic microsphere based on self-assembly of a pure biomembrane, characterized in that, It is prepared by the method according to any one of claims 1 to 3.

5. A drug, characterized in that, The drug contains the probiotic microspheres based on pure biological membrane self-assembly as described in claim 4.

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