Preparation method and application of probiotic-loaded sulfydryl hyaluronic acid-based hydrogel

Abstract

The invention belongs to the technical field of probiotic protection, and discloses a preparation method and application of probiotic-loaded sulfydryl hyaluronic acid-based hydrogel, sulfydryl hyaluronic acid is prepared by taking cysteamine hydrochloride as a substrate, and connection of L-glutamine is further catalyzed through a cross-linking agent, so that the sulfydryl hyaluronic acid-based hydrogel is obtained. The cross-linking agent is a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide or a mixture of N-hydroxysuccinimide and 1-hydroxybenzotriazole (HOBT), and the cross-linking agent is a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole (HOBT). The hydrogel prepared by the preparation method disclosed by the invention is used as a probiotic carrier, so that the influence of complex gastrointestinal tracts is effectively resisted, the hydrogel accurately reaches an intestinal injury part and is split, and the targeted intestinal controllable release of probiotics is realized; meanwhile, the L-glutamine is introduced and has a synergistic effect with the probiotics to relieve specific intestinal diseases, so that the L-glutamine can be used as a probiotic supplement for the specific intestinal diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic protection, and in particular to a preparation method and application of a thiol hyaluronic acid-based hydrogel loaded with probiotics. Background Art

[0002] Driven by the national "Healthy China" strategy, probiotics have become a favorite in people's pursuit of health thanks to their numerous beneficial functions, including regulating intestinal flora balance, participating in immune responses, and inhibiting the growth and reproduction of intestinal pathogens. However, existing technologies for the application and delivery of probiotics still face many challenges that need to be addressed.

[0003] Due to the extremely complex human gastrointestinal ecosystem, the functional effects of some probiotics are not ideal, and their health benefits in the human body need further confirmation. The number, vitality, and adhesion ability of probiotics in the human intestine are important foundations for achieving their beneficial effects. However, during oral delivery, probiotics face challenges such as harsh gastrointestinal conditions and rapid transit through the digestive tract. These challenges include weak intestinal adhesion, low oral bioavailability, and unclear effects, which seriously hinder the beneficial effects of probiotics.

[0004] Encapsulation provides a suitable microenvironment for probiotics, isolating the bacteria from the relatively harsh external environment, thereby improving the survival advantage of probiotics in overcoming chemical barriers in the digestive tract. Existing technologies usually use protein or protein-polysaccharide carriers to load probiotics, which lack targeted responsiveness to specific intestinal microenvironments (such as the common reducing environment in inflammatory areas). This results in the inability of loaded probiotics to accurately locate and enrich in the diseased part of the intestine, and cannot meet the needs of precise microecological intervention for specific intestinal diseases, thereby limiting the application effect of probiotics in the treatment of intestinal diseases.

[0005] While hyaluronic acid, a natural polysaccharide carrier, possesses excellent biocompatibility and gelling ability, its inherent physical properties (such as mechanical strength and anti-swelling properties) are insufficient and it is prone to rapid degradation in the digestive tract. This results in insufficient protection of probiotics from hyaluronic acid gels, necessitating modification or modification to improve bioavailability. Furthermore, existing carriers primarily function to encapsulate and protect probiotics, acting as a "transport vehicle" for them. They typically lack direct biological activity and are unable to actively participate in the regulation of intestinal diseases, limiting their overall effectiveness.

[0006] Through searching, no patent publication documents related to the patent application of the present invention have been found. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a method for preparing and applying a thiol-hyaluronic acid-based hydrogel loaded with probiotics and having specific biological activity.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] A method for preparing a thiol hyaluronic acid-based hydrogel loaded with probiotics comprises the following steps: activating hyaluronic acid with a cross-linking agent, adding an organic matter to carry out a cross-linking reaction, and obtaining a final product after the reaction is completed; and further treating the product, freeze-drying it, and redissolving it in a bacterial solution to obtain the thiol hyaluronic acid-based hydrogel loaded with probiotics.

[0010] Further, the following steps are included:

[0011] (1) Probiotics are statically cultured in MRS liquid culture medium, and the cultured bacterial liquid is centrifuged to obtain bacterial sludge; the bacterial sludge obtained by centrifugation is resuspended in phosphate buffer solution to form a bacterial suspension;

[0012] (2) adding a crosslinking agent to the hyaluronic acid solution, stirring at room temperature for 1 to 2 hours, adding cysteamine hydrochloride in the dark, adjusting the pH to 4.5 to 5.0, and continuing to stir and react at room temperature for 1 hour to obtain a thiol hyaluronic acid solution;

[0013] (3) adding a crosslinker to the thiol hyaluronic acid solution again, stirring at room temperature in the dark for 1 to 2 hours, adding L-glutamine, adjusting the pH to 4.5 to 5.0, stirring continuously at room temperature in the dark for 24 hours, and obtaining a thiol hyaluronic acid / L-glutamine mixed solution;

[0014] (4) dialyzing the thiol hyaluronic acid / L-glutamine mixed solution against a dilute hydrochloric acid solution with a pH of 3.5 for 48 h, and freeze-drying under vacuum to obtain a thiol hyaluronic acid / L-glutamine cotton-like solid;

[0015] (5) Dissolving the thiol hyaluronic acid / L-glutamine cotton-like solid in the bacterial suspension and incubating to obtain the thiol hyaluronic acid / L-glutamine hydrogel loaded with probiotics.

[0016] Furthermore, the static culture condition in step (1) is static culture in a 37°C constant temperature incubator for 16 to 18 hours, and the number of viable bacteria reaches 1×10 8 CFU / mL or above;

[0017] Alternatively, the probiotics in step (1) include Bifidobacterium, Lactobacillus, Streptococcus, and Lactococcus;

[0018] Alternatively, in step (1), the bacterial sludge is obtained by centrifugation at 7000×g for 10 min;

[0019] Alternatively, the pH of the phosphate buffer solution in step (1) is 7.2, and the viable bacterial count of the bacterial suspension after resuspension reaches 1×10 9 CFU / mL or above;

[0020] Alternatively, Bifidobacterium spp. is cultured anaerobically during static culture.

[0021] Furthermore, the preparation method of the hyaluronic acid solution in step (2) is as follows: dissolving hyaluronic acid or hyaluronate with a molecular weight of 100 to 200 kDa in distilled water to obtain a hyaluronic acid solution with a mass concentration of 0.4%;

[0022] Alternatively, the molar concentration of cysteamine hydrochloride in step (2) is 5 mM;

[0023] Alternatively, the cross-linking agent in step (2) and step (3) is a mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), or the cross-linking agent is a mixture of N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBT); the molar concentration of the cross-linking agent in step (2) is 5 mM, and the molar concentration of the cross-linking agent in step (3) is 1.25 mM.

[0024] Furthermore, the molar concentration of cysteamine hydrochloride in step (2) is 5 mM, the reaction process needs to be protected from light, and the stirring speed is 800-1000 r / min.

[0025] Furthermore, the molar concentration of L-glutamine in step (3) is 6.25 mM, the reaction is continuously shielded from light, and the stirring speed is 800-1000 r / min.

[0026] Furthermore, during the dialysis in step (4), a dialysis interception tube or a dialysis bag is used for dialysis, the molecular weight of the dialysis interception tube or the dialysis bag is 3.5 kDa, and the dialysate is replaced every 10 to 12 hours; and the vacuum freeze-drying time in step (4) is 24 to 48 hours.

[0027] Furthermore, step (5) needs to be performed under aseptic conditions in a clean bench; the hydrogel is incubated in a 37°C incubator for 1 hour to obtain the hydrogel;

[0028] Alternatively, the mass concentration of the mercapto hyaluronic acid / L-glutamine cotton-like solid in (5) is 4% after being dissolved in the bacterial suspension.

[0029] The probiotic-loaded thiol hyaluronic acid-based hydrogel prepared by the preparation method described above.

[0030] Application of the mercapto hyaluronic acid-based hydrogel as described above in biomedical materials and / or controlled drug release and / or food chemistry.

[0031] The advantages and positive effects achieved by the present invention are:

[0032] 1. The present invention uses hyaluronic acid with good biocompatibility as raw material and prepares hydrogel by adding chemical crosslinking agent and L-glutamine for simple chemical synthesis reaction. Using it as a probiotic carrier can effectively resist the effects of gastric acid and bile salts. In the simulated gastrointestinal continuous digestion experiment, the probiotics were 7.81×10 10 The CFU / mL decreased to 3.27×10 9 The CFU / mL only dropped by 1 order of magnitude, achieving the encapsulation protection of probiotics.

[0033] 2. The present invention provides a method for preparing a thiol hyaluronic acid hydrogel by cross-linking L-glutamine. The method comprises activating the carboxyl groups of hyaluronic acid with an activator, adding a cross-linking agent and cysteamine hydrochloride to prepare thiol hyaluronic acid, repeating the process, adding L-glutamine to form chemical cross-links, and dialyzing, freeze-drying, and redissolving to obtain a thiol hyaluronic acid / L-glutamine hydrogel. The method of the present invention cross-links L-glutamine, which has the function of promoting the repair of intestinal mucosal epithelial tissue, on the basis of thiol hyaluronic acid. The constructed composite hydrogel has a synergistic effect with the probiotics, thereby increasing the efficacy against intestinal diseases.

[0034] 3. The thiolated hyaluronic acid hydrogel prepared by the present invention has good mechanical properties, stability and biocompatibility, and is responsive to reduction stimuli. It can accurately reach the site of intestinal damage and lyse and release probiotics, thereby achieving targeted intestinal controlled release of probiotics.

[0035] 4. The preparation method of the present invention does not require any special equipment, and the required raw materials are easily available and can be obtained from commercial channels, making it easier to process and obtain.

[0036] 5. The present method utilizes modified hyaluronic acid to construct a cross-linked system. Common cross-linking agents for hyaluronic acid modification are compatible with the present system, and the cross-linked system significantly improves reaction stability. The probiotic-loaded hydrogel prepared by the present invention exhibits excellent in vivo safety and improves the tolerability of probiotics in gastrointestinal fluids, making it suitable for use as a delivery system for intestinal-targeted, controlled cleavage and release.

[0037] 6. The hydrogel prepared by the present invention can be used as a probiotic carrier to effectively resist the influence of a complex gastrointestinal tract, accurately reach the site of intestinal damage and lyse it, thereby realizing the targeted intestinal controlled release of probiotics; at the same time, the introduction of L-glutamine and the synergistic effect of probiotics can alleviate specific intestinal diseases, and can be used as a probiotic supplement for specific intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Graphs showing the effects of the secondary crosslinker concentration and L-glutamine concentration on the swelling (a, c) and degradation behavior (b, d) of the thiol hyaluronic acid / L-glutamine hydrogel in the present invention;

[0039] Figure 2 Graph showing the relationship between G' and G" of different concentrations of mercapto hyaluronic acid / L-glutamine hydrogels of the present invention as a function of amplitude (a), frequency (b), and temperature (c);

[0040] Figure 3 The microstructure of the mercapto hyaluronic acid / L-glutamine hydrogel and its loaded probiotics in the present invention;

[0041] Figure 4 Graph showing the release of mercapto hyaluronic acid / L-glutamine hydrogel in the present invention in different concentrations of dithiothreitol;

[0042] Figure 5 This is a graph showing the survival of the probiotics of the present invention after continuous incubation in simulated gastrointestinal fluid;

[0043] Figure 6 Pathological images of H&E staining of colon tissue of mice with DSS-induced enteritis induced by the probiotic-loaded thiol hyaluronic acid-based hydrogel of the present invention. The scale bar is 100 μm.

[0044] Figure 7 This figure shows the effect of the probiotic-loaded thiol hyaluronic acid-based hydrogel on the intestinal cytokine levels in mice with DSS-induced enteritis. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0046] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0047] A method for preparing a thiol hyaluronic acid-based hydrogel loaded with probiotics comprises the following steps: activating hyaluronic acid with a cross-linking agent, adding an organic matter to carry out a cross-linking reaction, and obtaining a final product after the reaction is completed; and further treating the product, freeze-drying it, and redissolving it in a bacterial solution to obtain the thiol hyaluronic acid-based hydrogel loaded with probiotics.

[0048] Preferably, the method comprises the following steps:

[0049] (1) Probiotics are statically cultured in MRS liquid culture medium, and the cultured bacterial liquid is centrifuged to obtain bacterial sludge; the bacterial sludge obtained by centrifugation is resuspended in phosphate buffer solution to form a bacterial suspension;

[0050] (2) adding a crosslinking agent to the hyaluronic acid solution, stirring at room temperature for 1 to 2 hours, adding cysteamine hydrochloride in the dark, adjusting the pH to 4.5 to 5.0, and continuing to stir and react at room temperature for 1 hour to obtain a thiol hyaluronic acid solution;

[0051] (3) adding a crosslinker to the thiol hyaluronic acid solution again, stirring at room temperature in the dark for 1 to 2 hours, adding L-glutamine, adjusting the pH to 4.5 to 5.0, stirring continuously at room temperature in the dark for 24 hours, and obtaining a thiol hyaluronic acid / L-glutamine mixed solution;

[0052] (4) dialyzing the thiol hyaluronic acid / L-glutamine mixed solution against a dilute hydrochloric acid solution with a pH of 3.5 for 48 h, and freeze-drying under vacuum to obtain a thiol hyaluronic acid / L-glutamine cotton-like solid;

[0053] (5) Dissolving the thiol hyaluronic acid / L-glutamine cotton-like solid in the bacterial suspension and incubating to obtain the thiol hyaluronic acid / L-glutamine hydrogel loaded with probiotics.

[0054] Preferably, the static culture condition in step (1) is static culture in a 37°C constant temperature incubator for 16 to 18 hours, and the number of viable bacteria reaches 1×10 8 CFU / mL or above;

[0055] Alternatively, the probiotics in step (1) include Bifidobacterium, Lactobacillus, Streptococcus, and Lactococcus;

[0056] Alternatively, in step (1), the bacterial sludge is obtained by centrifugation at 7000×g for 10 min;

[0057] Alternatively, the pH of the phosphate buffer solution in step (1) is 7.2, and the viable bacterial count of the bacterial suspension after resuspension reaches 1×10 9 CFU / mL or above;

[0058] Alternatively, Bifidobacterium spp. is cultured anaerobically during static culture.

[0059] Preferably, the preparation method of the hyaluronic acid solution in step (2) is: dissolving hyaluronic acid or hyaluronate with a molecular weight of 100 to 200 kDa in distilled water to obtain a hyaluronic acid solution with a mass concentration of 0.4%;

[0060] Alternatively, the molar concentration of cysteamine hydrochloride in step (2) is 5 mM;

[0061] Alternatively, the cross-linking agent in step (2) and step (3) is a mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), or the cross-linking agent is a mixture of N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBT); the molar concentration of the cross-linking agent in step (2) is 5 mM, and the molar concentration of the cross-linking agent in step (3) is 1.25 mM.

[0062] Preferably, the molar concentration of cysteamine hydrochloride in step (2) is 5 mM, the reaction process needs to be protected from light, and the stirring speed is 800-1000 r / min.

[0063] Preferably, the molar concentration of L-glutamine in step (3) is 6.25 mM, the reaction is continuously shielded from light, and the stirring speed is 800-1000 r / min.

[0064] Preferably, the dialysis in step (4) is performed using a dialysis interception tube or a dialysis bag, the molecular weight of the dialysis interception tube or the dialysis bag is 3.5 kDa, and the dialysate is replaced every 10 to 12 hours; and the vacuum freeze-drying time in step (4) is 24 to 48 hours.

[0065] Preferably, step (5) is performed under aseptic conditions in a clean bench; the hydrogel is incubated in a 37°C incubator for 1 hour to obtain the hydrogel;

[0066] Alternatively, the mass concentration of the mercapto hyaluronic acid / L-glutamine cotton-like solid in (5) is 4% after being dissolved in the bacterial suspension.

[0067] The probiotic-loaded thiol hyaluronic acid-based hydrogel prepared by the preparation method described above.

[0068] Application of the mercapto hyaluronic acid-based hydrogel as described above in biomedical materials and / or controlled drug release and / or food chemistry.

[0069] Specifically, the relevant preparation and detection are as follows:

[0070] 1. Experimental Materials

[0071] Lactobacillus fermentum CECT 5716 was purchased from Shanghai Baishi Biotechnology Co., Ltd.

[0072] Hyaluronic acid, cysteamine hydrochloride, and dithiothreitol (DTT) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and L-glutamine were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0073] 2. Experimental equipment

[0074] Incubator; dialysis bag; vacuum freeze dryer; magnetic stirrer.

[0075] Example 1

[0076] A method for preparing a probiotic-loaded thiol hyaluronic acid-based hydrogel comprises the following steps:

[0077] (1) 0.4 g (5 mM) of hyaluronic acid with a molecular weight of 100 kDa and 0.575 g (5 mM) of NHS were added to 100 mL of distilled water and stirred to dissolve. 0.958 g (5 mM) of EDC was added to the solution and stirred at room temperature for 2 h. The pH was adjusted to 4.5-5.0. 0.568 g (5 mM) of cysteamine hydrochloride was added in the dark and stirred to dissolve to obtain a thiol hyaluronic acid solution.

[0078] (2) 0.1438 g (1.25 mM) NHS and 0.2395 g (1.25 mM) EDC were added to the thiol hyaluronic acid solution, and the pH was adjusted to 4.5-5.0. 0.9135 g (6.25 mM) L-glutamine was added under dark conditions, and the mixture was stirred to dissolve. The mixture was stirred continuously for 24 hours in the dark to obtain a thiol hyaluronic acid / L-glutamine mixed solution;

[0079] (3) The thiol hyaluronic acid / L-glutamine mixed solution was dialyzed against a dilute hydrochloric acid solution at pH 3.5 for 48 hours, with the dialyzate replaced every 12 hours to remove unreacted by-products. The dialyzed solution was refrigerated at -80°C for 24 hours and then freeze-dried in a vacuum freeze dryer for 24-48 hours to obtain a thiol hyaluronic acid / L-glutamine cotton-like solid.

[0080] (4) The activated Lactobacillus fermentum CECT 5716 was inoculated into MRS liquid culture medium and cultured at 37°C for 18 h. At this time, the number of viable bacteria was 1×10 8 When the cell count is above 400 CFU / mL, the cells are collected by centrifugation (7000 rpm, 10 minutes, 4°C), washed 2-3 times with physiological saline, and resuspended in an equal volume of PBS buffer (pH 7.2) to obtain a bacterial suspension. Freeze-dried thiol hyaluronic acid / L-glutamine is re-dissolved in the bacterial suspension to a final concentration of 4% (w / v) and incubated at 37°C for 40 minutes to obtain the probiotic-loaded thiol hyaluronic acid / L-glutamine hydrogel.

[0081] Comparative Example 1

[0082] The preparation steps of Comparative Example 1 are the same as those of Example 1, except that the molar concentrations of the second cross-linking agent in Comparative Example 1 are 1.25 mM, 2.5 mM, 3.75 mM and 5.0 mM, respectively, to study the effect of cross-linking agent concentration on gel swelling and degradation properties.

[0083] Comparative Example 2

[0084] The preparation steps of Comparative Example 2 are the same as those of Example 1, except that the molar concentrations of L-glutamine in Comparative Example 2 are 2.5 mM, 3.75 mM, 5.0 mM, 6.25 mM, 7.5 mM and 10 mM, respectively, to study the effect of L-glutamine concentration on the swelling and degradation properties of the gel.

[0085] Comparative Example 3

[0086] The preparation steps of Comparative Example 3 are the same as those of Example 1, except that the final mass concentrations of thiol hyaluronic acid / L-glutamine in Comparative Example 3 are 3%, 4%, 5% and 6% (w / v), respectively, to study the effect of thiol hyaluronic acid / L-glutamine concentration on gel properties.

[0087] The relevant tests are as follows:

[0088] 1. The concentration of the second crosslinker in Example 1 affects the performance of the thiol hyaluronic acid / L-glutamine hydrogel. Preferably, the optimal molar concentration of the second crosslinker is 1.25 mM. The water absorption trend of the hydrogels prepared with different concentrations of the second crosslinker all showed a rapid increase at first and then a gradual decrease, approaching equilibrium in about 4 hours ( Figure 1 a) After 10 hours of incubation, the hydrogel prepared with 1.25mM secondary crosslinker had the highest swelling rate, 40.0%, higher than 38.1% for 2.5mM, 36.3% for 3.75mM, and 33.5% for 5mM. The swelling rate of the 1.25mM hydrogel group was significantly higher than that of the 3.75mM and 5mM high concentration groups (p < 0.05). Figure 1 As shown in (b), the degradation rate of the hydrogel prepared with 1.25 mM secondary crosslinker (91.0%) was higher than that of the 2.5 mM (84.1%), 3.75 mM (79.9.3%), and 5 mM (71.6.5%). The degradation rate of the 1.25 mM hydrogel was significantly different from that of the 3.75 mM and 5 mM hydrogels (p < 0.05). These results indicate that hydrogels prepared with a 1.25 mM secondary crosslinker molar concentration exhibited superior stability and degradation.

[0089] 2. The concentration of L-glutamine in Example 1 affects the performance of the mercapto hyaluronic acid / L-glutamine hydrogel. Preferably, the optimal molar concentration of L-glutamine is 6.25 mM. Figure 1As shown in (c), after reaching swelling equilibrium (10 h), the highest swelling rates were observed at L-glutamine concentrations of 6.25 mM, 7.5 mM, and 10 mM, reaching 37.0%, 37.4%, and 38.2%, respectively, with no significant differences (p>0.05). Furthermore, the swelling rate at 6.25 mM was significantly higher than that at 2.5 mM (27.0%), 3.75 mM (30.2%), and 5.0 mM (33.5%). At 45 minutes of degradation, the degradation rate of all hydrogels exceeded 60%. The degradation rate of the hydrogels increased with increasing L-glutamine addition, reaching 85.4% at a molar concentration of 6.25 mM, significantly higher than those at 2.5 mM (63.2%), 3.75 mM (70.4%), and 5.0 mM (78.4%). However, there was no significant difference in the degradation of hydrogels with molar concentrations of 6.25 mM, 7.5 mM and 10 mM, so the molar concentration of L-glutamine was selected to be 6.25 mM for the construction of thiol hyaluronic acid / L-glutamine hydrogel.

[0090] 3. The concentration of mercapto hyaluronic acid / L-glutamine in Example 1 affects the rheological properties of the hydrogel. Preferably, the optimal final concentration of mercapto hyaluronic acid / L-glutamine is 4% (w / v). Figure 2 As shown in the figure, with the increase of hydrogel concentration, the elastic modulus (G') hardly changes significantly, and the viscous modulus (G") shows a significant increasing trend. In order to analyze the viscoelastic properties of hydrogels, amplitude sweeps, frequency sweeps and temperature sweeps were performed on hydrogel systems with different concentrations. The hydrogel was subjected to amplitude sweeps at a constant angular frequency of 10 rad / s and a temperature of 37°C, with a dynamic strain range of 0.01 to 10%. The results are shown in the figure. Figure 2 (a) shows that G' did not change significantly, while G" of hydrogels with different concentrations showed an upward trend. In comparison, G" of hydrogels with 5% (w / v) and 6% (w / v) concentrations showed a significant increase. However, the trend of hydrogels with 3% concentration was not stable. The hydrogel was subjected to frequency sweep under the conditions of constant strain of 1% and temperature of 37℃, with variable angular frequency (ω) ranging from 0.1 to 100 rad / s. The results are shown in Figure 2. Figure 2 (b) As shown. In all concentrations of hydrogel, G' value has a very slight increase, and G" shows an increasing trend. Compared with 5% (w / v) and 6% (w / v) concentrations, the hydrogel with 4% (w / v) concentration tends to be stable, indicating that the hydrogel structure stability of this concentration is better. To further analyze the thermal stability of the hydrogel during storage and gastrointestinal delivery in vivo, the hydrogel was subjected to temperature scanning. The results are shown as follows Figure 2As shown in (c), the dynamic modulus of the hydrogels at all concentrations showed no significant change, and the overall trend was stable, indicating that the hydrogels possessed good thermal stability within a certain temperature range. Considering factors such as mechanical properties, a 4% (w / v) concentration of mercapto hyaluronic acid / L-glutamine hydrogel was selected for construction.

[0091] 4. The thiol hyaluronic acid / L-glutamine hydrogel prepared in Example 1 exhibits a three-dimensional network structure, a rough surface, a compact overall structure, a relatively neat morphology, thick pore walls, and complete and dense pores. After loading probiotics, the probiotics are evenly distributed in the hydrogel, and the surface morphology of the probiotics does not change significantly. The microstructure of the thiol hyaluronic acid / L-glutamine hydrogel and its loaded probiotics is shown in FIG. Figure 3 .

[0092] 5. The reduction responsiveness of the probiotic-loaded thiol hyaluronic acid / L-glutamine hydrogel in Example 1 was analyzed. Figure 4 As shown. The release of probiotics in gels in different media is different. In the PBS buffer solution of the control group, the strain release is less than 20% after 70min, indicating that the hydrogel has good stability under physiological conditions, can effectively reduce the loss of strains in normal tissues, and ensure that it plays the highest probiotic role after reaching the designated position of the intestine. In a high concentration of DTT solution, the disulfide bond (-SS-) inside the gel is quickly disconnected, and the strain is continuously released. The release rate is close to 100% at about 70min, while the strain release rate is less than 50% in a low concentration of DTT solution. This result shows that the reducing agent concentration can be used as a method for regulating the release rate of probiotics in hydrogels. The higher the concentration of the reducing medium, the faster the hydrogel cracks, and the higher the strain release rate. The above results show that the hydrogel of the present invention can quickly respond to reducing stimuli, and then control the release of lactobacilli, which plays an important role in delivering probiotics to the targeted intestine and achieving controlled release.

[0093] The reduction responsiveness assay method was as follows: 1 mL of freshly prepared hydrogel was briefly rinsed with physiological saline (i.e., 0.9% sodium chloride aqueous solution) and then immersed in 9 mL of PBS buffer (pH 7.4), 5 mM DTT solution, and 10 mM DTT solution, respectively. The hydrogel was incubated on a shaker at 37°C until completely lysed, and the number of released probiotics was calculated by plate counting.

[0094] Probiotic lysis and release rate (%) = Y / X × 100%, where X is the total number of viable bacteria and Y is the number of viable bacteria after hydrogel lysis.

[0095] 6. A simulated in vitro gastrointestinal digestion experiment was conducted on the probiotics-loaded thiol hyaluronic acid / L-glutamine hydrogel prepared in Example 1 (hereinafter referred to as the probiotics-loaded thiol hyaluronic acid / L-glutamine hydrogel).

[0096] (1) Simulated gastric conditions

[0097] The test method is as follows: 1 mL of fresh hydrogel is placed in 9 mL of simulated gastric fluid (SGF) with a pH of 2 (2.0 g of sodium chloride is dissolved in 7 mL of hydrochloric acid and distilled water is added to 1000 mL. The pH is adjusted to 1.2 with 0.1 mol / L hydrochloric acid, and pepsin is added to make its concentration reach 3.2 g / L. It is filtered and sterilized through a 0.22 μm filter membrane). It is cultured in a constant temperature shaking incubator at 37 ° C at a speed of 150 r / min for 180 minutes. The gel is then removed and placed in 9 mL of dithiothreitol solution (10 mM) until it is completely lysed. Subsequently, 1 mL of the solution is taken for gradient dilution and plate count is performed to determine the survival rate of the probiotics.

[0098] The stability of the hydrogel in gastric fluid is the prerequisite for its protective effect. After the hydrogel was incubated in simulated gastric fluid for 180 minutes, the number of probiotics increased from the original 8.59×10 10 The CFU / mL decreased to 7.26×10 9 The results show that the hydrogel of this embodiment has a relatively stable structure in an environment simulating gastric digestion, has strong gastric acid resistance, and can relatively effectively resist the effects of gastric acid, thereby maintaining the activity of probiotics.

[0099] (2) Simulating intestinal digestion conditions

[0100] The test method is as follows: 1 mL of fresh hydrogel is placed in 9 mL of simulated intestinal fluid (SIF) with a pH of 6.8 (SIF, 6.8 g of potassium dihydrogen phosphate is dissolved in 250 mL of distilled water, and then 77 mL of 0.2 mol / L sodium hydroxide and 10 g of trypsin are added. After mixing, the pH is adjusted to 6.8 ± 0.1 with 0.2 mol / L sodium hydroxide or 0.2 mol / L hydrochloric acid, and the volume is made up to 1000 mL with distilled water. It is sterilized by filtration through a 0.22 μm filter membrane) and cultured in a constant temperature shaking incubator at 37 ° C at a speed of 150 r / min for 180 min. The gel is then taken out and placed in 9 mL of dithiothreitol solution (10 mM) until it is completely lysed. Subsequently, 1 mL of the solution is taken for gradient dilution and plate count is performed to determine the survival rate of the probiotics.

[0101] After the hydrogel was incubated in simulated intestinal fluid for 180 min, the number of probiotics increased from the original 8.59×10 10 The CFU / mL decreased to 6.38×10 10 CFU / mL, with a survival rate of 74.3%. These results demonstrate that the probiotics loaded into the hydrogel of this example are relatively stable in an environment simulating intestinal digestion, have the potential for targeted release in specific areas of intestinal disease, and can be used as a probiotic supplement for specific intestinal diseases.

[0102] (3) Simulating continuous gastrointestinal digestion

[0103] 1 mL of fresh hydrogel was placed in 9 mL of simulated gastric fluid and incubated at 150 rpm in a 37°C constant-temperature shaking incubator for 180 minutes. The hydrogel was then transferred to 90 mL of simulated intestinal fluid and incubated at 150 rpm in a 37°C constant-temperature shaking incubator for 180 minutes. The hydrogel was then placed in 9 mL of 10 mM dithiothreitol solution until completely lysed. 1 mL of the solution was then serially diluted and plate counted to determine the survival rate of the probiotic bacteria.

[0104] The survival of probiotics in simulated gastrointestinal fluid (continuous) is shown in Figure 5 The number of free strains after incubation in simulated continuous gastrointestinal fluid increased from 3.21×10 11 The CFU / mL decreased to 3.13×10 6 CFU / mL, while the bacterial count after hydrogel loading increased from 7.81×10 10 The CFU / mL only decreased to 3.27×10 9 The CFU / mL was significantly higher than that of free strains, indicating that the hydrogel of this example has good stability and a high protective effect on strains in an environment simulating continuous gastrointestinal digestion.

[0105] 7. The effect of the probiotic-loaded thiol hyaluronic acid / L-glutamine hydrogel prepared in Example 1 on relieving enteritis was studied. The specific experimental method is: after the mice were adapted to the SPF environment, male C57BL / 6J mice weighing about 22g were randomly divided into 6 groups: ①Control group (as a control group; 8 mice); ②CECT 5716 group (probiotic suspension, 8 mice); ③HA-SH group (thiol hydrogel, the thiol hyaluronic acid solution prepared in step (1) of Example 1 was dialyzed and freeze-dried in step (3), dissolved in PBS buffer (pH 8.0) at a final mass concentration of 4% (w / v), and incubated at 37°C for 40min to prepare; 8 mice); ④HA-SH-P group (loaded CECT 5716 thiol hydrogel, the thiol hyaluronic acid solution prepared in step (1) of Example 1 was dialyzed and freeze-dried in step (3), and then prepared in step (4); 8); ⑤ HA-SH-L group (thiol hyaluronic acid / L-glutamine hydrogel, the thiol hyaluronic acid / L-glutamine cotton-like solid prepared in step (3) of Example 1 was dissolved in PBS buffer (pH 8.0) at a final concentration of 4% (w / v) and incubated at 37°C for 40 minutes; 8); ⑥ HA-SH-LP group (thiol hyaluronic acid / L-glutamine hydrogel loaded with CECT 5716, prepared in step (4) of Example 1; 8). The bacterial concentration was 1010 CFU / mL.

[0106] At the start of the experiment, all groups received a modeling-grade dextran sulfate sodium salt (DSS) solution (final concentration 3% (w / v)) in their regular drinking water. Seven days after modeling, the DSS solution was replaced with drinking water, and mice were gavaged at a rate of 10 μL / g for five consecutive days, with free access to food and water. The efficacy of the hyaluronic acid-based hydrogel in alleviating enteritis in mice was comprehensively analyzed based on changes in physiological indicators, expression of intestinal inflammatory factors, changes in intestinal barrier permeability, and high-throughput sequencing of colonic fecal flora.

[0107] The efficacy of the hydrogel system in alleviating enteritis was evaluated using a DSS-induced enteritis mouse model. The study found that the colon tissue structure of mice in the HA-SH-LP group was intact, and the morphology and number of goblet cells and crypts were normal ( Figure 6 ); For the spleen index, the spleen index of mice in the HA-SH-LP hydrogel group was the lowest, at 0.26%, which was significantly lower than 0.41% in the control group (p<0.001), 0.31% in the CECT5716 group (p<0.01), and 0.28% in the HA-SH-P group (p<0.05). The mRNA levels of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the above three groups of mice were significantly reduced in sequence, among which the relative expression levels in the HA-SH-LP group were the lowest, at 0.60, 0.57, and 0.46, respectively, which were significantly lower than those in the control group (p<0.001). At the same time, the mRNA levels of the anti-inflammatory cytokine IL-10 in each group were significantly increased, with the highest level in the HA-SH-LP group at 3.79, which was significantly higher than 2.81 in the HA-SH-P group and 2.16 in the CECT5716 group (p<0.05). These results demonstrate that the CECT 5716-loaded thiol hyaluronic acid / L-glutamine hydrogel exhibits the best efficacy in alleviating DSS-induced enteritis in mice, outperforming both free CECT 5716 strains and CECT 5716-loaded thiol hydrogels. This suggests that L-glutamine and probiotics act synergistically to improve intestinal inflammation. For example, Xiao et al. found that HA-SH-encapsulated rhamnosus bacteria can alleviate enteritis and reduce spleen index and relative mRNA expression of inflammatory factors in mice with enteritis. However, this performance is significantly lower than that of the HA-SH-LP hydrogel prepared by the present method.

[0108] At the same time, by comparing the ①Control group, ②CECT 5716 group, ③HA-SH group, ④HA-SH-P group, ⑤HA-SH-L group, and ⑥HA-SH-LP group, it can be seen that in the method of the present invention, step (2) (i.e., (2) adding 0.1438 g (1.25 mM) NHS and 0.2395 g (1.25 mM) EDC to the thiol hyaluronic acid solution, adjusting the pH to 4.5-5.0, adding 0.9135 g (6.25 mM) L-glutamine under light-proof conditions, stirring and dissolving, and continuously stirring for 24 hours in the dark to obtain a thiol hyaluronic acid / L-glutamine mixed solution;) and step (4) (i.e., (4) inoculating the activated fermented Lactobacillus CECT 5716 into the MRS liquid culture medium and culturing it at 37°C for 18 hours, at which time the number of viable bacteria is 1×10 8 When the concentration of CFU / mL is above 400, the cells are collected by centrifugation (7000 rpm, 10 minutes, 4°C), washed 2-3 times with physiological saline, and resuspended in an equal volume of PBS buffer (pH 7.2) to obtain a bacterial suspension. Freeze-dried thiol hyaluronic acid / L-glutamine is re-dissolved in the bacterial suspension to a final concentration of 4% (w / v) and incubated at 37°C for 40 minutes to obtain a probiotic-loaded thiol hyaluronic acid / L-glutamine hydrogel. These two steps act synergistically, synergistically improving the properties of the prepared thiol hyaluronic acid-based hydrogel.

[0109] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing a probiotic-loaded thiol hyaluronic acid-based hydrogel, characterized in that: Hyaluronic acid is activated with a cross-linking agent and then an organic matter is added to carry out a cross-linking reaction. After the reaction is completed, a final product is obtained; the product is further processed, freeze-dried, and redissolved in a bacterial solution to obtain a thiol hyaluronic acid-based hydrogel loaded with probiotics.

2. The preparation method according to claim 1, wherein: The steps include: (1) Probiotics are statically cultured in MRS liquid culture medium, and the cultured bacterial liquid is centrifuged to obtain bacterial sludge; the bacterial sludge obtained by centrifugation is resuspended in phosphate buffer solution to form a bacterial suspension; (2) adding a crosslinking agent to the hyaluronic acid solution, stirring at room temperature for 1 to 2 hours, adding cysteamine hydrochloride in the dark, adjusting the pH to 4.5 to 5.0, and continuing to stir and react at room temperature for 1 hour to obtain a thiol hyaluronic acid solution; (3) adding a crosslinker to the thiol hyaluronic acid solution again, stirring at room temperature in the dark for 1 to 2 hours, adding L-glutamine, adjusting the pH to 4.5 to 5.0, stirring continuously at room temperature in the dark for 24 hours, and obtaining a thiol hyaluronic acid / L-glutamine mixed solution; (4) dialyzing the thiol hyaluronic acid / L-glutamine mixed solution against a dilute hydrochloric acid solution with a pH of 3.5 for 48 h, and freeze-drying under vacuum to obtain a thiol hyaluronic acid / L-glutamine cotton-like solid; (5) Dissolving the thiol hyaluronic acid / L-glutamine cotton-like solid in the bacterial suspension and incubating to obtain the thiol hyaluronic acid / L-glutamine hydrogel loaded with probiotics.

3. The method for preparing the probiotic-loaded thiol hyaluronic acid-based hydrogel according to claim 2, characterized in that: The static culture condition in step (1) is to culture in a 37°C constant temperature incubator for 16 to 18 hours until the viable bacteria count reaches 1×10 8 CFU / mL or above; Alternatively, the probiotics in step (1) include Bifidobacterium, Lactobacillus, Streptococcus, and Lactococcus; Alternatively, in step (1), the bacterial sludge is obtained by centrifugation at 7000×g for 10 min; Alternatively, the pH of the phosphate buffer solution in step (1) is 7.2, and the viable bacterial count of the bacterial suspension after resuspension reaches 1×10 9 CFU / mL or above; Alternatively, Bifidobacterium spp. is cultured anaerobically during static culture.

4. The preparation method according to claim 2, wherein: The preparation method of the hyaluronic acid solution in step (2) is as follows: dissolving hyaluronic acid or hyaluronate with a molecular weight of 100 to 200 kDa in distilled water to obtain a hyaluronic acid solution with a mass concentration of 0.4%; Alternatively, the molar concentration of cysteamine hydrochloride in step (2) is 5 mM; Alternatively, the cross-linking agent in step (2) and step (3) is a mixture of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, or the cross-linking agent is a mixture of N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole; the molar concentration of the cross-linking agent in step (2) is 5 mM, and the molar concentration of the cross-linking agent in step (3) is 1.25 mM.

5. The preparation method according to claim 2, wherein: The molar concentration of cysteamine hydrochloride in step (2) is 5 mM, the reaction process needs to be protected from light, and the stirring speed is 800-1000 r / min.

6. The preparation method according to claim 2, wherein: The molar concentration of L-glutamine in step (3) is 6.25 mM, the reaction is continuously shielded from light, and the stirring speed is 800-1000 r / min.

7. The preparation method according to claim 2, wherein: During the dialysis in step (4), a dialysis interception tube or a dialysis bag is used for dialysis, the molecular weight of the dialysis interception tube or the dialysis bag is 3.5 kDa, and the dialysate is replaced every 10 to 12 hours; and the vacuum freeze-drying time in step (4) is 24 to 48 hours.

8. The preparation method according to any one of claims 2 to 7, characterized in that: Step (5) requires aseptic operation in a clean bench; incubate in a 37°C incubator for 1 hour to obtain a hydrogel; Alternatively, the mass concentration of the mercapto hyaluronic acid / L-glutamine cotton-like solid in (5) is 4% after being dissolved in the bacterial suspension.

9. The probiotic-loaded thiol hyaluronic acid-based hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the mercapto hyaluronic acid-based hydrogel according to claim 9 in biomedical materials and / or controlled drug release and / or food chemistry.

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