Biofilm state probiotic microcapsule and application thereof in improving tolerance of probiotics

Abstract

The invention discloses a biofilm state probiotic microcapsule and application thereof in improving the tolerance of probiotics, and belongs to the technical field of microorganism preservation and maintenance. The biofilm state probiotic microcapsule is prepared by the following method: mixing a probiotic bacterial suspension with a polysaccharide solution, then contacting the mixed solution with a cross-linking agent solution, and carrying out cross-linking to obtain a polysaccharide cross-linked gel microcapsule, adjusting the pH value of the complex culture medium to 5-9, then adding the polysaccharide cross-linked gel microcapsule, and culturing to obtain the biofilm state probiotic microcapsule. The microcapsule has excellent tolerance to gastric juice and cholate, so that the problem of low survival rate of probiotics in a gastrointestinal tract environment is solved.

Description

Technical Field

[0001] This invention belongs to the field of microbial preservation and maintenance technology, specifically relating to a biofilm-coated probiotic microcapsule and its application in improving probiotic tolerance. Background Technology

[0002] Probiotics (such as Lactobacillus and Bifidobacterium) have various potential health effects, including regulating gut microbiota and improving barrier function. However, their effectiveness depends on a sufficient number of live bacteria reaching the gut. Orally administered probiotics are easily destroyed by gastric acid and bile salts during gastrointestinal passage. To improve survival rates, gel encapsulation technologies such as calcium alginate are widely used. However, ordinary gel encapsulation may still swell and degrade under extreme pH or bile salt stress, and current technologies lack precise control over the microenvironment and biofilm formation within the gel, resulting in limited protective effects. Therefore, addressing the problems of low survival rates of free probiotics and ordinary encapsulated probiotics during processing (e.g., freeze-drying), storage, and in the gastrointestinal environment, insufficient gel mechanical strength, and difficulty in controlling the induction of biofilms in existing technologies, this paper proposes a probiotic microcapsule that induces biofilm formation through pH-controlled reculturing and constructs a "biofilm-gel" composite barrier to improve probiotic tolerance. This approach is of significant importance and value. Summary of the Invention

[0003] This invention provides a biofilm-coated probiotic microcapsule, prepared by the following method: Probiotic suspension was mixed with polysaccharide solution, and then the mixture was contacted with cross-linking agent solution to cross-link and obtain polysaccharide cross-linked gel microcapsules. The pH of the complex culture medium was adjusted to 5-9, and then the polysaccharide cross-linked gel microcapsules were added and cultured to obtain biofilm-coated probiotic microcapsules.

[0004] In the above technical solution, the probiotics include Lactobacillus and / or Bifidobacterium; preferably at least one of Lactobacillus rhamnosus GG (LGG), Lactobacillus rhamnosus LRa05, Lactobacillus rhamnosus WKA55, Lactobacillus plantarum Lp90, Lactobacillus plantarum CW006, Lactobacillus plantarum Lp05, Lactobacillus gasseri LG08, Lactobacillus acidophilus LA85, Lactobacillus acidophilus LA05, and Bifidobacterium breve BBr60.

[0005] In the above technical solution, the polysaccharide is at least one of sodium alginate, pectin, and carrageenan.

[0006] In the above technical solution, the bacterial content in the probiotic suspension is at least 1×10⁻⁶. 8 CFU / mL; the mass-volume concentration of the polysaccharide solution is 0.3-5%; the volume ratio of the probiotic suspension to the polysaccharide solution is 1:(10-30).

[0007] In the above technical solution, the crosslinking agent solution is an aqueous solution containing divalent metal ions; preferably, it is at least one of calcium chloride aqueous solution, calcium lactate aqueous solution, and strontium chloride aqueous solution; the mass-volume concentration of the crosslinking agent solution is 0.5-8%.

[0008] In the above technical solution, the volume ratio of the mixture to the crosslinking agent solution is 1:(5~20); preferably 1:10.

[0009] In the above technical solution, crosslinking is carried out by dripping, spraying, or peristaltic pump dripping, and the crosslinking time is 10~60 min.

[0010] In the above technical solution, the mass-to-volume ratio of the polysaccharide cross-linked gel microcapsules to the complex culture medium is 1:(5~20), g / mL; preferably 1:10, g / mL.

[0011] In the above technical solution, the complex culture medium is MRS broth or other culture medium that can support the growth of probiotics; the culture conditions are: anaerobic culture at 30~45℃ for 12~72 h.

[0012] In the above technical solution, the pH of the compound culture medium is adjusted by acid and base; the acid includes inorganic acid and / or organic acid, the inorganic acid includes hydrochloric acid, phosphoric acid or sulfuric acid, the organic acid includes lactic acid, citric acid, acetic acid or malic acid; the base includes inorganic base and / or organic base, the inorganic base includes sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate, and the organic base includes triethanolamine or ammonia.

[0013] This invention provides the application of the above-mentioned biofilm-coated probiotic microcapsules in improving probiotic tolerance; the tolerance is processing tolerance or gastrointestinal digestive tolerance; the processing tolerance refers to tolerance to high and low temperature environments during processing; the gastrointestinal digestive tolerance refers to tolerance to gastric acid and bile salts.

[0014] This invention provides a freeze-dried bacterial powder, which is prepared by freeze-drying the above-mentioned biofilm-coated probiotic microcapsules.

[0015] The beneficial effects of this invention are as follows: This invention involves reculturing probiotic microcapsules at a specific initial pH. By adjusting the initial pH of the reculturing medium, quorum sensing and biofilm formation are regulated, thereby enhancing the tolerance of polysaccharide-crosslinked probiotics and preparing a biofilm-coated probiotic microcapsule. This microcapsule exhibits excellent tolerance to simulated gastric juice (pH=2) and bile salts (0.6%), overcoming the problem of low survival rate of probiotics in the gastrointestinal environment. This is of significant importance and value in improving the bioavailability of probiotics.

[0016] In this invention, pH regulation can promote the generation of extracellular matrix and couple it with the gel network to form a denser "cell cluster-matrix-gel" composite structure, enhance mechanical strength and barrier effect, and make the structure of microcapsules more dense and stable.

[0017] This invention optimizes microcapsule performance simply by adjusting the initial pH of the complex culture medium, without the need for additional expensive inducers or complex equipment; it is applicable to a variety of probiotics such as Lactobacillus and Bifidobacterium, and the encapsulation of polysaccharides and calcium sources can be flexibly selected, making it easy to scale up industrially. Attached Figure Description

[0018] Figure 1 The image shows a laser confocal scanning microscope (CLSM) image of the biofilm-coated probiotic microcapsules described in Example 1; where A is extracellular protein staining and B is eDNA staining.

[0019] Figure 2 SEM images of the polysaccharide cross-linked gel microcapsules (A) and the biofilm-coated probiotic microcapsules (B) in Example 1. Detailed Implementation

[0020] In this invention, pH-controlled reculture is a core step. Polysaccharide cross-linked gel microcapsules are added to a reculture medium containing divalent metal ions and cultured. Adjusting the initial pH of the reculture medium to a slightly acidic or slightly alkaline range induces probiotics to activate quorum sensing signals and secrete extracellular proteins and extracellular DNA, forming a dense biofilm matrix within the gel network, thus creating biofilm-coated probiotic microcapsules.

[0021] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way. Example 1

[0022] The following steps were taken to prepare biofilm-coated probiotic microcapsules under pH 5.0 conditions: (1) Activation of microbial strains Lactobacillus rhamnosus GG was inoculated into MRS broth and anaerobically cultured at 37°C for 24 h. The bacterial cells were collected by centrifugation and washed with sterile physiological saline. Then, physiological saline was added to make the bacterial concentration 1×10⁻⁶. 8 CFU / mL was used to obtain a bacterial suspension.

[0023] (2) Preparation of polysaccharide solution Dissolve sodium alginate in deionized water and stir until completely dissolved to prepare a sodium alginate solution with a concentration of 0.6% (w / v).

[0024] (3) Encapsulation and crosslinking The bacterial suspension was mixed with sodium alginate solution at a volume ratio of 1:20, and then added dropwise to a 1.0% (w / v) calcium chloride solution for cross-linking for 30 min. The mixture was then washed with sterile distilled water to obtain polysaccharide cross-linked gel microcapsules.

[0025] (4) pH-controlled reculture MRS broth containing 1.0% (w / v) calcium chloride was prepared, and its initial pH was adjusted to 5.0 with hydrochloric acid (0.1 mol / L). Polysaccharide cross-linked gel microcapsules were added at a ratio of 1:10 (g / mL), and the mixture was anaerobically cultured at 37°C for 48 h.

[0026] (5) Collection The microcapsules of probiotics were obtained by filtering with a filter screen and then washing with sterile distilled water. Example 2

[0027] This embodiment describes the preparation of biofilm-coated probiotic microcapsules at pH 9.0. The preparation steps are the same as those in Example 1 above. The difference from Example 1 is that in step (4), sodium hydroxide (0.1 mol / L) is used to adjust the initial pH to 9.0. Example 3

[0028] This embodiment describes the preparation of biofilm-coated probiotic microcapsules under pH 5.0 conditions. The preparation steps are the same as in Example 1 above, except that pectin is used as the polysaccharide. Example 4

[0029] This embodiment describes the preparation of biofilm-coated probiotic microcapsules at pH 4.0. The preparation steps are the same as those in Example 1 above. The difference from Example 1 is that in step (4), hydrochloric acid (0.1 mol / L) is used to adjust the initial pH to 4.0. Example 5

[0030] This embodiment describes the preparation of biofilm-coated probiotic microcapsules at pH 10.0. The preparation steps are the same as those in Example 1 above. The difference from Example 1 is that in step (4), sodium hydroxide (0.1 mol / L) is used to adjust the initial pH to 10.0.

[0031] I. Performance Testing The biofilm-coated probiotic microcapsules prepared in Examples 1-5 were used as samples, and the bacterial suspension and polysaccharide cross-linked gel microcapsules in Example 1 were used as control samples, respectively, and were denoted as Comparative Example 1 and Comparative Example 2.

[0032] Simulated gastrointestinal digestion: The samples were sequentially placed in simulated gastric fluid (pH=2) for 2 h, and then transferred to simulated intestinal fluid containing bile salts (0.6%) for 1 h. After the treatment, the number of viable bacteria was counted.

[0033] Freeze-drying and storage: Freeze-dried bacterial powder was prepared by freeze-drying the biofilm microcapsules, and the storage survival rate was determined after being placed at 37°C for 8 weeks.

[0034] All the above indicators are expressed in CFU (Cholesterol Fuel Count), and the calculation formula is as follows: Freeze-dried survival rate (%) = (Number of viable bacteria after freeze-drying / Number of viable bacteria before freeze-drying) × 100%; Storage survival rate (%) = (Number of viable bacteria after storage / Number of viable bacteria after freeze-drying) × 100%; Simulated gastrointestinal survival rate (%) = number of viable bacteria after digestion / number of viable bacteria before digestion × 100%.

[0035] The experimental results are shown in Table 1: Table 1. Results of the survival test of probiotics Note: This experiment also tested the survival rate of probiotics in biofilm-coated probiotic microcapsules prepared at pH 1, 2, 3 and 11 (preparation steps refer to Example 1 above). The test results showed that the probiotics in the capsules had no survival rate, so the relevant data are not listed in Table 1 above.

[0036] As shown in Table 1, the loss of viable bacteria in the samples under reculture conditions at pH 5.0 and pH 9.0 was significantly less than that in the comparative samples. Specifically, the survival rate of free bacteria (bacterial suspension) was significantly reduced after treatment with gastric acid and bile salts, to the point of being undetectable. Furthermore, the tolerance and structural stability of probiotics in ordinary gel microcapsules were also lower than those in the example samples.

[0037] II. Verification of biological membrane state To verify that the probiotic microcapsules prepared in this invention (Example 1) possess a biofilm state, their CLSM and SEM images were measured, as shown below. Figure 1 and Figure 2 As shown: Figure 1 Green fluorescence was clearly observed in A, indicating the presence of extracellular proteins. Figure 1 Blue fluorescence corresponding to DNA was detected in the microcapsules. The extensive co-localization of green (protein) and blue (DNA) signals directly demonstrates that the spatial integration of these two main matrix components forms a protective structure that encapsulates the bacterial community—a biofilm. The encapsulated LGG exhibited dense cell aggregation within the microcapsules, a key feature of biofilm formation.

[0038] Figure 2 Image A shows the SEM image of the polysaccharide cross-linked gel microcapsules. The surface of the microcapsules is dense and relatively smooth, with no identifiable biofilm structure, indicating that there are no biofilm-like features on the surface of the microcapsules. Figure 2 In the study, microcapsule surfaces were observed to be densely covered with biofilms; this indicates that LGG has proliferated and aggregated to form microcolonies, and the images show a large amount of extracellular polymeric material aggregated between cells, which is consistent with the formation of biofilm-like structures.

[0039] Comprehensive observations using CLSM and SEM under both dehydrated and dried conditions ultimately demonstrated biofilm formation. By comparing the two physical states (hydrated and dried), direct microscopic evidence of biofilm formation in probiotic-loaded gel systems was systematically provided.

[0040] This invention also provides adaptation examples of different polysaccharides, cross-linking agents, and bacterial strains, based on Example 1, as follows: Sodium alginate can be replaced with pectin or carrageenan (0.5-4.0% w / v); the cross-linking agent calcium chloride solution can be replaced with calcium lactate aqueous solution or strontium chloride aqueous solution (0.5-8.0% w / v); the bacterial strain *Lactobacillus rhamnosus* GG can be replaced with one or more of the following: *Lactobacillus rhamnosus* LRa05, *Lactobacillus rhamnosus* WKA55, *Lactobacillus plantarum* Lp90, *Lactobacillus plantarum* CW006, *Lactobacillus plantarum* Lp05, *Lactobacillus gasseri* LG08, *Lactobacillus acidophilus* LA85, *Lactobacillus acidophilus* LA05, and *Bifidobacterium breve* BBr60; the pH-controlled reculture conditions can be adjusted to: reculture at pH 3-10 for 12-72 h. Specific implementation examples are shown in Table 2 below. All other preparation steps are the same as in Example 1. Table 2 Other Feasible Implementation Cases The biofilm-coated probiotic microcapsules prepared based on the conditions in Table 2 above were subjected to a probiotic survival test using the methods described in Part I of the performance test. The digestibility and survival rate were determined, and the test results are shown in Table 2 above. As shown in Table 2, the microcapsule products obtained in the above embodiments all have excellent probiotic digestion and survival rates, indicating that the technical solution provided by the present invention is feasible.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A biofilm-coated probiotic microcapsule, characterized in that, It is prepared by the following method: Probiotic suspension was mixed with polysaccharide solution, and then the mixture was contacted with cross-linking agent solution to cross-link and obtain polysaccharide cross-linked gel microcapsules. The pH of the complex culture medium was adjusted to 5-9, and then the polysaccharide cross-linked gel microcapsules were added and cultured to obtain biofilm-coated probiotic microcapsules.

2. The biofilm-coated probiotic microcapsule according to claim 1, characterized in that, The probiotics include at least one of the genera Lactobacillus and Bifidobacterium.

3. The biofilm-coated probiotic microcapsule according to claim 1, characterized in that, The polysaccharide is at least one of sodium alginate, pectin, and carrageenan.

4. The biofilm-coated probiotic microcapsule according to claim 1, characterized in that, The probiotic suspension contains at least 1×10⁻⁶ bacteria. 8 CFU / mL; the mass-volume concentration of the polysaccharide solution is 0.3-5%; the volume ratio of the probiotic suspension to the polysaccharide solution is 1:(10-30).

5. The biofilm-coated probiotic microcapsule according to claim 1, characterized in that, The crosslinking agent solution is at least one of calcium chloride aqueous solution, calcium lactate aqueous solution, and strontium chloride aqueous solution; the mass-volume concentration of the crosslinking agent solution is 0.5-8%.

6. The biofilm-coated probiotic microcapsule according to claim 1, characterized in that, Crosslinking is carried out by dripping, spraying, or peristaltic pump dripping, with a crosslinking time of 10-60 min.

7. The biofilm-coated probiotic microcapsule according to claim 1, characterized in that, The mass-to-volume ratio of the polysaccharide cross-linked gel microcapsules to the complex culture medium is 1:(5~20), g / mL.

8. The biofilm-coated probiotic microcapsule according to claim 1, characterized in that, The recombinant culture medium is MRS broth or other culture medium that can support the growth of probiotics; the culture conditions are: anaerobic culture at 30~45℃ for 12~72 h.

9. The use of the biofilm-coated probiotic microcapsules according to any one of claims 1 to 8 in improving probiotic tolerance.

10. The application of the biofilm-coated probiotic microcapsules according to claim 9 in improving probiotic tolerance, characterized in that, The tolerance is either processing tolerance or gastrointestinal digestive tolerance.

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