Thiolated sodium alginate hydrogel and methods of making and using the same

Abstract

The application discloses a kind of sulfhydrylated sodium alginate hydrogel, and is prepared by the following method: (1) EDC, NHS are added to sodium alginate solution, and react in the dark;Cysteine is added, and react in the dark;Dialysis, freeze-drying, and obtain sulfhydrylated sodium alginate;(2) sulfhydrylated sodium alginate is placed in PBS buffer solution, and obtain sulfhydrylated sodium alginate hydrogel.The sulfhydrylated sodium alginate hydrogel is used as a carrier in the preparation of probiotic preparation for targeted delivery to the intestine.The application also discloses a kind of probiotic preparation, which is composed of sulfhydrylated sodium alginate hydrogel and probiotic bacteria or its bacterial liquid.The sulfhydrylated sodium alginate hydrogel of the application shrinks in acidic environment, swells in alkaline condition, has pH responsiveness;It has reduction responsiveness;It has better mucin adhesion.The sulfhydrylated sodium alginate hydrogel of the application has great application prospect as a carrier in delivering probiotic bacteria.

Description

Technical Field

[0001] This invention relates to thiolated sodium alginate hydrogels, their preparation methods, and their applications, belonging to the field of modified sodium alginate technology. Background Technology

[0002] Probiotics are live microorganisms that offer certain health benefits to the host after ingestion. Oral administration is one of the effective ways to supplement intestinal probiotics; however, stomach acid and digestive enzymes can negatively impact probiotics. Most probiotics cannot maintain their original activity during digestion, leading to reduced bioactivity and the death of a large number of bacteria, thus affecting the effectiveness of oral probiotics. Furthermore, the existing gut microbiota can compete with ingested probiotics, reducing their residence time in the intestines.

[0003] A series of encapsulation methods for oral delivery of probiotics have been developed in the prior art, such as hydrogels, liposomes, and membranes. Although existing probiotic delivery carriers have made great progress, they still cannot meet current needs in terms of prolonging the residence time of probiotics in the intestine. An ideal oral delivery carrier for probiotics should have the following characteristics: (1) low biotoxicity and good biocompatibility; (2) the ability to protect probiotics in the environment of gastric acid and digestive enzymes, thereby improving the survival rate of probiotics; (3) the ability to release probiotics under intestinal conditions to achieve targeted release; and (4) the ability to appropriately prolong the residence time of probiotics in the intestine.

[0004] Biopolymers are natural alternatives to synthetic polymers, and can be effectively obtained from natural resources. Due to their unique biological and physical properties, such as cell adhesion, degradability, and bioactivity, they have attracted widespread attention. Sodium alginate, as a natural linear polysaccharide, has a large number of carboxyl and hydroxyl groups in its sugar chain, exhibits good pH responsiveness, and can undergo various chemical modifications, showing broad application prospects. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a thiolated sodium alginate hydrogel. The present invention obtains a thiolated sodium alginate hydrogel by modifying sodium alginate, which can be used as a carrier to deliver probiotics to the intestines.

[0006] This invention is achieved through the following technical solution:

[0007] A thiolated sodium alginate hydrogel was prepared by the following method:

[0008] (I) Preparation of thiol-modified sodium alginate

[0009] (1) Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to a sodium alginate solution with a concentration of 0.5% to 2% (mass volume concentration, unit g / ml) and a pH of 4.5 to 5.5, adjust the pH to 4.5 to 5.5, then add N-hydroxysuccinimide (NHS), and react in the dark under ice bath conditions for 50 to 70 min, keeping the pH of the solution at 4.5 to 5.5 during the reaction;

[0010] (2) Add cysteine ​​(Cys) to the above reaction solution, stir at room temperature and react in the dark for 5-7 hours, and keep the pH of the solution at 4.0-5.0 during the reaction.

[0011] The weight ratio of sodium alginate, EDC, NHS, and Cys is 0.5–2:3.5–4.5:0.5–2:4.0–6.0, preferably 1.0:4.0:1.0:5.0.

[0012] (3) Adjust the pH of the reaction solution to 5.8-6.5 and stop the reaction at room temperature in the dark for 25-35 minutes;

[0013] (4) The solution after the reaction was dialyzed with deionized water. The molecular weight of the dialysis sample was 3.5k. The water was changed every 6 hours and the dialysis was performed for 24 hours. The dialysis sample was obtained after the dialysis.

[0014] (5) The dialyzed sample was freeze-dried to obtain thiolated sodium alginate, which was then stored in a sealed container at -20℃.

[0015] (II) Preparation of thiolated sodium alginate hydrogel: Place thiolated sodium alginate in PBS buffer and let it stand at room temperature for more than 30 minutes to obtain thiolated sodium alginate hydrogel.

[0016] The thiolated sodium alginate hydrogel prepared using the above method is used as a carrier in the preparation of probiotic formulations that can be directionally delivered to the intestines.

[0017] A probiotic preparation comprising a thiolated sodium alginate hydrogel and probiotics or their bacterial culture.

[0018] Furthermore, the probiotics are selected from existing bacteria with probiotic functions, such as Bifidobacterium animalis subsp. Lactis.

[0019] The preparation method of the probiotic preparation is as follows: place sodium thiosulfate and probiotic culture in PBS buffer and let stand at room temperature for more than 30 minutes to obtain the probiotic preparation.

[0020] The thiolated sodium alginate of this invention is formed through the conjugation of cysteine ​​and sodium alginate. Under normal temperature and pressure, it can self-crosslink in PBS buffer to form a thiolated modified sodium alginate hydrogel without the need for additional acid or divalent ions. Experiments have shown that the thiolated sodium alginate hydrogel of this invention shrinks under acidic conditions and swells under alkaline conditions, exhibiting pH responsiveness; it can gradually degrade and disappear in dithiothreitol (DTT) solution, exhibiting reduction responsiveness; and it has better mucin adhesion. Probiotic preparations using the thiolated sodium alginate hydrogel of this invention as a carrier can smoothly pass through gastric juice to the intestines. The thiol groups on the surface of the thiolated sodium alginate hydrogel can form disulfide bonds with intestinal mucus, thereby prolonging the retention time of probiotics in the intestines. The thiolated sodium alginate hydrogel of this invention has great application potential as a carrier for probiotic delivery.

[0021] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0022] Figure 1 Fourier transform infrared spectra of sodium alginate and thiolated sodium alginate.

[0023] Figure 2 Photograph of thiolated sodium alginate hydrogel.

[0024] Figure 3 Schematic diagram of swelling rate determination results for sodium alginate hydrogel and thiolated sodium alginate hydrogel.

[0025] Figure 4 Schematic diagram of the weight change rate of thiolated sodium alginate hydrogel in DTT solution.

[0026] Figure 5 Schematic diagram of the adhesion rate of sodium alginate hydrogel and thiolated sodium alginate hydrogel to mucin.

[0027] Figure 6 Schematic diagram showing the survival rate of free probiotics and probiotic preparations after incubation in SGF.

[0028] Figure 7 Schematic diagram of plate count results for probiotics in feces.

[0029] Figure 8 Schematic diagram of plate count results of probiotics in intestinal tissue and its contents. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0031] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0032] Example 1: Thiolized Sodium Alginate Hydrogel

[0033] The steps are as follows:

[0034] (I) Preparation of thiol-modified sodium alginate

[0035] Weigh 1.0g of sodium alginate using a balance, and dissolve it completely in 100mL of deionized water under magnetic stirring. Slowly add 0.1mol / L hydrochloric acid to the solution to adjust the pH of the sodium alginate solution to 5.0.

[0036] Weigh 4.0g of EDC and add it to the sodium alginate solution above. Adjust the pH to 5.0 with 0.1mol / L hydrochloric acid. Then add 1.0g of NHS and react in the dark under ice bath conditions for 1 hour. During the reaction, keep the pH of the solution between 4.5 and 5.5.

[0037] Weigh 5.0g of Cys and add it to the above reaction solution. Stir and react at room temperature in the dark for 6 hours, keeping the pH of the solution between 4.0 and 5.0 during the reaction.

[0038] Add 1 mol / L sodium hydroxide solution to adjust the pH of the solution to 6.0, and stop the reaction by keeping it at room temperature in the dark for 0.5 h.

[0039] The reacted solution was poured into a dialysis bag (KD = 3.5k) and dialyzed with deionized water. The water was changed every 6 hours, and the dialysis lasted for 24 hours.

[0040] The dialysis sample was freeze-dried at -50℃ and 10mbar for 48h to obtain thiolated sodium alginate, which was then stored in a sealed container at -20℃.

[0041] Fourier transform infrared spectra of sodium alginate and thiolated sodium alginate are as follows: Figure 1 As shown. By Figure 1 It can be seen that 256cm -1 The peak is the stretching vibration generated by the hydroxyl group on the side chain of sodium alginate, at 1603 cm⁻¹. -1The peak at 1415 cm⁻¹ is caused by the asymmetric stretching of the carboxyl groups in sodium alginate. -1 The peak is caused by the symmetrical stretching of the carboxyl groups in sodium alginate. Compared to sodium alginate, the Fourier transform infrared spectrum of thiolated sodium alginate after cysteine ​​modification is at 1671 cm⁻¹. -1 and 2558cm -1 Different characteristic peaks appear at various locations, with the peak for the thiol group located at 2500 cm⁻¹. -1 Up to 2600cm -1 Between these values, the stretching peak of amide C=O is located at 1630 cm⁻¹. -1 Up to 1690cm -1 The presence of these components demonstrates the successful cross-linking of thiol groups.

[0042] (II) Preparation of thiolized sodium alginate hydrogel

[0043] Weigh 0.07 g of the prepared thiolated sodium alginate and place it in 2 mL of PBS buffer (0.01 mol / L, pH 7.4), immersing it completely. Let it stand at room temperature for 30 min to obtain a thiolated sodium alginate hydrogel (see photo). Figure 2 As shown), the concentration was 3.5% (w / v, g / ml).

[0044] Example 2: Effect of pH Changes on Thioylated Sodium Alginate Hydrogel

[0045] According to the Chinese Pharmacopoeia, the preparation method for simulated gastric juice (SGF) is as follows: Take 16.4 mL of 4 mol / L hydrochloric acid, dilute with water, add 10 g of pepsin, mix well, dilute with water to approximately 1000 mL, adjust the pH to 1.0, and bring the volume to 1000 mL. The preparation method for simulated intestinal juice (SIF) is as follows: Take 500 mL of 0.5 mol / L potassium dihydrogen phosphate solution, add 10 g of trypsin, mix well, dilute with water to approximately 1000 mL, adjust the pH to 6.8 with NaOH solution, and bring the volume to 1000 mL.

[0046] Weigh 0.07 g of sodium alginate and add it to 2 mL of 0.2 mol / L calcium chloride (CaCl2) solution to prepare sodium alginate hydrogel.

[0047] Take 1.0 g of thiolated sodium alginate hydrogel (prepared in Example 1) and 1.0 g of sodium alginate hydrogel, and place them separately in 50 mL of SGF and incubate at 37 °C for 1 h. Remove them and immediately place them in 50 mL of SIF, and continue incubating at 37 °C for 2 h. Measure the swelling rate of the hydrogels at 40 min, 60 min, 100 min, 130 min, and 180 min. To ensure accurate results, each group was performed in triplicate.

[0048] Swelling rate determination: After removing the hydrogel, the surface moisture was absorbed with filter paper, the hydrogel was weighed, and the swelling rate of the hydrogel was calculated using the following formula to determine the changes of the hydrogel during the simulated digestion process.

[0049]

[0050] In the formula, SR is the swelling ratio (%) of the hydrogel, and W h W represents the mass (g) of the hydrogel after soaking. d The mass (g) of the hydrogel before soaking.

[0051] The results are as follows Figure 3 As shown. By Figure 3 It can be seen that in SGF, both sodium alginate hydrogel and thiolized sodium alginate hydrogel exhibit shrinkage, with thiolized sodium alginate hydrogel showing stronger shrinkage and a significant difference. In SIF, both sodium alginate hydrogel and thiolized sodium alginate hydrogel exhibit swelling, with thiolized sodium alginate hydrogel showing stronger swelling and a significant difference.

[0052] Conclusion: During oral administration, drastic pH changes in the digestive tract are not the only challenge faced after taking hydrogels. The prolonged digestion process and digestive enzymes secreted by the digestive tract also affect the performance of hydrogels as delivery carriers. Compared to sodium alginate hydrogels, thiolated sodium alginate hydrogels exhibited stronger shrinkage in SGF and stronger swelling in SIF. This indicates that thiolated sodium alginate hydrogels have better pH responsiveness than sodium alginate hydrogels.

[0053] Example 3: Effect of reduction conditions on thiolated sodium alginate hydrogel

[0054] Weigh DTT and prepare 15 mmol / L and 25 mmol / L DTT solutions, and prepare them fresh each time you need them.

[0055] Thiol-modified sodium alginate hydrogel (prepared in Example 1) was immersed in 10 mL of DTT solution of different concentrations for 180 min. The hydrogel was weighed every 30 min, and the change in mass was calculated using a formula. Immersion in PBS buffer was used as a control.

[0056]

[0057] In the formula, WR is the weight change rate of the hydrogel (%), W s W0 represents the mass (g) of the thiolated sodium alginate hydrogel after soaking, and W0 represents the mass (g) of the thiolated sodium alginate hydrogel before soaking.

[0058] The results are as follows Figure 4 As shown. By Figure 4As can be seen, when immersed in 15 mmol / L and 25 mmol / L DTT solutions, the thiolated sodium alginate hydrogel first swells, then gradually degrades (in 15 mmol / L DTT solution, the weight change rate is less than 100% after about 100 min; in 25 mmol / L DTT solution, the weight change rate drops to 0 after about 122 min), until it completely disappears. However, it does not degrade in PBS buffer.

[0059] Conclusion: Thioylated sodium alginate hydrogels are formed through disulfide bond cross-linking, and DTT can diffuse into the hydrogel matrix through the pores. The reducing properties of DTT cause the disulfide bond network inside the thioylated sodium alginate hydrogel to break, and the disulfide bonds are reduced to form thiol groups. As a result, the thioylated sodium alginate hydrogel cannot maintain its original morphology and gradually degrades until it disappears completely. This indicates that thioylated sodium alginate hydrogels have reduction responsiveness, a characteristic that ensures that the ingested hydrogel degrades in the reducing environment of the colon.

[0060] Example 4: Mucin adhesion amount of thiolated sodium alginate hydrogel

[0061] The adhesion of mucin to hydrogels was detected using the periodic acid-Schiff (PAS) colorimetric method.

[0062] The periodate oxidation reagent was prepared by mixing 7 mL of acetic acid solution (7%, v / v) and 10 μL of periodic acid solution (50%, w / v, g / ml).

[0063] Standard solutions of mucin at different concentrations (25 μg / mL, 50 μg / mL, 125 μg / mL, 250 μg / mL, 375 μg / mL, 500 μg / mL) were prepared.

[0064] Add 0.2 mL of periodate oxidation reagent to the mucin standard solution, heat in a water bath at 37°C for 2 h, then add 0.2 mL of Schiff reagent, let stand at room temperature for 30 min, and measure the absorbance at 555 nm using an ELISA reader to obtain a standard curve of mucin concentration.

[0065] Weigh out the mucin and dissolve it in SGF to prepare a mucin solution (500 μg / mL). Place 1.0 g of thiolated sodium alginate hydrogel and sodium alginate hydrogel separately into 10 mL of the mucin solution and incubate in a 37°C water bath for 8 h. Centrifuge at 10000 r / min for 10 min, add 0.2 mL of periodate oxidizing reagent, heat in a 37°C water bath for 2 h, add 0.2 mL of Schiff reagent, and let stand at room temperature for 30 min. Measure the absorbance at 555 nm and use it to calculate the amount of mucin adhered to the hydrogel using the mucin concentration standard curve. Calculate the adhesion rate using the standard curve.

[0066] The results are as follows Figure 5 As shown. By Figure 5 It is evident that the adhesion rate of thiolated sodium alginate hydrogel to mucin is 80%, while that of sodium alginate hydrogel to mucin is only 37%, showing a significant difference.

[0067] Conclusion: Within the same time frame, thiolated sodium alginate hydrogel absorbed more mucin than sodium alginate hydrogel. The formation of disulfide bonds between the thiol groups on the thiol-modified sodium alginate hydrogel and intestinal mucus occurs through thiol-disulfide bond exchange reactions or through the oxidation of thiol groups. Thiol groups can form disulfide bonds with mucin, and these strong covalent bonds enhance the mucosal adhesion of the thiolated sodium alginate hydrogel, increasing the adhesion between thiolated sodium alginate and mucin.

[0068] Example 5: Protective effect of thiolated sodium alginate hydrogel as a delivery carrier in simulated gastric digestion.

[0069] To evaluate the protective and release capabilities of thiolated sodium alginate hydrogels for probiotics, experiments were conducted under continuous simulated gastrointestinal digestion conditions.

[0070] Bifidobacterium animalis subsp. Lactis was selected as the embedding model bacterium.

[0071] The method for preparing probiotic formulations (probiotics encapsulated in thiolated sodium alginate hydrogel) is as follows:

[0072] Remove the bacterial culture stored in the glycerol tube from the -80℃ freezer, dissolve it in a 37℃ water bath, and aseptically aspirate 50μL of the bacterial culture to a sterile test tube containing 5mL of RCM liquid medium. Quickly stopper the tube with a rubber stopper, place it in an anaerobic culture bag, and then place it in an anaerobic gas-generating bag. Seal the culture bag immediately and place it in a 37℃ constant temperature incubator for static incubation for 48h.

[0073] After obvious colony deposition appears in the test tube, remove the test tube from the culture bag, gently shake it to make the culture medium turbid, and use a pipette to draw 200 μL into a 96-well cell culture plate. Measure the absorbance of the bacterial solution at 600 nm to determine the growth density of the colony.

[0074] Transfer 50 μL of the bacterial culture from the test tube to a test tube containing 5 mL of RCM liquid medium for subculturing. Repeat the above steps for 2–3 subcultures to enhance the viability of the strain. Adjust the concentration to 2.0 × 10⁻⁶. 8 CFU / ml was used as a bacterial culture for the following experiments.

[0075] Weigh 0.07g of thiolated sodium alginate (prepared in Example 1), add 1ml of bacterial culture, and then add 1mL of PBS buffer (concentration 0.01mol / L, pH 7.4). Let stand at room temperature for 30min to obtain the probiotic preparation.

[0076] One mL of unencapsulated bacterial culture (free probiotics) and the probiotic preparation prepared above were placed separately into 10 mL of SGF. The mixtures were then placed in an anaerobic environment containing an anaerobic gas-generating bag, sealed, and incubated at 37°C for 1 hour. Simulated gastric fluid was serially diluted and plated on RCM agar for counting. The plate count results of the unencapsulated probiotics were used as a control.

[0077] The results are as follows Figure 6 As shown. By Figure 6 It can be seen that the colony count of the unencapsulated probiotics after incubation in SGF was 1.4 × 10⁻⁶. 3 The probiotic preparation, after incubation in SGF, had a colony count of 1.5 × 10⁻⁶ CFU / mL. 8 CFU / mL is 1.1 × 10⁻⁶ for unencapsulated probiotics. 5 times.

[0078] Conclusion: Probiotics have poor tolerance to acid. Encapsulation with thiolated sodium alginate hydrogel significantly improved the survival rate of probiotics. Thiolated sodium alginate hydrogel, as a delivery carrier, provides good protection for probiotics under acidic conditions.

[0079] Example 6: Effect of thiolated sodium alginate hydrogel as a delivery carrier in vivo experiments on mice.

[0080] C57BL / 6 mice (5 weeks old, female, average weight 16g) were randomly divided into three groups: a blank control group, a free probiotic group, and a probiotic preparation group. The probiotic preparation group was fed 0.2ml of probiotic preparation (prepared in Example 5; 0.07g of thiolated sodium alginate was weighed, added to 1ml of bacterial suspension, and then 1ml of PBS buffer was added, and the mixture was allowed to stand at room temperature for 30min). The free probiotic group was fed 0.2ml of bacterial suspension (prepared in Example 5, concentration 2.0×10⁻⁶) daily. 8 (CFU / ml) once daily. The blank control group was fed 0.2 ml of PBS buffer once daily. Each group was fed normal feed. Feeding continued for 7 days. Mouse feces were collected on days 0, 7, and 10. After collecting mouse feces on day 10, the mice were euthanized by cervical dislocation, and the intestines and intestinal contents were collected.

[0081] Intestinal tissue, contents, and feces were thoroughly homogenized in sterile PBS buffer. The homogenate was then serially diluted with sterile PBS buffer and plated on BBL culture solids for counting.

[0082] Plate count results of probiotics in feces as follows Figure 7 As shown. Plate count results of probiotics in intestinal tissue and its contents are as follows. Figure 8 As shown. By Figure 7 It can be seen that after feeding with probiotic preparations, the number of probiotic colonies in the feces on the 7th and 10th days were 4.7 × 10⁻⁶. 9 3.1×10 9 CFU / g; After feeding with free probiotics, the colony counts of probiotics in the feces on days 7 and 10 were 2.0 × 10⁻⁶. 9 1.6×10 9 CFU / g; After feeding with PBS buffer, the colony counts of probiotics in the feces on days 7 and 10 were 1.1 × 10⁻⁶. 9 1.2×10 9 CFU / g. (From) Figure 8 It was observed that on day 10, the number of Bifidobacterium colonies in the intestines of mice in the PBS group, the free probiotic group, and the probiotic preparation group were 2.3 × 10⁻⁶. 8 3.1×10 8 7.1×10 8 CFU / g.

[0083] Conclusion: Before the experiment, there was no significant difference in the number of Bifidobacteria in the intestines of the three groups of mice. After 7 days of continuous feeding, the number of Bifidobacteria colonies in the feces of mice administered probiotic preparations and mice with free probiotics increased. The number of Bifidobacteria colonies in the feces of mice administered probiotic preparations was significantly higher than that in the free probiotic group and the blank control group. This indicates that after administration of probiotic preparations, the probiotic preparations successfully reached the intestines (thiolized sodium alginate hydrogel has a protective effect on probiotics during digestion) and increased the number of Bifidobacteria in the mouse intestines, leading to an increase in the number of Bifidobacteria in the feces.

[0084] After stopping oral administration of probiotics, and following three days of normal feeding, the number of Bifidobacterium colonies in the feces of mice in both the probiotic preparation group and the free probiotic group decreased to some extent. The decrease in colony count was greater in the free probiotic group, indicating that probiotics have a shorter retention time in the intestines and quickly return to the pre-administration colony level after oral supplementation. While the number of probiotics encapsulated in thiolated sodium alginate hydrogel also decreased after three days of normal feeding, it still remained at a relatively high level.

[0085] To further determine the changes in the retention time of probiotics in the intestine, the number of probiotics in the intestines and intestinal contents of mice was measured three days after probiotic administration was stopped. Probiotics encapsulated in thiolated sodium alginate hydrogels showed a higher colony count than the free probiotic group and the blank control group. The thiol groups on the surface of the thiolated sodium alginate hydrogel can form disulfide bonds with intestinal mucus, thereby prolonging the retention time of probiotics in the intestine.

[0086] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing thiolized sodium alginate hydrogel, characterized in that, Includes the following steps: (I) Preparation of thiol-modified sodium alginate (1) Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to sodium alginate solution, adjust the pH to 4.5-5.5, then add N-hydroxysuccinimide, and react in the dark under ice bath conditions for 50-70 min, keeping the pH of the solution at 4.5-5.5 during the reaction. (2) Add cysteine ​​to the above reaction solution, stir at room temperature and react in the dark for 5-7 h, and keep the pH of the solution at 4.0-5.0 during the reaction; The weight ratio of sodium alginate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cysteine ​​is 0.5–2:3.5–4.5:0.5–2:4.0–6.

0. (3) Adjust the pH of the reaction solution to 5.8-6.5 and stop the reaction at room temperature in the dark for 25-35 minutes; (4) The solution after the reaction was dialyzed with deionized water. The molecular weight of the dialysis sample was 3.5 k. The dialysis sample was obtained after the dialysis. (5) The dialysis sample was freeze-dried to obtain thiolated sodium alginate; (II) Preparation of thiolated sodium alginate hydrogel: 0.07 g of thiolated sodium alginate was placed in 2 mL of PBS buffer with a concentration of 0.01 mol / L and a pH of 7.

4. The mixture was completely immersed and allowed to stand at room temperature for 30 min to obtain thiolated sodium alginate hydrogel.

2. The method for preparing thiolated sodium alginate hydrogel according to claim 1, characterized in that: In step (i) (1), the concentration of sodium alginate solution is 0.5% to 2%, in g / ml, and the pH is 4.5 to 5.

5.

3. The method for preparing thiolated sodium alginate hydrogel according to claim 1, characterized in that: In steps (i) and (2), the amounts of sodium alginate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and cysteine ​​are 1.0 g, 4.0 g, 1.0 g, and 5.0 g, respectively.

4. The method for preparing thiolated sodium alginate hydrogel according to claim 1, characterized in that: In steps (i) and (4), the water is changed every 6 hours, and the dialysis time is 24 hours.

5. The thiolized sodium alginate hydrogel prepared by any one of claims 1 to 4.

6. The use of the thiolated sodium alginate hydrogel of claim 5 as a carrier in the preparation of probiotic formulations for targeted delivery to the intestine.

7. A probiotic preparation, characterized in that: It consists of the thiolated sodium alginate hydrogel as described in claim 5, and probiotics or their bacterial solutions.

8. The probiotic preparation according to claim 7, characterized in that: The probiotics are selected from Bifidobacterium animalis subsp. lactis.

9. The method for preparing the probiotic preparation according to claim 7 or 8, characterized in that: The probiotic preparation is obtained by placing thiolated sodium alginate and probiotic culture in PBS buffer and letting it stand at room temperature for more than 30 minutes.

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