Single-cell probiotic packaging method of thiolated guluronic acid oligosaccharide and probiotic microcapsule

Abstract

The invention discloses a single-cell probiotic packaging method of thiolated guluronic acid oligosaccharide and a probiotic microcapsule, and relates to the technical field of biological medicines. The packaging method comprises the following steps: mixing a thiolated guluronic acid oligosaccharide solution with a probiotic suspension to form a probiotic-polysaccharide mixed solution; and adding a calcium chloride solution into the probiotic-polysaccharide mixed solution, and stirring to form the single-cell microcapsule encapsulating the probiotics. The packaging method can protect a single strain from being damaged by an external adverse environment to the maximum extent, and the survival rate is increased. Meanwhile, the obtained single-cell microcapsule can improve the digestion of the probiotics, the survival rate of the probiotics under the oxidative stress environment and the antibiotic combination, has high stability, and prolongs the intestinal residence time of the probiotics. An innovative solution is provided for the problems that in the existing medicine field, probiotic preparations cannot improve the gastrointestinal stress resistance of probiotics, and antibiotics lose efficacy when combined.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a single-cell probiotic encapsulation method of sulfhydrylated guluronate oligosaccharide and a probiotic microcapsule. BACKGROUND

[0002] Probiotics have shown great application potential and play a key role in the prevention and treatment of various diseases due to their dynamic interaction with the host's digestive system, immune system and metabolic network. However, probiotics face many challenges in practical application, which seriously restricts the full play of their efficacy. The adhesion ability of probiotics themselves in the intestinal tract is limited, and it is difficult to stably colonize in the intestinal tract. At the same time, the gastrointestinal environment is complex and harsh, and factors such as gastric acid, protease and bile salts will damage probiotics, leading to reduced activity and stability. Especially under pathological conditions, the oxidative stress environment in the intestinal tract further aggravates the survival difficulties of probiotics, greatly limiting their long-term survival and effective colonization, and thus affecting the effect of probiotics in preventing and treating diseases.

[0003] To solve the above problems, polysaccharide-based delivery systems are widely used in microbial encapsulation. At present, natural polysaccharide matrices such as xanthan gum, fucoidan and chondroitin sulfate are mainly used to embed and protect probiotics. However, these natural polysaccharide microcapsules have obvious defects. Their porous structure makes the encapsulation efficiency low, and they cannot effectively block the penetration of external harmful factors. Moreover, they are easily degraded in acid and alkaline environments, and cannot provide long-term and stable protection for probiotics in the complex and variable gastrointestinal environment.

[0004] To improve the encapsulation effect, layer-by-layer self-assembly technology and other processes have been tried to be applied to probiotic encapsulation. Although this process improves the encapsulation performance to some extent, it usually requires multiple coating operations, which not only leads to a complex and tedious preparation process, prolongs the production cycle, but also causes low material utilization and increases production costs. More importantly, in the embedding process of multiple coatings, the probiotic strains may be damaged by physical or chemical factors, affecting their activity and function, and thus limiting the large-scale application of this process.

[0005] Low-molecular-weight polyguluronate (PG) is a product of controlled degradation of alginate, which has unique gel properties and can form a stable structure to provide a good protective environment for probiotics. At the same time, low-molecular-weight polyguluronate also has good prebiotic activity, which can promote the growth and reproduction of probiotics. Its rigid backbone structure enables it to maintain excellent stability in extreme environments, such as the acid-base environment and oxidative stress environment of the gastrointestinal tract, and is not easily degraded. In addition, it has excellent cation chelating ability and natural advantages of specific adhesion in the intestinal stress area. However, there are still limitations in using low-molecular-weight polyguluronate alone to embed probiotics. By modifying the polysaccharide with thiol groups, a new delivery material with good biocompatibility can be obtained. Thiolated homopolymer low-molecular-weight polyguluronate can quickly form disulfide cross-linking with cysteine residues on the surface of probiotics, and anchor in the intestinal mucus layer through a dynamic cross-linking mechanism, thereby enhancing the retention and protection effect.

[0006] However, there is currently a lack of efficient microcapsule preparation methods suitable for thiolated low-molecular-weight polyguluronate-embedded probiotics. The existing technology cannot achieve universal packaging of different strains (such as gram-positive and negative bacteria, bacilli and cocci, etc.), which limits its diversified application. Therefore, the present application aims to develop a new probiotic delivery material based on the probiotic properties and biocompatibility of low-molecular-weight polyguluronate, and to construct a universal, efficient, and repeatable single-cell microencapsulation method for probiotics, in order to overcome the limitations of existing microbial delivery systems on strain specificity. SUMMARY

[0007] The technical problem to be solved by the present application is at least one of the technical problems mentioned in the background art, and aims to provide a new polysaccharide delivery material, thiolated low-molecular-weight polyguluronate, which has both prebiotic properties and biocompatibility, in order to improve the stress resistance and adhesion of probiotics. Based on the material of thiolated low-molecular-weight polyguluronate, a single-cell microencapsulation system suitable for different strains is constructed to improve the application value of probiotics.

[0008] To solve the above problems, the present application proposes the following technical solutions: In a first aspect, the present application provides a single-cell probiotic encapsulation method of thiolated low-molecular-weight polyguluronate, comprising: mixing a thiolated low-molecular-weight polyguluronate solution and a probiotic suspension to form a probiotic-polysaccharide mixed solution; adding a calcium chloride solution to the probiotic-polysaccharide mixed solution and stirring to form single-cell microcapsules encapsulating probiotics.

[0009] The further technical scheme is that the concentration of the mercapto-guluronic acid oligosaccharide solution is 0.5-5 mg / mL, the concentration of the probiotic suspension is not less than 1×10 9 CFU / mL, and the concentration of the calcium chloride solution is 0.1-1 mg / mL.

[0010] The further technical scheme is that the stirring is carried out at room temperature, the stirring speed is 150-300 rpm, and the stirring time is 1-30 min.

[0011] The further technical scheme is that the mercapto group substitution degree of the mercapto-guluronic acid oligosaccharide is 1600-1700 μmol / g, and the mercapto-guluronic acid oligosaccharide is obtained by coupling reaction of guluronic acid oligosaccharide and L-cysteine through carboxyl and amino.

[0012] The further technical scheme is that the preparation method of the mercapto-guluronic acid oligosaccharide comprises the following steps: dissolving guluronic acid oligosaccharide in a phosphate buffer solution, and adding a crosslinking agent to activate carboxyl on a molecular chain of the guluronic acid oligosaccharide; then adding L-cysteine, stirring at room temperature, purifying and freeze-drying the product after the reaction is completed, and obtaining the mercapto-guluronic acid oligosaccharide.

[0013] The further technical scheme is that the crosslinking agent comprises at least one of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.

[0014] The further technical scheme is that the probiotic in the probiotic suspension comprises at least one of gram-positive bacteria, gram-negative bacteria, bacilli or coccobacilli, preferably at least one of Escherichia coli (E. coli) EcN ), Lactobacillus plantarum (L. plantarum) Lactiplantibacillus plantarum ), and Lactococcus lactis (L. lactis) Lactococcus lactis ).

[0015] In the second aspect, the application provides a probiotic microcapsule, which is prepared by the single-cell probiotic encapsulation method of the mercapto-guluronic acid oligosaccharide, and the probiotic microcapsule is a single-cell encapsulation structure.

[0016] The application further provides application of the probiotic microcapsule in preparation of a medicine or health product for preventing or treating intestinal flora imbalance, inflammatory bowel disease, metabolic syndrome or enhancing immunity of the body.

[0017] In a third aspect, the present application also provides a probiotic encapsulation material, which is thiolated guluronate oligosaccharide having a thiol substitution degree of 1600-1700 μmol / g, is obtained by coupling modification of guluronate oligosaccharide with L-cysteine, and is used for encapsulating probiotics by the single-cell probiotic encapsulation method of the thiolated guluronate oligosaccharide.

[0018] Compared with the prior art, the present application can achieve the following technical effects: Based on the cross-linking of guluronate oligosaccharide and metal ions to form a three-dimensional network gel structure, the thiolated guluronate oligosaccharide solution and the calcium ion solution are mixed, and after stirring, the probiotic single-cell microcapsules with uniform and dense microstructure are formed. The encapsulation method of the present application can maximize the protection of individual strains from external adverse environments and improve the survival rate. At the same time, the obtained single-cell microcapsules have a larger specific surface area, which greatly increases the contact and adhesion opportunities with the intestinal mucosa, and is beneficial to the colonization and functional exertion of probiotics in the intestinal tract. This precise single-cell encapsulation strategy not only improves the protection performance of the probiotic delivery system, but also provides a new technical path for precise regulation of microbial functions.

[0019] The encapsulation material provided by the present application is thiolated guluronate oligosaccharide, which is constructed by modifying guluronate oligosaccharide with L-cysteine and has good biocompatibility. Guluronate oligosaccharide has a rigid skeleton and a unique molecular “pocket” structure that can provide more precise steric hindrance effect, significantly enhancing the protection performance of probiotics. Experiments show that, compared with guluronate oligosaccharide and medical enteric material methacrylic acid-methyl methacrylate copolymer (Eudragit® L100), the probiotics encapsulated based on thiolated guluronate oligosaccharide can covalently bind with the free thiol of proteins in the intestinal mucus layer through the dynamic and rapid cross-linking characteristics of disulfide bond, significantly enhancing the anchoring ability of probiotic single-cell microcapsules on the surface of intestinal mucosa, prolonging the retention time of probiotics in the intestinal tract, and thus improving the intestinal colonization efficiency of probiotics. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 The response surface method is used to optimize the synthesis conditions of thiolated guluronate oligosaccharide (sPG) in the present application. A, B and C are three-dimensional surface optimization, and D is a residual normal graph.

[0022] Figure 2 Physical and chemical characterization of L-cysteine (Cys), glucuronic acid oligosaccharide (PG) and sPG in the application. Wherein A is the Fourier transform infrared spectrum of Cys, PG and sPG, B is the nuclear magnetic resonance spectrum of PG and sPG, and C is the zeta potential determination result of PG and sPG.

[0023] Figure 3 Biocompatibility of thiolated glucuronic acid. Wherein, A is the cytotoxicity of sPG to RAW264.7 and HCoEpiC cell lines, B is the growth influence of different concentrations of PG and sPG on probiotics, and C is the hemolysis experiment result of different concentrations of sPG.

[0024] Figure 4 Single-cell microcapsules of probiotics encapsulated by thiolated glucuronic acid oligosaccharide in the application. Wherein A is the encapsulation rate of gram-negative bacteria Escherichia coli Nissle 1917 (EcN), B is the growth curve, C is the transmission electron microscope image of EcN microcapsules, D is the transmission electron microscope image of gram-positive bacteria Lactobacillus plantarum (Lp) microcapsules, and E is the transmission electron microscope image of gram-positive bacteria Lactococcus lactis (Ll) microcapsules. Lactiplantibacillus plantarum )microcapsules. Lactococcus lactis )microcapsules.

[0025] Figure 5 Survival of different encapsulated probiotics in different environments. Wherein A is a simulated artificial gastric juice environment, B is a simulated artificial intestinal juice environment, C is a simulated oxidative stress environment, and D is a simulated antibiotic combination environment.

[0026] Figure 6 Intestinal adhesion of probiotic single-cell microcapsules observed by 3D fluorescence imaging technology in the application: A is a normal mouse, and B is a DSS-induced colitis mouse. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the following described embodiments are only part of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0028] It should be understood that the terms "comprises" and "comprising," when used in this specification and the following claims, indicate the presence of the described features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0029] It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present embodiments. As used in this specification and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0030] In the present embodiments, "room temperature" means 0-50°C; in some embodiments, room temperature means 4-40°C; in some embodiments, room temperature means 16-35°C; in some embodiments, room temperature means 20-35°C.

[0031] Example 1: Thiolated guluronate oligosaccharide and preparation thereof The present embodiments provide a probiotic encapsulating material, which is thiolated guluronate oligosaccharide (sPG). Understandably, the degree of substitution of thiol is a key parameter for measuring the number of thiol (-SH) groups on the guluronate oligosaccharide molecule, which directly determines the final physicochemical properties (encapsulation effect, protection through the gastrointestinal tract) and biological functions (adhesion and localization in the intestinal tract) of the material, and thus fundamentally affects the efficiency of the entire probiotic delivery system. Too low a degree of substitution can result in insignificant modification effect, failing to fully exert the advantages of cross-linking and adhesion with mucus proteins in the intestinal tract. Too high a degree of substitution can excessively change the natural structure of the polysaccharide, which in turn interferes with its recognition and utilization by probiotics, resulting in a decrease in prebiotic activity.

[0032] In the present embodiments, the preparation conditions of thiolated guluronate oligosaccharide are optimized by response surface methodology. Three factors, namely the amount of crosslinking agent, the amount of L-cysteine (Cys) and the reaction time, are selected, and the free thiol content of sPG is used as the response value. A response surface design test is performed according to three factors and three levels, a fitting equation is obtained, and the optimal process parameters are verified. The experimental scheme and results of response surface analysis are shown in Table 1.

[0033] Guluronate oligosaccharide (PG) was dissolved in phosphate buffer at pH 6.0 at 10 mg / mL, and 50-200 mg of crosslinking agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added in sequence to form an active intermediate by activating the carboxyl group. Then 25-400 mg of Cys was added, and the reaction was stirred at 200-300 rpm for 12-24 h at room temperature to couple PG with Cys. After the reaction was completed, the solution was purified by dialysis, and after lyophilization, it was stored at -20°C. The content of sPG thiol group was calculated by measuring the absorbance of the solution at 412 nm using Ellman's reagent method.

[0034] Table 1 Experimental scheme for response surface optimization of sPG preparation (1) According to the test results, multiple regression analysis was performed to establish a binary regression equation of the corresponding variables and the response value sPG free thiol content, as follows: Y = -1684.26 - 1.72X1 + 1.01X2 + 310.64X3 + 0.02X1X2 + 0.15X1X3 - 0.02X2X3 + 0.01X1 2 -8.79X3 2 X1: crosslinking agent addition amount; X2: Cys addition amount; X3: reaction time (2) The results of variance analysis are shown in Table 2, and the model has significance (P < 0.0001). p <0.0001>.

[0035] Table 2 Variance analysis of regression coefficients of the fitted quadratic equation (3) The preparation conditions were optimized using Design-Expert 8.0.6 software to obtain the order of factors affecting the free thiol content: crosslinking agent addition amount > Cys addition amount > reaction time. Under the optimized preparation conditions, the predicted response value of sPG free thiol content was 1615.89 μmol / g, and the experimental value of sPG prepared in the laboratory was 1645.04 ± 18.54 μmol / g (Table 3). The p value between the experimental value and the predicted value was 0.053, and there was no significant difference (P > 0.05) between the experimental value and the predicted value. p > 0.05).

[0036] Table 3 Experimental and predicted response values under optimized conditions It is understandable that the thiol groups can combine with each other to form firm disulfide bonds (-S-S-) under mild oxidation conditions or through thiol-disulfide exchange reactions. The higher the degree of substitution, the more sites available for cross-linking on the molecular chain, thus enabling the construction of a more compact and stable three-dimensional network structure. In turn, the mechanical strength of the microcapsules is higher, and they can better resist the physical shear force generated by the peristalsis of the gastrointestinal tract, preventing premature rupture of the microcapsules. At the same time, it can effectively block the penetration of harmful substances such as gastric acid, bile salts, digestive enzymes, and provide more persistent protection for the internal probiotics. The thiol substitution degree of the best thiolated guluronate oligosaccharide constructed in this embodiment is 1645.04 ± 18.54 μmol / g, and in other embodiments, a thiol substitution degree in the range of 1600-1700 μmol / g can have similar effects.

[0037] Example 2 Physicochemical characterization of thiolated guluronate oligosaccharide The structure of sPG was characterized by Fourier transform infrared spectroscopy. Cys, PG and sPG obtained in Example 1 were freeze-dried into powder and mixed with KBr according to the proportion, ground and pressed into a tablet. The Fourier transform infrared spectrometer was used to obtain the spectral signal from wave number 650-4000 cm -1 The recorded spectral signal. According to Figure 2 , among them, Figure 2 In A, the wide peak of Cys at 2965 cm -1 nearby indicates the presence of -NH2, and the peak value at 2548 cm -1 can be attributed to the stretching of S-H in thiol. In sPG, the stretching of S-H shifts from 2548 cm -1 to 2555 cm -1 , and this stretching becomes less intense. The characteristic peaks of PG also exist in the infrared spectrum of sPG, but compared with PG, a new characteristic peak appears at 1741 cm -1 nearby, which is related to the stretching vibration of carbonyl, indicating the formation of amide bond.

[0038] PG and sPG powders were dissolved in D2O. The 1D NMR spectrum of sPG was collected using a superconducting nuclear magnetic resonance spectrometer. According to Figure 2 The results of B nuclear magnetic resonance hydrogen spectrum also prove that Cys is successfully connected to PG.

[0039] PG and sPG powders were dissolved in deionized water to prepare a solution with a concentration of 100 μg / mL. The dynamic light scattering instrument was used to determine the zeta potential of each sample. PG and sPG are both strongly negatively charged, Figure 2 C shows that the surface charge density of the particles is high, and the system has good stability. The statistical results show that there is a significant difference between the two ( p<0.05), the surface negative charge density of sPG was significantly higher than that of PG, which proved that the thiolated modification further enhanced the electrostatic repulsion and dispersion stability.

[0040] Biocompatibility of thiolated guluronate The effect of sPG on the viability of RAW264.7 and HCoEPiC cells was detected by CCK-8 method. Cells were seeded in 96-well plates at a density of 1 × 10 5 cells / well, and cultured for 24 h to adhere to the wall. Then, the cells were cultured with sPG medium containing 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL of sPG for another 24 h. CCK-8 reagent was added to each well, and incubated for 1-4 h. The absorbance was detected by a microplate reader at 450 nm. The cell wells without samples were used as negative controls to calculate the relative cell viability percentage at each concentration. According to the cell viability curve, the IC50 value was calculated. Figure 3 A. Within the concentration range of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL, the sPG did not show obvious cytotoxicity to the two cell lines, and the cell viability was maintained at a high level.

[0041] According to the growth curve of free Escherichia coli Nissle 1917 (EcN), the growth activity of EcN co-incubated with different concentrations of PG and sPG was measured by UV-Vis spectrophotometer, and the OD 600 values were recorded every 2 h. According to the growth curve of free EcN, the IC50 value was calculated. Figure 3 B. Compared with the blank control, PG and sPG at concentrations of 2, 4, and 6 mg / mL did not significantly inhibit the growth of EcN within the 20 h monitoring period; the OD 600 curves of each treatment group were almost overlapped with the free EcN control, and all entered the logarithmic phase at 2-3 h, and reached the platform (OD 600 ≈1.0) at 8-10 h. The results suggested that PG and sPG had no significant effect on the growth activity of EcN within the measured concentration range, indicating that these two polysaccharides had no inhibitory effect on the probiotic bacteria EcN.

[0042] Fresh blood was collected from the ear veins of healthy New Zealand white rabbits. The blood was then diluted with saline at a ratio of 4:5. Three types of samples were prepared for this experiment: a negative control (physiological saline), a positive control (Triton X), and test samples (sPG at different concentrations). Each sample (1 mL) was immersed in a 37°C water bath for 30 min. Afterward, 20 μL of diluted rabbit blood was added to each tube. The tubes were further incubated in a 37°C water bath for 1 h. After incubation, the samples were centrifuged at 500 × g for 5 min. The supernatant was carefully removed, and the absorbance was measured at 545 nm. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = (OD) t -OD nc ) / (OD pc -OD nc ) × 100%.

[0043] OD t The absorbance and OD of the samples after treatment with different sPG concentrations nc This is the absorbance of the negative control, OD. pc This is the absorbance of the positive control.

[0044] according to Figure 3 The results showed that the hemolysis rate in the sPG concentration groups of 1.25-20 mg / mL was <5%, which was not different from the PBS negative control. No obvious hemolysis was observed, indicating that sPG is safe for rabbit erythrocytes.

[0045] Example 4: Encapsulation method for single-cell probiotics containing thiolated guluronic acid oligosaccharides This embodiment uses the Gram-negative bacillus Escherichia coli Nissle 1917 (EcN) as an example. The activated probiotics were cultured to the logarithmic growth phase, centrifuged to collect the cells, washed, and resuspended in sterile physiological saline to obtain 1×10⁻⁶ cells. 9 CFU / mL probiotic suspension. sPG solution (1 mg / mL) and calcium chloride solution (0.2 mg / mL) were added to the bacterial suspension and stirred at 200 rpm to form probiotic single-cell microcapsules Ca-sPG@EcN. PG was prepared as a control using the same method to create probiotic single-cell microcapsules Ca-PG@EcN. Gram-positive bacilli were also used. Lactiplantibacillus plantarum ( L. plantarum ) and Gram-positive cocci Lactococcus lactis ( L. lactis Using representative strains, the encapsulation method of thiolated guluronic acid oligosaccharides for single-cell probiotics provided in this invention was verified to be universal in different types of probiotics.

[0046] The unencapsulated bacterial suspension was centrifuged at 4°C, 4000 rpm for 5 min, and the supernatant was collected. The suspension of the microcapsule-encapsulated group was centrifuged at 4°C, 1000 rpm for 5 min, and the supernatant was collected. The centrifugation conditions were optimized by pre-experiment to completely precipitate the microcapsules without rupture, ensuring that only unencapsulated or free bacteria were contained in the supernatant. Add 90 μL of the above supernatant to each well of a 96-well plate, and then add 10 μL of CCK-8 reagent. Incubate at 37°C in the dark for 2 h. Measure the absorbance (OD value) at 450 nm wavelength with a microplate reader, and record OD n (blank control group) and OD t (microcapsule-encapsulated group), respectively. Five replicate wells were set for each sample, and the average value was taken. Convert the OD value to the encapsulation efficiency using the following formula: Encapsulation efficiency (%) = [(OD n - OD t ) / OD n ] x 100%.

[0047] wherein OD t is the OD test value obtained in different microcapsule-encapsulated groups, and OD nc is the OD test value obtained in the control group.

[0048] According to Figure 4 A, the encapsulation efficiency of different formulations showed significant differences. sPG showed excellent encapsulation performance, with an encapsulation efficiency of 90% in only 1 min, and the encapsulation efficiency could be rapidly increased to 99% and remained stable when the encapsulation time was extended to 5 min. This result indicates that thiolated guluronate oligosaccharide has a significant advantage in completing efficient single-cell encapsulation in a very short time, providing an innovative design strategy for probiotic delivery systems.

[0049] The EcN, Ca-PG@EcN, Ca-sPG@EcN, and sPG@EcN groups were incubated at 37°C, and the OD 600 was measured every 2 h with a microplate reader to monitor the growth of the strains and draw a growth curve to ensure that encapsulation does not affect the normal physiological activity of probiotics. Figure 4 B shows that the growth dynamics of the Ca-PG@EcN, Ca-sPG@EcN, and sPG@EcN groups are basically consistent with those of free EcN. It is worth noting that the probiotics in the Ca-sPG@EcN and sPG@EcN groups show more excellent growth state in the late growth stage, which clearly proves that sPG not only does not inhibit the normal proliferation of probiotics, but also may provide additional nutritional support for the strains. This finding further verifies the biocompatibility and potential biological functionality of sPG as a new delivery material.

[0050] The surface morphology of EcN, Ca-sPG@EcN, and Ca-PG@EcN was observed using transmission electron microscopy (TEM). Bacterial solution was added to a 300-mesh carbon-coated copper grid and allowed to stand for 3 minutes. Finally, excess liquid was absorbed using filter paper and the cells were dried under a heat lamp for 3 minutes for TEM observation. Figure 4 The C10 transmission electron microscopy image shows a distinct coating around the bacterial cells, indicating that sPG and PG were successfully coated on the bacterial surface. According to... Figure 4 D and 4E, the same encapsulation method is applied to Gram-positive bacilli. L. plantarum and Gram-positive cocci L. lactis All methods can achieve the encapsulation effect, indicating that the single-cell probiotic encapsulation method of thiolated guluronic acid oligosaccharides in the embodiments of the present invention has good cross-strain universality and high reproducibility, and can be used as a broad-spectrum probiotic delivery platform.

[0051] Example 5: Survival of probiotics encapsulated in different materials in different environments This implementation uses the medical enteric-coated material methacrylate-methyl methacrylate copolymer (Eudragit) ® L100 was used as the positive control shell material to prepare L100@EcN microcapsules. EcN, Ca-PG@EcN, Ca-sPG@EcN, and L100@EcN were placed in simulated gastric digestive fluid (SGF, containing 0.2% NaCl and 3.2 g / L pepsin, pH 2.0) and cultured anaerobically at 37℃ for 0.5 h, 1 h, 1.5 h, and 2 h, respectively. After culturing, the bacterial suspension was washed multiple times with PBS and resuspended, and bacterial viability was detected by the CCK-8 assay. Figure 5 A. After SGF exposure, the survival rate of each group decreased in a gradient over time. The positive control L100@EcN dropped to approximately 65% ​​at 0.5 h and only 35% at 2 h, demonstrating that traditional enteric polymers offer limited gastric digestive protection for probiotics. In contrast, Ca-PG@EcN increased the 2-h survival rate to approximately 60%, while Ca-sPG@EcN performed best: the survival rate was close to 100% at 0.5 h and remained at approximately 80% at 2 h, significantly higher than the other three groups. p < 0.01). This result directly confirms that sPG can provide excellent barrier protection for probiotics in the extreme gastric acid environment, and its performance is significantly better than that of the enteric material L100.

[0052] The growth activity of EcN, Ca-PG@EcN, Ca-sPG@EcN and L100@EcN in simulated intestinal fluid (SIF, trypsin 10 mg / mL, 6.8 mg / mL KH2PO4, pH 7.0) was measured using a UV-Vis spectrophotometer, and the OD values were recorded every 2 h with reference to the growth curve of free Escherichia coli Nissle 1917 (EcN). 600 Figure 5 B, in simulated intestinal fluid, free EcN hardly grew. The OD value of the Ca-PG@EcN group reached 0.6 at 6 h, with a 3-fold increase in survival; the OD value of the Ca-sPG@EcN group reached 0.8 at 4 h, with a 4-fold increase in survival, indicating that Ca-sPG can promote the growth of probiotics in a simulated intestinal environment. 600 600

[0053] After EcN, Ca-PG@EcN, Ca-sPG@EcN and L100@EcN were treated in 1 mM H2O2, 37°C anaerobic environment for 0 h, 1 h, 2 h, 3 h respectively, after culture, the bacterial liquid was washed and resuspended with PBS for multiple times, and the bacterial activity was detected by CCK-8 method. As a control group, the survival rate of free EcN bacteria decreased most sharply under H2O2 attack, indicating that EcN bacteria were easily damaged when directly exposed to an oxidative environment, and the survival rate was very low after 3 h. L100@EcN as a positive control also only remained about 20%, suggesting that the anti-oxidative stress ability of traditional enteric polymer was limited; the Ca-PG@EcN curve was obviously flat, with a 3 h survival rate of about 40%, indicating that unmodified PG could provide a certain barrier. In contrast, the survival rate of Ca-sPG@EcN was always the highest during the entire exposure period, still maintaining about 82% after 3 h, which was significantly better than that of L100@EcN and Ca-PG@EcN (P<0.01), confirming that thiol modification endows the material with excellent oxidative barrier function, enabling probiotics to maintain high activity in extreme oxidative environments. p <0.01), confirming that thiol modification endows the material with excellent oxidative barrier function, enabling probiotics to maintain high activity in extreme oxidative environments.

[0054] After EcN, Ca-PG@EcN, Ca-sPG@EcN and L100@EcN were treated in 1% penicillin-streptomycin antibiotic, respectively, for 0 h, 1 h, 2 h, 3 h, after culture, the bacterial liquid was washed and resuspended with PBS for multiple times, and the bacterial activity was detected by CCK-8 method. Under the stress of 1% penicillin-streptomycin, the survival rate of free EcN decreased sharply to below 2% within 3 h, indicating that it was almost impossible to resist the killing effect of high-concentration antibiotics. The positive control L100@EcN still only remained about 12% in survival rate, indicating that the physical barrier effect of conventional enteric coating against antibiotics was limited and difficult to effectively block the penetration of small molecule antibiotics. ​​​

[0055] In contrast, Ca-PG@EcN showed better protection, with a 3 h survival rate of about 45%, significantly higher than that of free bacteria and L100@EcN, demonstrating that the calcium ion coordination cross-linking structure enhances the protection of the bacterial body. Ca-sPG@EcN maintained the highest survival level throughout the 3 h exposure period, with a final survival rate of up to 88%, significantly better than L100@EcN and Ca-PG@EcN (P < 0.01). This excellent result is due to the dual cross-linking network of sPG constructed in its shell: on the one hand, a stable matrix is formed by calcium ion coordination, and on the other hand, a more compact and dynamically stable barrier layer is formed by thiol-disulfide exchange reaction, effectively blocking the penetration of antibiotic molecules and greatly improving the survival ability of EcN in extreme antibiotic environments. p <0.01). This excellent result is due to the dual cross-linking network of sPG constructed in its shell: on the one hand, a stable matrix is formed by calcium ion coordination, and on the other hand, a more compact and dynamically stable barrier layer is formed by thiol-disulfide exchange reaction, effectively blocking the penetration of antibiotic molecules and greatly improving the survival ability of EcN in extreme antibiotic environments.

[0056] Example 6 Intestinal adhesion of probiotics encapsulated by different materials This implementation collects healthy 6-week-old C57BL / 6 male mice and the same strain of male mice with induced intestinal stress by 3% dextran sulfate sodium (DSS) drinking water for one week. One side of the colon is sutured and ligated, and the colon mucus is stained with 100 μL of WGA-FITC fluorescent dye for 1 h. The colon tissue is cut open, and 100 μL of 1 x 10 9 CFU / mL of mCherry-EcN, PG@mCherry-EcN and sPG@mCherry-EcN containing mCherry fluorescent plasmid are added to the mucus surface, and naturally settled at 37°C for 1 h, washed 3 times, and the unadhered samples are washed away. The adhesion rate of probiotics in normal intestinal tissue and intestinal stress area is compared using 3D imaging technology to evaluate the active tropism of the system to the intestinal stress area.

[0057] According to Figure 6 In the normal intestinal tract of mice, free EcN (red) and WGA-FITC labeled intestinal mucosa (green) are co-localized in a limited area, and the adhesion is weak; Ca-sPG@EcN coated with Ca-sPG shows a large range of fluorescent co-localization area in the merged image, indicating that its combination with the intestinal mucosa is significantly enhanced, and the adhesion is excellent. The traditional packaging material L100@EcN also shows a certain adhesion, but it is significantly weaker than Ca-sPG@EcN.

[0058] According to Figure 6In the DSS-induced colitis mouse model, free EcN adhesion is further weakened; in contrast, Ca-sPG@EcN still maintains a strong co-localization signal, indicating that it can still adhere efficiently in the inflammatory intestinal environment. This is mainly due to the fact that sPG can be rapidly and dynamically cross-linked through disulfide bonds, enhancing interaction with mucin, while its own negative charge helps to target and enrich in the positively charged inflammatory area, thereby exhibiting excellent and stable intestinal adhesion capacity in both normal and colitis states.

[0059] The above experimental results show that the thiolated guluronate oligosaccharide with high biocompatibility, safety and stability is successfully prepared by the precisely optimized process. Based on the characteristics of rapid covalent anchoring of the thiolated guluronate oligosaccharide to the cysteine residues on the surface of probiotic bacteria and chelation with cations, the thiolated guluronate oligosaccharide and the single-cell probiotic bacteria encapsulation method are provided. The prepared probiotic bacteria microcapsules can provide comprehensive and effective protection for probiotic bacteria, significantly improve the survival ability of probiotic bacteria under various adverse conditions such as simulated gastrointestinal environment, oxidative stress and antibiotic stress, have high stability, can enhance the anchoring and retention of probiotic bacteria in the intestinal mucosa layer, thereby effectively promoting the colonization of probiotic bacteria in the intestinal tract and prolonging the intestinal retention time of probiotic bacteria. The present application provides an innovative solution to the problems of existing probiotic bacteria preparations, such as inability to improve the gastrointestinal stress resistance of probiotic bacteria and failure when used in combination with antibiotics.

[0060] In the above embodiments, the description of each embodiment focuses on different aspects, and the parts not described in detail in a certain embodiment can be referred to the related description of other embodiments.

[0061] The above is a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present application, and these modifications or replacements should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides, characterized in that, include: A probiotic-polysaccharide mixed solution was formed by mixing a thiolated guluronic acid oligosaccharide solution with a probiotic suspension. Calcium chloride solution was added to the probiotic-polysaccharide mixture and stirred to form single-cell microcapsules encapsulating the probiotics.

2. The method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides as described in claim 1, characterized in that, The concentration of the thiolated guluronic acid oligosaccharide solution is 0.5-5 mg / mL, and the concentration of the probiotic suspension is not less than 1×10⁻⁶ mg / mL. 9 CFU / mL, wherein the concentration of the calcium chloride solution is 0.1-1 mg / mL.

3. The method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides as described in claim 1, characterized in that, The stirring is carried out at room temperature, at a stirring speed of 150-300 rpm, and for a stirring time of 1-30 min.

4. The method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides as described in claim 1, characterized in that, The thiolated guluronic acid oligosaccharide has a thiol substitution degree of 1600-1700 μmol / g and is obtained by coupling guluronic acid oligosaccharide and L-cysteine ​​through a carboxyl-amino coupling reaction.

5. The method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides as described in claim 4, characterized in that, The method for preparing the thiolated guluronic acid oligosaccharide includes: Dissolve guluronic acid oligosaccharides in phosphate buffer and add a cross-linking agent to activate the carboxyl groups on their molecular chains; L-cysteine ​​was then added, and the mixture was stirred at room temperature to obtain thiolated guluronic acid oligosaccharides.

6. The method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides as described in claim 5, characterized in that, The crosslinking agent includes at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

7. The method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides as described in claim 1, characterized in that, The probiotic suspension contains at least one of Gram-positive bacteria, Gram-negative bacteria, bacilli, or cocci.

8. A probiotic microcapsule, characterized in that, The probiotic microcapsules are prepared by the single-cell probiotic encapsulation method of thiolated guluronic acid oligosaccharides according to any one of claims 1-7, wherein the probiotic microcapsules have a single-cell encapsulation structure.

9. The use of the probiotic microcapsules as described in claim 8 in the preparation of drugs or health products for the prevention or treatment of intestinal flora imbalance, inflammatory bowel disease, metabolic syndrome or to enhance the body's immunity.

10. A probiotic encapsulation material, characterized in that, The probiotic encapsulation material is a thiolated guluronic acid oligosaccharide with a thiol substitution degree of 1600-1700 μmol / g, obtained by coupling modification of guluronic acid oligosaccharide with L-cysteine, and used to encapsulate probiotics by the method of any one of claims 1-7.

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