Thiolated oxidized guar gum / sodium alginate microspheres, and preparation method and application thereof

Abstract

The application discloses sulfhydrylated oxidized guar gum / sodium alginate microspheres and a preparation method and application thereof, and belongs to the field of biomedical materials. The sulfhydrylated oxidized guar gum and a sodium alginate base material are used to prepare and construct microspheres with pH response, colon targeting and intestinal adhesion by an emulsion gel method, the microsphere technology is used to embed probiotics, and the survival rate of the probiotics in gastric juice is improved. Through in-vitro verification, the method of embedding the microspheres successfully prevents the direct contact of the probiotics with a harsh environment, improves the ability of the probiotics to resist strong acid in the gastrointestinal tract, and enhances the adhesion and colonization effect of the probiotics in the intestinal tract. Through an animal experiment of simulating colitis, the LGG microspheres have the best treatment effect on colitis. Compared with a large number of side effects caused by antibiotic therapy, the treatment method has a certain clinical application prospect as a means with biological safety to treat inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a thiolated oxidized guar gum / sodium alginate microsphere, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Inflammatory bowel disease (IBD) is a chronic intestinal disease characterized by symptoms such as abdominal pain, diarrhea, rectal bleeding, and weight loss, seriously threatening human health. IBD has become a global public health problem. Although the pathogenesis of this disease is not fully understood, there is a potential link between the gut microbiota and inflammatory features. Probiotics can exert beneficial effects on the body by modulating immune function and producing organic acids and antimicrobial compounds. However, after oral administration, probiotics are susceptible to adverse factors such as the low pH of gastric acid and bile, leading to a decreased survival rate upon reaching the intestines. It is difficult to ensure that a sufficient quantity of probiotics colonizes the intestines and exerts their beneficial effects. Currently, microspheres are one of the most effective technologies for encapsulating probiotics. Microspheres can significantly enhance the tolerance of probiotics to harsh environments, thereby increasing the number of live bacteria reaching the intestines. Although encapsulating probiotics in simulated gastric juice with microspheres can improve survival rates, their adhesion in the intestines is poor. Therefore, there is an urgent need to develop novel drug delivery systems with resistance to gastric acid, intestinal targeting, and intestinal adhesion and colonization capabilities. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a thiolated oxidized guar gum / sodium alginate microsphere, its preparation method, and its application. The present invention uses thiolated oxidized guar gum and sodium alginate as substrates, and constructs microspheres with gastric acid resistance, colon targeting, and intestinal adhesion through an emulsification gelation method. Probiotics are encapsulated using microsphere technology to improve the survival rate of probiotics in gastric juice and their intestinal adhesion.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] The first aspect of this invention provides a method for preparing thiolated oxidized guar gum / sodium alginate microspheres, comprising the following steps:

[0007] S1. OGG is obtained by TEMPO oxidation of GG, and SOGG is generated by grafting L-cys onto the OGG molecular chain through amidation.

[0008] S2. Prepare a mixture containing SOGG, SA, and calcium carbonate. Add the bacterial suspension to the mixture and stir well to obtain an aqueous phase. Prepare a liquid paraffin solution containing Span 80. Dissolve Span 80 completely to obtain an oil phase. Slowly add the aqueous phase to the oil phase, emulsify, and stir to form W / O droplets. Add glacial acetic acid and continue stirring to solidify. Demulsify, let stand, centrifuge, remove the oil phase, and collect the thiolated oxidized guar gum / sodium alginate microspheres, abbreviated as LGG microspheres.

[0009] In a second aspect, the present invention provides a thiolated oxidized guar gum / sodium alginate microsphere, wherein the thiolated oxidized guar gum / sodium alginate microsphere is prepared by the above-described preparation method.

[0010] A third aspect of the present invention provides the application of the above-mentioned thiolated oxidized guar gum / sodium alginate microspheres in the field of biomedical materials.

[0011] The beneficial effects of this invention are as follows:

[0012] Currently, there are few reports on research regarding the use of modified GG encapsulated with probiotics for intestinal targeting. GG is a natural high-molecular-weight polysaccharide that is inexpensive, biocompatible, and non-toxic. This invention prepares SOGG through an amidation reaction, providing a new approach to the application of GG.

[0013] This invention prepares SOGG with thiol groups by oxidizing the natural high-molecular-weight polysaccharide GG in a TEMPO system and then modifying it by grafting L-cys via amide bonds. LGG microspheres are constructed by cross-linking SOGG and SA with LGG using an emulsification-gel method. These LGG microspheres exhibit good biocompatibility. This invention utilizes the self-oxidation of the thiol groups in SOGG to form disulfide bonds and the interaction between SA and Ca. 2+ Utilizing the properties of ionogelation, microspheres with a dual-network cross-linking were prepared. These microspheres exhibit intestinal adhesion and gastric acid resistance, improving the survival rate of LGG in the stomach. The sulfhydryl groups on the microspheres act as a link between probiotics and intestinal mucus, increasing the colonization and adhesion of probiotics in the intestine, thus facilitating their beneficial effects. This invention provides a theoretical basis for the application of probiotics in the dairy, beverage, and pharmaceutical industries.

[0014] In vitro experiments simulating gastrointestinal fluid have verified that the LGG microspheres prepared in this invention improve the survival rate and intestinal adhesion of LGG in gastric acid, thus providing protection for LGG.

[0015] In vivo simulation experiments, using a mouse model of colitis induced by 3% DSS, demonstrated that compared with free LGG treatment for colitis, the LGG microspheres of this invention can significantly alleviate DSS-induced colonic shortening, mouse weight loss, increased disease activity index, and increased spleen coefficient, and reduce colitis cell infiltration, indicating that LGG microspheres can effectively alleviate colitis and have a good therapeutic effect. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 This diagram illustrates the formation mechanism and functional principle of the LGG microspheres prepared in Example 1 of the present invention.

[0018] Figure 2 The graph shows the effect of sodium hypochlorite dosage on GG, where a represents the effect of NaClO dosage on the oxidation degree of GG; and b represents the transmittance of GG and OGG.

[0019] Figure 3 The infrared spectra are for SOGG characterization; where a is the infrared spectrum of GG, OGG and the product after OGG acidification; b is the infrared spectrum of GG, OGG and SOGG.

[0020] Figure 4 For GG, OGG, SOGG and L-cys 1 HNMR spectrum;

[0021] Figure 5 The diagram shows the screening conditions for thiol grafting rate; where a represents the effect of EDC / NHS on thiol content; b represents the effect of different reaction pH on thiol content; and c represents the effect of different feed ratios on thiol content.

[0022] Figure 6 The diagram shows the screening of preparation conditions for LGG microspheres; where a represents the effect of SOGG concentration on encapsulation efficiency; b represents the effect of SA:CaCO3 ratio on encapsulation efficiency; c represents the effect of water-oil volume ratio on encapsulation efficiency; and d represents the effect of glacial acetic acid dosage on encapsulation efficiency.

[0023] Figure 7 The images show the morphological appearance of LGG microspheres; where a is the lyophilized LGG microspheres, b is the morphological image of the microspheres under a conventional optical microscope, and c and d are scanning electron microscope images of LGG microspheres at different magnification ratios.

[0024] Figure 8Figure 1 shows the tolerance of LGG microspheres to simulated gastrointestinal fluid; where a represents the tolerance of free LGG to simulated gastrointestinal fluid; and b represents the tolerance of LGG microspheres to simulated gastrointestinal fluid.

[0025] Figure 9 To assess the tolerance of LGG microspheres in a simulated continuous gastrointestinal environment;

[0026] Figure 10 The results show the adhesion test results between LGG and the membrane; where a represents the membranes of four different polysaccharides; and b represents the adhesion between LGG and different membranes.

[0027] Figure 11 The effect of LGG microspheres on mouse body weight;

[0028] Figure 12 Pathological images of mouse heart, liver, spleen, lung, and kidney after H&E staining.

[0029] Figure 13 This shows the changes in mouse body weight during the experiment;

[0030] Figure 14 The DAI score of the mice during the experiment;

[0031] Figure 15 For each group of mice, (a) colon length; (b) HE staining and histological score (200×); Note a: Compared with the blank group, ###P<0.0001; Note b: Compared with the model group, ***P<0.005, ****P<0.0001;

[0032] Figure 16 The spleen coefficient of mice; Note a: Compared with the blank group, ###P<0.0001; Note b: Compared with the model group, ****P<0.0001;

[0033] Figure 17 MPO enzyme activity; Note a: Compared with the blank group, ###P<0.0001; Note b: Compared with the model group, ****P<0.0001;

[0034] Figure 18 Immunohistochemistry of (a) IL-10, (b) TNF-α and (c) IL-6 in mouse colon (200×); Note a: Compared with the blank group, **P<0.05; Note b: Compared with the model group, #P<0.01, ##P<0.05, ###P<0.001. Detailed Implementation

[0035] Given the low survival rate of probiotics in gastric juice and their poor adhesion in the intestine, this invention proposes a thiolated oxidized guar gum / sodium alginate microsphere, its preparation method, and its application.

[0036] A typical embodiment of the present invention provides a method for preparing thiolated oxidized guar gum / sodium alginate microspheres, comprising the following steps:

[0037] S1. OGG is obtained by TEMPO oxidation of GG, and SOGG is generated by grafting L-cys onto the OGG molecular chain through amidation.

[0038] S2. Prepare a mixed solution containing SOGG, SA and calcium carbonate. Add bacterial suspension to the mixed solution and stir well to obtain an aqueous phase. Prepare a liquid paraffin solution containing Span80. Dissolve Span80 completely to obtain an oil phase. Slowly add the aqueous phase to the oil phase, emulsify, stir to form W / O droplets, add glacial acetic acid and continue stirring to solidify. Demulsify, let stand, centrifuge, remove the oil phase, and collect the microspheres.

[0039] This invention prepares probiotic-loaded microspheres by cross-linking SOGG and SA through ionic and disulfide bond cross-linking. The free thiol groups in SOGG possess adhesive properties, which can solve the problem of probiotic adhesion and colonization in the intestine. The self-oxidation of thiol groups in SOGG to form disulfide bonds enables cross-linking for microsphere preparation. SA and Ca... 2+ The ions formed by chelation do not dissociate in the stomach but cleave into calcium hydroxide in the alkaline environment of the intestine, thus protecting probiotics in the stomach. However, single SA microspheres have many pores and poor mechanical properties, resulting in poor protection of probiotics. Therefore, SA is compounded and cross-linked with SOGG to form a dual-network hydrogel, thereby reducing the porous structure of the SA hydrogel. Simultaneously, GG can be degraded by colonic bacteria, achieving a colon-targeted release of probiotics. Intestinal mucus is composed of glycoprotein mucin with many cysteine ​​residues and is rich in thiol groups. Thiolized polymers can form disulfide bonds with cysteine ​​residues on the surface proteins of probiotics. Thiol polymers can also form disulfide bonds with the mucus gel layer through thiol or disulfide bond exchange reactions. Therefore, thiol polymers can act as a bridge connecting probiotics and mucus, enabling long-term adhesion between probiotics and mucus. This invention compoundes SOGG with SA to form dual-network microspheres with ion chelation and disulfide bonds, achieving the functions of protecting probiotics and promoting intestinal adhesion and colonization.

[0040] In some embodiments of this implementation, the bacterium is Lactobacillus rhamnosus CICC 6141, purchased from the China Industrial Microbial Culture Collection Center.

[0041] In some embodiments of this implementation, the TEMPO oxidation reaction includes the following steps:

[0042] Dissolve GG in deionized water, add NaBr and TEMPO, and place the solution in an ice-water bath after dissolution. Adjust the pH to 10-10.5, and add NaClO solution dropwise over 10 minutes, maintaining the pH at 10-10.5, until all NaClO is used up and the pH of the reaction no longer changes, thus terminating the oxidation reaction. Precipitate with alcohol, centrifuge the precipitate, and wash it. Redissolve the separated precipitate in water, dialyze, and freeze-dry to obtain OGG.

[0043] The synthetic route for OGG is shown below:

[0044]

[0045] In some embodiments of this implementation, the molecular weight of GG is 200-250 kDa, and the aqueous solution is mostly turbid. The presence of numerous hydroxyl groups in the GG molecular chain and the intramolecular hydrogen bond self-crosslinking are the main reasons for the poor solubility of GG.

[0046] In some embodiments of this implementation, the ratio of GG to deionized water is 0.2-0.8g:190-210mL.

[0047] In some embodiments of this implementation, the amount of NaBr added is 0.25-0.27 g / g.

[0048] In some embodiments of this implementation, the amount of TEMPO added is 18-22 mg / g, GG.

[0049] In some embodiments of this implementation, the effective chlorine content in the NaClO solution is 10%; the amount of NaClO used is 20-25 mmol / g GG. In the TEMPO oxidation system, NaClO acts as an oxidant, and its amount determines the degree of oxidation of GG. The TEMPO oxidation system can oxidize the C6 hydroxyl group of GG to a carboxyl group. At a dosage of 20-25 mmol / g GG, the TEMPO oxidation system can achieve an oxidation degree of over 90% for the C6 hydroxyl group of GG. When the amount of NaClO is further increased, the oxidation degree remains essentially unchanged.

[0050] In some embodiments of this implementation, the amidation reaction includes the following steps:

[0051] Prepare an OGG aqueous solution with a mass fraction of 0.15-0.25%, add EDC to activate the carboxyl groups, and then add NHS to fix the carboxyl groups after 15-25 min. Stir the reaction at room temperature in the dark for 30-50 min. Add L-cys to the reaction solution, adjust the pH, and stir continuously at room temperature in the dark for 24 h. After the reaction is completed, dialyze to remove unreacted reagents. Freeze-dry the dialyzed liquid to obtain SOGG.

[0052] The synthetic route for SOGG is shown below:

[0053]

[0054] In some embodiments of this implementation, the pH is 5.0-6.0. The reaction pH affects the thiol content in SOGG. As the reaction pH increases, the thiol content in SOGG initially increases and then decreases. The thiol content reaches its highest level at pH = 5.5.

[0055] In some embodiments of this implementation, the EDC:NHS mass ratio is 4-5:1. The EDC / NHS ratio also affects the thiol content in SOGG. EDC activates carboxyl groups, while NHS fixes them. The thiol content is highest when the EDC / NHS ratio is 5:1. This may be because the NHS-fixed carboxyl groups combine with the activated carboxyl groups, competing with L-cys.

[0056] In some embodiments of this implementation, the mass ratio of OGG to L-cys is 1:5-10. The OGG / L-cys mass ratio also affects the thiol content in SOGG. As the amount of L-cys added increases, the thiol content in SOGG first increases and then decreases. The free thiol content is highest when the feed ratio is 1:7.

[0057] In some embodiments of this implementation, the ratio of OGG to EDC is 1:5.

[0058] In some embodiments of this implementation, the concentration of the bacterial suspension is 2 × 10⁻⁶. 9 cfu / mL; the volume ratio of bacterial suspension to the mixture is 1:5.

[0059] In some embodiments of this implementation, the volume fraction of Span80 in the liquid paraffin solution is 1.5%-2.5%, more preferably 2%.

[0060] In some embodiments of this implementation, the concentration of SOGG in the aqueous phase is 0.25%-0.75% by mass. The concentration of SOGG affects the encapsulation efficiency of probiotics in the microspheres. The encapsulation efficiency of the microspheres first increases and then decreases with increasing SOGG concentration. The encapsulation efficiency of the microspheres is highest when the SOGG concentration is 0.5%.

[0061] In some embodiments of this implementation, the mass ratio of SA to CaCO3 in the aqueous phase is 1:3-5. The encapsulation effect of the microspheres is closely related to the mass ratio of SA to CaCO3. Therefore, both the SA / CaCO3 mass ratio and the encapsulation effect of the microspheres will affect the encapsulation effect. At a ratio of 1:4, the encapsulation effect of the microspheres is better.

[0062] In some embodiments of this implementation, the volume ratio of the aqueous phase to the oil phase is 1:2-4. Emulsification plays a crucial role in the preparation of microspheres. The water-to-oil volume ratio can affect the particle size of the microspheres and thus their quality. As the volume of the oil phase gradually increases, the encapsulation efficiency of the microspheres shows a trend of first increasing and then decreasing. A volume ratio of 1:3 yields microspheres of good quality.

[0063] In some embodiments of this implementation, the amount of glacial acetic acid added is 0.25-0.35 mL, more preferably 0.3 mL. Glacial acetic acid reacts with calcium carbonate to release Ca. 2+ Acetic acid plays a crucial role in the gelation of SA (superoxide dismutase). Insufficient glacial acetic acid hinders SA gelation and makes microsphere preparation difficult; excessive use results in excessively acidic pH, which can kill probiotics. A dosage of 0.25-0.35 mL yields microspheres with good quality and a high number of viable bacteria after encapsulation.

[0064] In some embodiments of this implementation, considering the combined effects of SOGG concentration, SA / CaCO3 mass ratio, water-oil volume ratio, and glacial acetic acid dosage on the encapsulation efficiency of LGG microspheres, the optimal preparation process conditions for LGG microspheres were obtained through orthogonal experiments as follows: in the aqueous phase, the SOGG concentration was 0.5%, the SA to calcium carbonate mass ratio was 1:3, the aqueous phase to oil phase volume ratio was 1:3, and the amount of glacial acetic acid added was 0.3 mL.

[0065] Another typical embodiment of the present invention provides a thiolated oxidized guar gum / sodium alginate microsphere, wherein the thiolated oxidized guar gum / sodium alginate microsphere is prepared by the above-described preparation method.

[0066] In some embodiments of this implementation, the thiolated oxidized guar gum / sodium alginate microspheres have a smooth, regular spherical structure with an average particle size of 260-350 μm.

[0067] Another typical embodiment of the present invention provides the application of the above-mentioned thiolated oxidized guar gum / sodium alginate microspheres in the field of biomedical materials.

[0068] In some embodiments of this implementation, the application is used in the preparation of drugs for treating colitis.

[0069] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0070] Example 1

[0071] 1. Experimental materials

[0072] 1.1 Main Symbols

[0073] The symbols used in the experiment are shown in Table 1.

[0074] Table 1 Main Symbols

[0075]

[0076]

[0077] 1.2 Main Reagents

[0078] The main materials and reagents used in the experiment are shown in Table 2.

[0079] Table 2 Main Materials and Reagents

[0080]

[0081]

[0082] The strain was Lactobacillus rhamnosus CICC 6141, purchased from the China Industrial Microbial Culture Collection Center.

[0083] Cells: RAW264.7 macrophages were cultured in our laboratory.

[0084] 1.3 Main Instruments

[0085] The main instruments used in the experiment are shown in Table 3.

[0086] Table 3 Main Instruments

[0087]

[0088]

[0089] 2. Test methods

[0090] 2.1 Synthesis of OGG

[0091] (1) Weigh 0.5g GG and stir in 200mL of deionized water until completely dissolved. Add 0.13g NaBr (0.26g / g, based on GG) and 10mg TEMPO (20mg / g, based on GG). Continue stirring until TEMPO and NaBr are completely dissolved.

[0092] (2) Place the solution in an ice-water bath at 0℃, adjust the pH to 10.3 (±0.02), add NaClO solution dropwise over 10 minutes to initiate the TEMPO-mediated oxidation process, and maintain the pH of the reaction at 10.3 (±0.02) with 0.5M NaOH. Record the amount of NaOH solution consumed every 10 minutes until the pH of the reaction no longer changes.

[0093] (3) Add 20 mL of 95% ethanol to adjust the pH of the solution to 7 and terminate the reaction.

[0094] (4) Add 3 times the volume of ethanol to precipitate OGG. Wash the precipitate 3 times with 75% ethanol solution, redissolve it in water, transfer the solution to a dialysis bag and dialyze it in ultrapure water for 72 hours to remove unreacted reagents.

[0095] (5) The dialyzed OGG was placed in a -80℃ freezer overnight and then freeze-dried for 48 hours to obtain a cotton-like solid.

[0096] (6) Acidification of OGG (HOGG): 1 g / L OGG was dispersed in 99% acetic acid solution, 10% HCl (37%, v / v) was added, mixed for 20 min, centrifuged at 1000 g for 20 min, and dried to remove excess acid. The carboxylates on the OGG were converted to the acidic form (carboxylic acid) for subsequent characterization.

[0097] 2.2 Synthesis of SOGG

[0098] (1) Weigh 0.094g of OGG and stir overnight in distilled water to make a 0.2% solution. Add EDC to activate the carboxyl group, and after 20min, add NHS to fix the carboxyl group. Stir the reaction at room temperature in the dark for 40min.

[0099] (2) Add L-cys with different mass ratios (1:1, 1:3, 1:5, 1:7 and 1:10) to the reaction solution, adjust the reaction pH (4.0, 4.5, 5.0, 5.5 and 6.0), and stir continuously at room temperature for 24 hours in the dark.

[0100] (3) After the reaction was completed, the sample was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 7500 to remove unreacted reagents. The dialysis media used were: Day 1, deionized water with pH = 5; Day 2, deionized water with pH = 5 containing 1% NaCl; Day 3, deionized water with pH = 5. The water was changed every 12 hours to remove unreacted reagents.

[0101] (4) The dialysis liquid was transferred to a -80℃ freezer and frozen overnight, and then freeze-dried for 48 hours to obtain a cotton-like solid.

[0102] 2.3 Preparation of LGG microspheres

[0103] 2.3.1 Preparation of LGG bacterial suspension

[0104] In a clean bench, LGG bacteria were inoculated into 10 mL of MRS broth at a 2% inoculum and incubated at 37°C for 24 h. The activated LGG bacteria were then subcultured at a 2% inoculum in 200 mL of broth and incubated at 37°C for 14 h. This subculture process was repeated once more. The cultured bacterial solution was centrifuged at 4000 rpm for 10 min, and the supernatant was removed. The bacterial sludge was washed with sterile 0.85% NaCl solution three times until the supernatant became colorless. Finally, the bacterial sludge was resuspended in 0.85% physiological saline to prepare a bacterial culture concentration of 2 × 10⁻⁶. 9 Store cfu / mL at 4°C for later use.

[0105] 2.3.2 Preparation of LGG microspheres

[0106] Weigh out SOGG and SA separately and dissolve them in deionized water, stirring overnight until fully dissolved.

[0107] Aqueous phase: Prepare a mixed solution containing SOGG, SA and CaCO3, and stir thoroughly to make it homogeneous; mix the bacterial suspension in 2.3.1 with the mixed solution by vortex stirring at a volume ratio of 1:5 until homogeneous.

[0108] Oil phase: Liquid paraffin containing 2% by volume Span80, stirred at 45°C to fully dissolve Span80.

[0109] 5 mL of the aqueous phase was slowly added to 20 mL of the oil phase. The mixture was coarsely emulsified using a magnetic stirrer, followed by fine emulsification using a shear homogenizer at 5000 rpm for 10 min. After forming W / O droplets through mechanical stirring, 200 μL of glacial acetic acid was added, and stirring continued for 40 min to allow for complete solidification. Four times the volume of sterile buffer solution was added to break the emulsion. The mixture was allowed to stand for 2 h. The microspheres settled at the bottom of the beaker due to gravity, while the oil phase floated on the surface. The mixture was centrifuged (4000 rpm, 10 min) to remove the oil phase and surfactant. This process was repeated three times, and the microspheres were collected. The collected probiotic microspheres were stored at 4°C. Figure 1 This is a diagram illustrating the formation mechanism and functional principle of LGG microspheres.

[0110] 3. Optimization and Characterization

[0111] 3.1 Characterization of OGG

[0112] 3.1.1 Effect of sodium hypochlorite dosage on the oxidation degree of OGG

[0113] In the TEMPO oxidation system, NaClO acts as the oxidant, and its amount determines the degree of oxidation of GG. During the synthesis of OGG, a NaClO solution with pH = 10.3 was added to a 0.25% GG polysaccharide solution. The pH of the polysaccharide solution initially decreased. Then, 0.5M NaOH solution was added to maintain the pH of the GG polysaccharide solution at 10.3. When the pH of the solution no longer decreased, NaClO solution was continuously added, and neutralization with 0.5M NaOH solution was continued. This process was repeated until NaClO solution was added again, at which point the pH of the polysaccharide solution remained essentially unchanged. The relationship between the amount of NaClO solution consumed and the degree of oxidation (OD) was recorded.

[0114] 3.1.2 FTIR

[0115] The mass ratio of freeze-dried GG and OGG samples to dried potassium bromide was 1:100. The samples and potassium bromide were thoroughly mixed in an agate mortar and pestle, then pressed into tablets using a tablet press. Fourier transform infrared spectrometer was used to scan GG and OGG in infrared.

[0116] 3.2 Characterization of SOGG

[0117] 3.2.1 FTIR

[0118] Same experimental method as in 3.1.2.

[0119] 3.2.2 1 HNMR

[0120] Take 10 mg of GG and SOGG respectively and dissolve them thoroughly in 1 mL of D2O. 1 Its chemical structure was resolved by 1H NMR on a Bruker AM500 spectrometer.

[0121] 3.2.3 Determination of thiol content using the Ellman method

[0122] The basic principle of Ellman's reagent for quantitative detection of free thiol groups is as follows:

[0123]

[0124] DTNB has no UV absorption at 412 nm. When it reacts with a sample containing thiol groups, it generates 2-nitro-5-mercaptobenzoic acid (TNB). 2- TNB 2- The reaction exhibits strong ultraviolet absorption at 412 nm and has high specificity. In this experiment, the standard curve of the L-cys standard sample was determined by ultraviolet spectrophotometry, and then the thiol content in SOGG was determined using the standard curve.

[0125] 3.3 Optimization of LGG microsphere preparation process

[0126] The effects of different SOGG mass fractions (0, 0.5%, 1.0%, 1.5%, 2.0%), SA:CaCO3 mass ratios (1:1, 1:2, 1:3, 1:4, 1:5), water-oil volume ratios (1:1, 1:2, 1:3, 1:4, 1:5), and glacial acetic acid dosages (0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL) on the microsphere encapsulation efficiency were determined through single-factor experiments. The encapsulation rate of probiotics was used as the indicator, and an orthogonal L9(3)24-fold (3^2 / ... 4 The design of the SOGG-SA microsphere formulation L9(3) was studied to investigate the effects of four factors on the microsphere encapsulation efficiency. 4 The orthogonal experimental design is shown in Table 4.

[0127] Table 4. Factor Level Table for Orthogonal Experiment

[0128]

[0129] 3.3.1 Determination of microsphere encapsulation rate

[0130] 0.5 g of microspheres were added to 9 mL of lysis solution for microsphere lysis. The mixture was shaken at 37 °C and 230 rpm for 1 h. Samples were taken and counted using the plate count method. The number of viable bacteria initially added was counted using the same method.

[0131] The encapsulation efficiency of probiotics can be expressed as: Encapsulation efficiency (%) = (m1 / m0) * 100%

[0132] Where: m0 is the initial number of viable LGG bacteria added, cfu / mL; m1 is the number of viable LGG bacteria embedded in the microspheres, cfu / mL.

[0133] 3.3.2 Characterization of microsphere morphology and particle size

[0134] Microspheres were dispersed in water and observed using an inverted fluorescence microscope. The particle size of the microspheres was measured, and 100 microspheres were counted, with the average value taken. The microstructure of the freeze-dried microspheres was then observed using a scanning electron microscope.

[0135] 3.3.3 Tolerance of LGG microspheres to simulated gastrointestinal fluid

[0136] Preparation of artificial simulated gastric juice (SGF): 1g of pepsin was added to 100mL of deionized water, and the mixture was stirred evenly in a 37℃ water bath. The pH was adjusted to 1.2 with 4M hydrochloric acid, and sterilized with a 0.2μm sterile microporous filter membrane. It should be prepared and used immediately.

[0137] Preparation of artificial simulated intestinal fluid (SIF): Add 1g of trypsin to 100mL of deionized water, then add 0.65g of potassium dihydrogen phosphate, adjust the pH to 7.4 with sodium hydroxide solution, stir the above solution evenly, and then filter it through a 0.2μm sterile microporous membrane for later use.

[0138] Collect LGG samples during the logarithmic growth phase, centrifuge, wash and resuspend in physiological saline, and adjust the final concentration to approximately 2 × 10⁻⁶. 9 cfu / mL.

[0139] Tolerance to simulated gastric fluid: Free LGG bacterial suspension and LGG microsphere samples were respectively placed into test tubes containing 10 mL of simulated gastric fluid at 37℃ and treated at 80 rpm for 120 min. Encapsulation solution was added to the probiotic microspheres, and lysis was performed at 37℃ and 230 rpm for 1 h to completely release the probiotics. Viable bacteria were counted using the plate count method, and counts were performed after 48 h of incubation, repeated three times, and the average value was taken.

[0140] Tolerance to simulated intestinal fluid: Free LGG bacterial suspension and LGG microsphere samples were respectively placed into test tubes containing 10 mL of simulated intestinal fluid preheated at 37℃, and treated at 37℃ and 80 r / min for 4 h. The remaining experimental steps were the same as those for simulated gastric fluid.

[0141] Control group: Free LGG was added to 0.85% saline.

[0142] Tolerance to continuous gastrointestinal fluid (GIF): Free LGG and LGG microspheres were transferred to test tubes containing 10 mL of simulated gastric fluid preheated at 37°C and treated on a shaker at 80 rpm for 120 min at 37°C. After centrifugation at 4000 rpm for 10 min, the simulated gastric fluid was removed, and the tubes were washed three times with physiological saline. 10 mL of simulated intestinal fluid was added to the test tubes containing the free LGG bacterial suspension and LGG microspheres, and the tubes were treated at 37°C at 80 rpm for 4 h. After centrifugation, the simulated intestinal fluid was removed, the tubes were washed with physiological saline, and encapsulation lysis buffer was added. Lysis was performed for 1 h, and plate counting was performed. This process was repeated three times, and the average value was taken.

[0143] 3.3.4 Adhesion Test

[0144] 3.3.4.1 Adhesion of LGG to the membrane

[0145] (1) Preparation of SA membrane, SA+GG membrane, SA+OGG membrane, and SA+SOGG membrane

[0146] Membrane solutions (30 mL each) were prepared as follows: 1.8% SA, 1.8% SA + 1.0% GG, 1.8% SA + 1.0% OGG, and 1.8% SA + 1.0% SOGG. The solutions were stirred at room temperature for 6 hours to ensure complete dissolution. Then, 1 mL of glycerol was added to each solution, and the mixture was sonicated for 20 minutes to remove air bubbles. Each 30 mL solution was poured into a plastic petri dish (D = 9 cm) and dried in an oven at 60 °C for 24 hours. The dried membranes were then removed and placed in a desiccator at 50% humidity and 25 °C for 48 hours before being peeled off and used for later use.

[0147] (2) Determination of the adhesion of the composite film to LGG

[0148] Four types of composite films were cut into 1cm pieces. 2 Adhesives were found on a glass slide and irradiated under a UV lamp in a clean bench for 30 minutes. LGG bacterial suspension (0.2 mL, 2 × 10⁻⁶) was then added. 8 cfu / mL) and membrane (1cm) 2 Combine the samples, incubate at 37°C for 30 min, rinse three times with sterile physiological saline, collect the rinse solution and dilute serially, and determine the number of unadhered probiotic bacteria using the plate count method. Calculate the adhesion rate using the following formula.

[0149] Adhesion rate (%) = ((AB) / A) * 100%

[0150] A represents the total number of LGGs, cfu / mL; B represents the number of unattached LGGs, cfu / mL.

[0151] 3.3.4.2 In vitro simulated adhesion of probiotic microspheres to the intestine

[0152] The mucosal adhesion of the composite microparticles was determined using the eversion encapsulation method. The experimental steps are as follows:

[0153] Male C57BL / 6J mice were fasted for 24 hours before intestinal harvesting, with free access to water during this period. The mice were euthanized the following day, the abdomen was opened, the colon was removed, the intestine was flushed with physiological saline to remove intestinal contents, and a 6cm section of colon was cut and stored in PBS buffer. The colonic segment was everted into a sac using a glass rod and placed in 10mL of PBS solution containing 10mg of microspheres. The sac was slowly shaken at 37℃ for 30min. The sac was removed, centrifuged, and the microspheres were separated. The microspheres were then freeze-dried for 48h, weighed, and the mucosal adhesion was calculated using the following formula.

[0154] Mucosal adhesion (%) = ((CD) / C) * 100%

[0155] C represents the initial microsphere weight (mg); D represents the weight of microspheres that did not adhere to the colon (mg).

[0156] 3.3.5 Biocompatibility of Microspheres

[0157] 3.3.5.1 Cytotoxicity assay

[0158] The in vitro toxicity of SOGG-SA microspheres to RAW264.7 cells was evaluated using the MTT assay. 200 mg of blank microspheres were immersed in 1 mL of culture medium for 24 h, and the extract was collected by centrifugation. RAW264.7 cells were counted using a cell counting chamber at a resolution of 5 × 10⁻⁶ cells / mL. 4 Cells were seeded per well into 96-well plates and incubated for 24 hours. The extract was diluted with culture medium to different concentrations (200, 100, 50, 25, 12.5, 6.25 mg / mL). The supernatant was discarded, and 100 μL of the extract at each well was added, with 6 replicates per group, and the cells were incubated for 24 hours. 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for 4 hours. The supernatant was then aspirated, and 150 μL of LDMSO was added to each well. The cells were mixed thoroughly in the dark to fully dissolve the formazan. Untreated cells were used as a control. The absorbance was measured at 570 nm using a microplate reader, and cell viability was calculated.

[0159] Survival rate (%) = ((A) s -A0) / (A c -A0))*100%

[0160] In the formula A s : The absorbance value of the sample; A c A0: Absorbance of the untreated cell group; A0: Absorbance value of the blank well.

[0161] 3.3.5.2 Hemolysis test

[0162] Rat blood was collected and centrifuged at 1000 rpm for 10 min at 4℃. The supernatant was removed, and the blood was repeatedly washed with PBS to obtain red blood cells, which were then diluted to 5% (V / V). 200 mg of blank microspheres were placed in 1 mL of culture medium for 24 h. The extract was collected by centrifugation and then diluted. 0.5 mL of each diluted extract was mixed with 0.5 mL of red blood cells and centrifuged at 37℃, 100 rpm for 1 h. Positive control: 0.5 mL distilled water and 0.5 mL red blood cells; blank control: 0.5 mL PBS buffer and 0.5 mL red blood cells. The absorbance at 540 nm was measured using a microplate reader, and the hemolysis rate was calculated.

[0163] Hemolysis rate (%) = ((A) s -A n ) / (A p -A n ))*100

[0164] In the formula A s: The absorbance value of the sample; A n : Absorbance of the blank control group; A p : Absorbance value of distilled water group.

[0165] 3.3.5.3 In vivo toxicity test

[0166] Mice were housed in an animal facility for one week to acclimatize. To investigate the systemic toxicity of the prepared *Lactobacillus rhamnosus* delivery system, mice were administered 0.2 mL of microspheres daily by gavage for 7 days, with changes in mouse weight monitored regularly. The control group received an equal volume of physiological saline. After the experiment, the mice were euthanized, and organs such as the heart, liver, spleen, lungs, and kidneys were cleaned, rinsed with physiological saline to remove blood stains, fixed with 4% formaldehyde, and sent for observation under an optical microscope.

[0167] 4. Results and Discussion

[0168] 4.1 Characterization of OGG

[0169] 4.1.1 Effect of NaClO dosage on GG

[0170] Glycerol (GG) has a molecular weight of approximately 220 kDa, and its aqueous solutions are often cloudy. The presence of numerous hydroxyl groups in the GG molecular chain and the resulting intramolecular hydrogen bonding self-crosslinking are the main reasons for its poor solubility. Chemical modification introduces hydrophilic carboxyl and thiol groups into GG, thereby improving its water solubility. The solubility of GG aqueous solutions can be reflected by transparency; higher transparency indicates better solubility. The TEMPO oxidation system can oxidize the C6 hydroxyl group of GG to a carboxyl group. Figure 2 The results show that as the amount of NaClO increases, the GG solution gradually changes from turbid to clear; simultaneously, the degree of oxidation of GG also gradually increases. Oxidation degree calculations show that the TEMPO oxidation system can oxidize the C6 hydroxyl group of GG to over 90%. When the amount of NaClO further increases, the oxidation degree remains essentially unchanged. The transmittance of the oxidized GG solution also continuously increases. These results indicate that the introduction of the carboxyl group improves the solubility of GG because the hydrophilicity of the carboxyl group accelerates the wetting process of GG by water molecules.

[0171] 4.1.2 FTIR

[0172] Depend on Figure 3 Infrared spectral analysis of α shows that the main absorption peaks of GG, OGG, and the acidified products of OGG are similar, indicating that the main structures of GG, OGG, and HOGG are consistent, and the TEMPO oxidation system did not destroy the main structure. The C=O absorption peak in the carboxylic acid group after GG oxidation mainly appears at 1614 cm⁻¹. -1 This can be attributed to the C=O stretching vibrations of the asymmetric carboxylate (COO-) group and the symmetric COO- group. After acidification with OGG, the C=O peak shifted to 1734 cm⁻¹. -1This indicates that -COOH is generated, and the -COOH group has been successfully introduced into the GG macromolecule structure, thus OGG has been successfully prepared.

[0173] 4.2 Characterization of SOGG

[0174] 4.2.1 FTIR

[0175] SOGG is formed by an amide bond between the amino group of L-cysteine ​​hydrochloride and the carboxyl group of OGG. Figure 3 b. Observe SOGG at 2696cm -1 A -SH peak appears at 1654 cm⁻¹. -1 and 1541cm -1 The appearance of amide peaks at 1608 cm⁻¹ indicates the formation of C=O and -NH bonds in the amide group, respectively. This confirms that the amide reaction occurred and the grafting was successful. -1 The presence of a carboxyl peak indicates that carboxyl and thiol groups coexist in SOGG.

[0176] 4.2.2 1 HNMR

[0177] like Figure 4 As shown, compared with the spectra of natural GG and OGG, the spectrum of SOGG shows obvious characteristic peaks at 3.34 ppm and 2.85 ppm. The 3.35 ppm peak is the -COCH3- part of the cysteine ​​derivative, and the 2.85 ppm peak is close to the absorption site of the methylene proton of -CH2-SH. This result indicates that the thiol unit has been successfully grafted onto the sugar chain of GG, confirming the successful synthesis of SOGG.

[0178] 4.2.3 Determination of thiol content using the Ellman method

[0179] To accurately determine the degree of thiol grafting in SOGG, a standard curve for free thiol groups was first determined using the Ellman method. The linear regression equation for free thiol groups was: y = 12.857x - 0.0071, R0 2 =0.9993, indicating a good linear relationship between absorbance and thiol concentration. Free thiol groups act as adhesives, while disulfide bonds are the cross-linking agents during microsphere preparation.

[0180] Initially, the thiol content after the amidation reaction was 47.67 μmol / g, which was relatively low. This may be due to the long molecular chain of OGG, resulting in low molecular mobility and making it difficult for L-cys to attach to the OGG backbone. Since a higher thiol content leads to stronger adhesion of the resulting SOGG, the reaction conditions for SOGG synthesis were optimized using the thiol content as an indicator. To increase the thiol content, three factors significantly affecting the amidation reaction were further identified through literature review and preliminary experiments: the EDC / NHS ratio, the reaction pH, and the amount of L-cys added. The conditions for these three influencing factors were then optimized.

[0181] 4.2.3.1 Screening of Thiol Grafting Rate Conditions

[0182] 4.2.3.1.1 Effect of different EDC / NHS ratios on thiol content

[0183] The reaction was controlled at pH 5.5, with a feed ratio of 1:7. The EDC / NHS ratio was varied; EDC activated carboxyl groups, while NHS fixed them. Figure 5 As shown in Figure a, with the increase of the EDC / NHS ratio, the thiol content increased from 191.3 μmol / g to 332.18 μmol / g. The thiol content reached its highest value when the EDC / NHS ratio was 5:1. This may be because the carboxyl groups fixed by NHS combine with the activated carboxyl groups, competing with L-cys. Experiments without adding NHS did not yield a higher thiol content than when the EDC / NHS ratio was 5:1. Therefore, the amidation reaction was performed at an EDC / NHS ratio of 5:1 based on the thiol content.

[0184] 4.2.3.1.2 Effect of different pH values ​​on thiol content

[0185] The pH of the amidation reaction was changed while other conditions remained constant. Figure 5 As shown in b, when the reaction pH increases from 4.0 to 6.0, the thiol content first increases and then decreases. When pH = 5.5, the thiol content reaches its highest level of 204.4 μmol / g, indicating that pH 5.5 is favorable for the amidation reaction in this experiment.

[0186] 4.2.3.1.3 Effect of different feed ratios on thiol content

[0187] Different feed ratios correspond to the OGG / L-cys mass ratio. Changing the amount of L-cys while keeping other reaction conditions constant... Figure 5The results in c show that the thiol content initially increases and then decreases with increasing L-cys input. The highest free thiol content (252.09 μmol / g) is observed at a feed ratio of 1:7, which may be related to collisions between L-cys and OGG. Increased L-cys concentration leads to greater intermolecular collisions and more product formation; however, further increases in L-cys inhibit the reaction process, resulting in fewer products.

[0188] In summary, single-factor reaction condition screening was conducted using the thiol content of SOGG as an indicator. The optimal reaction conditions were determined to be EDC / NHS = 5:1, pH = 5.5, and a feed ratio of 1:7. The free thiol content was calculated to be 332.18 μmol / g, and the total thiol content was 618.128 μmol / g, indicating that 46.26% of the thiol groups underwent auto-oxidation to form disulfide bonds. The free thiol groups have adhesive properties, acting as a bridge between LGG and intestinal mucus. Simultaneously, the disulfide bonds can undergo auto-oxidation to form disulfide bonds, which can play a cross-linking role in the microsphere preparation process.

[0189] 4.3 LGG microspheres

[0190] 4.3.1 Screening of preparation conditions for LGG microspheres

[0191] 4.3.1.1 Effect of SOGG concentration on encapsulation efficiency

[0192] from Figure 6 As shown in Figure a, the encapsulation efficiency of the microspheres initially increases and then decreases with increasing SOGG concentration. The maximum encapsulation efficiency (57.67%) is achieved when the SOGG concentration is 0.5%. As the SOGG concentration increases, the encapsulation efficiency decreases. This is mainly because the viscosity of the polysaccharide solution increases with higher SOGG concentrations, making it difficult for LGG to disperse uniformly in the solution. Conversely, as the SOGG concentration decreases, the resulting microspheres have smaller particle sizes and lower mechanical strength, failing to effectively encapsulate probiotics and affecting the encapsulation effect. When the SOGG concentration increases from 0% to 2%, the microsphere size gradually increases. This is primarily due to the increased viscosity of the polysaccharide solution, which hinders emulsification during microsphere preparation, leading to larger microsphere sizes. In conclusion, the optimal SOGG concentration for preparing LGG microspheres is 0.5%.

[0193] 4.3.1.2 Effect of SA / CaCO3 ratio on encapsulation efficiency

[0194] The encapsulation effect of the microspheres is closely related to the mass ratio of SA to CaCO3. In H... + In its presence, CaCO3 will produce Ca 2+ The generated Ca 2+SA chelates with CaCO3 to form a gel, therefore the SA / CaCO3 mass ratio affects the microsphere encapsulation effect. Figure 6 As can be seen from b, the microsphere encapsulation initially increases and then decreases with increasing mass ratio. When the mass ratio is 1:1, the CaCO3 concentration is low, and some SA remains unencapsulated in Ca. 2+ Cross-linking into a gel. At this stage, the microspheres are difficult to shape, have a large particle size and tend to stick together, have thin walls, and low mechanical strength, thus resulting in poor protection of probiotics in the stomach. With increasing mass ratio, the Ca in the reaction... 2+ The increased concentration of Ca allows for more complete cross-linking with SA, thus gradually increasing the encapsulation rate of probiotics. Simultaneously, the particle size of the formed microspheres gradually decreases, becoming spherical, compact, and non-adhesive, which enhances the protection of probiotics. When the mass ratio is 1:4, the encapsulation rate of probiotic microspheres reaches its highest level of 43.38%. However, with further increases in the mass ratio, Ca... 2+ Excess CaCO3 cannot react completely with H+. + During the reaction, the particle size of the microspheres began to increase, resulting in poor spherical shape, and the encapsulation efficiency began to decrease. In summary, the optimal SA / CaCO3 ratio for preparing LGG microspheres is 1:4.

[0195] 4.3.1.3 Effect of water-oil ratio on encapsulation rate

[0196] Emulsification plays a crucial role in the preparation of microspheres. The water-to-oil volume ratio can affect the particle size of the microspheres, and thus their quality. Figure 6 As shown in c, the encapsulation efficiency of microspheres initially increases and then decreases as the oil phase volume gradually increases. When the water-to-oil volume ratio is 1:1, larger dispersed droplets are generated in the oil phase, resulting in microspheres with large particle size, thin walls, low mechanical strength, and easy breakage, thus leading to a lower encapsulation efficiency. This may be due to the lower emulsifier content, resulting in a relatively higher microsphere content. During stirring, the microspheres collide and compress with each other, making it difficult for them to form spheres. When the water-to-oil volume ratio is 1:3, the encapsulation efficiency reaches its highest level of 34.41%, with well-formed microspheres and no adhesion. With an increase in the oil phase, the microsphere dispersion space becomes more spacious, allowing for sufficient emulsification of the aqueous phase. The microsphere particle size also gradually decreases, which is beneficial for improving the encapsulation efficiency. If the microsphere particle size is too small, the probiotics will be exposed outside the microspheres, and under mechanical action such as stirring and centrifugation, the microspheres are easily broken, ultimately leading to a lower encapsulation efficiency. In conclusion, the optimal water-to-oil volume ratio for LGG microsphere preparation is 1:3.

[0197] 4.3.1.4 Effect of glacial acetic acid dosage on encapsulation efficiency

[0198] Glacial acetic acid reacts with calcium carbonate to release Ca. 2+Acetic acid plays a crucial role in the gelation of salicylic acid (SA). Insufficient glacial acetic acid hinders SA gelation and makes microsphere preparation difficult; excessive use results in an overly acidic pH, which can kill probiotics. Therefore, it is essential to determine the optimal amount of glacial acetic acid that promotes microsphere formation without killing the probiotics. Figure 6 As shown in Figure d, the encapsulation efficiency initially increases and then decreases with increasing glacial acetic acid dosage. When the amount of glacial acetic acid added is 0.2 mL, the small amount results in excess calcium carbonate, leading to larger microspheres, weaker mechanical strength, and thus a lower encapsulation efficiency. When the amount of glacial acetic acid added is 0.3 mL, the encapsulation efficiency reaches its maximum of 81.56%, at which point the microspheres are uniform in size, well-formed, and have high mechanical strength. As the amount of glacial acetic acid gradually increases, the encapsulation efficiency begins to decrease, indicating a decrease in the number of viable bacteria after encapsulation. This is because excessive unreacted glacial acetic acid lowers the pH of the entire system, causing most probiotics to die. In conclusion, the optimal amount of glacial acetic acid for preparing LGG microspheres is 0.3 mL.

[0199] 4.3.1.5 Orthogonal Experiment

[0200] This orthogonal experiment was conducted based on the four single-factor experiments mentioned above. Factors significantly affecting the encapsulation efficiency of LGG microspheres—SOGG concentration, SA / CaCO3 mass ratio, water-oil volume ratio, and glacial acetic acid dosage—were selected as independent variables. The encapsulation efficiency of the microspheres was measured, and the optimal preparation process conditions for LGG microspheres were obtained through the orthogonal experiment. The encapsulation efficiency was used as the indicator; a larger range indicates a greater influence of the factor on the encapsulation efficiency. Table 5 shows that the order of influence of the four factors on the encapsulation efficiency is: D (acetic acid dosage) > C (water-oil volume ratio) > A (SOGG concentration) > B (SA / CaCO3 mass ratio). Therefore, A1B1C2D2 is the optimal formulation. However, since this formulation is not listed in the orthogonal experiment list, further verification is needed.

[0201] Table 5. Screening of microsphere formulations for L9(3) 4 Orthogonal experiment results

[0202] Test No. A B C D Encapsulation rate % 1 <![CDATA[A1]]> <![CDATA[B1]]> <![CDATA[C1]]> <![CDATA[D1]]> 79.59 2 <![CDATA[A1]]> <![CDATA[B2]]> <![CDATA[C2]]> <![CDATA[D2]]> 84.88 3 <![CDATA[A1]]> <![CDATA[B3]]> <![CDATA[C3]]> <![CDATA[D3]]> 69.61 4 <![CDATA[A2]]> <![CDATA[B1]]> <![CDATA[C2]]> <![CDATA[D3]]> 70.94 5 <![CDATA[A2]]> <![CDATA[B2]]> <![CDATA[C3]]> <![CDATA[D1]]> 77.83 6 <![CDATA[A2]]> <![CDATA[B3]]> <![CDATA[C1]]> <![CDATA[D2]]> 78.61 7 <![CDATA[A3]]> <![CDATA[B1]]> <![CDATA[C3]]> <![CDATA[D2]]> 84.36 8 <![CDATA[A3]]> <![CDATA[B2]]> <![CDATA[C1]]> <![CDATA[D3]]> 68.48 9 <![CDATA[A3]]> <![CDATA[B3]]> <![CDATA[C2]]> <![CDATA[D1]]> 80.37 K1 234.08 234.89 226.68 237.79 K2 227.38 231.19 236.19 247.85 K3 233.21 228.59 231.8 209.03 <![CDATA[k1]]> 78.03 78.30 75.56 79.26 <![CDATA[k2]]> 75.79 77.06 78.73 82.62 <![CDATA[k3]]> 77.74 76.20 77.27 69.68 R 2.23 2.10 3.17 12.94 Superior level <![CDATA[A1]]> <![CDATA[B1]]> <![CDATA[C2]]> <![CDATA[D2]]>

[0203] The validation experiments of the optimal formulation showed that the A1B1C2D2 combination had a high encapsulation rate, reaching (88.67±6.8)%, which was significantly higher than that of other groups (P<0.05). Furthermore, the microspheres exhibited good sphericity and small particle size. Therefore, the microspheres of the A1B1C2D2 combination were selected for further experiments. Using the encapsulation rate as the indicator, the optimal formulation for LGG microspheres was determined as follows: SOGG mass fraction 0.5%, SA / CaCO3 mass ratio 1:3, water-oil volume ratio 1:3, and glacial acetic acid dosage 0.3 mL. This was used for subsequent validation experiments.

[0204] 4.3.2 Appearance and Morphology of LGG Microspheres

[0205] The morphology of wet microspheres and freeze-dried microspheres was observed using a conventional optical microscope and a scanning electron microscope. The results are as follows: Figure 7 As shown. By Figure 7 As can be seen from this, the freeze-dried LGG microspheres appear as a white powder to the naked eye. Figure 7 b. Observation under a conventional optical microscope revealed that the LGG microspheres have a smooth, regular spherical structure with an average particle size of 294.46±31.8μm.

[0206] Depend on Figure 7 c. Figure 7 Scanning electron microscopy revealed that after freeze-drying, the surface of the microspheres developed wrinkles and depressions, and the microspheres became spindle-shaped, a common phenomenon in freeze-drying. Further magnified observation of specific areas of the microspheres showed that some probiotics were slightly raised and embedded inside the microspheres, while others adhered to the surface, indicating that a small number of probiotics were present on the surface of the microspheres.

[0207] 4.3.3 Tolerance of LGG microspheres to simulated gastrointestinal fluid

[0208] To investigate the survival ability of free LGG and LGG microspheres under simulated gastrointestinal conditions. Figure 8 Results showed that after 2 hours of incubation in simulated gastric fluid, the number of viable bacteria in free LGG microspheres decreased significantly, with a reduction of nearly 7 logs in colony count. After 4 hours of incubation in simulated intestinal fluid, the reduction in colony count was negligible, only about 0.2 logs. Numerous studies have shown that the vast majority of probiotics significantly lose their viability upon entering the human gastrointestinal tract. Subsequent investigations investigated the tolerance of LGG microspheres in simulated gastrointestinal fluid, such as... Figure 8 As shown in b, after incubation in simulated gastric and intestinal fluids for 2 hours and 4 hours respectively, the number of viable LGG microbes after encapsulation decreased slightly by approximately 0.7 Log and 0.2 Log, respectively, demonstrating strong tolerance to the simulated gastrointestinal fluids. This indicates that the microspheres provide some protection for the probiotics. Before investigating the tolerance of LGG microspheres to simulated gastrointestinal fluids, to avoid any impact of the encapsulation process on the activity of the probiotics themselves, the activity of the probiotics before and after encapsulation was studied. The growth curves of LGG after encapsulation showed no significant difference. This indicates that the encapsulation process of the microspheres has almost no effect on the activity of LGG.

[0209] 4.3.4 Tolerance of LGG microspheres in simulated continuous gastrointestinal fluid

[0210] The effects of continuous gastrointestinal fluid on free LGG and LGG microspheres were further simulated. Figure 9This further demonstrates the protective effect of microspheres on LGG. Compared with the saline group, free LGG showed almost no viable bacteria in continuously simulated gastrointestinal fluid (P<0.0001). In contrast, the probiotics encapsulated in LGG microspheres showed only a 0.5 log reduction in bacterial count compared to the control group after continuous incubation in gastrointestinal fluid for 6 hours. These results confirm that microspheres play a crucial protective role for LGG in gastrointestinal fluid, minimizing damage to probiotics caused by gastric acid.

[0211] 4.3.5 Adhesion Test

[0212] 4.3.5.1 Adhesion of LGG to the membrane

[0213] To investigate the adhesion of SA / CaCO3 microspheres to LGG, composite films were prepared by combining SA with GG, OGG, and SOGG, respectively. This indirectly verifies the adhesion of SOGG to LGG. The experimental results of the adhesion of LGG to the four different films are as follows: Figure 10 It can be seen that... Figure 10 As can be seen, the mechanical properties of the single SA film are inferior to the other three groups. The SA / SOGG film exhibits the best mechanical properties, which may be due to the enhanced mechanical strength of the film resulting from the self-oxidation of disulfide bonds in SOG. Figure 10 As shown in b, the SA membrane, SA / GG membrane, SA / OGG membrane, and SA / SOGG membrane all exhibit some adhesion to LGG. Among them, the SA / SOGG membrane shows a higher adhesion rate to LGG than the other three groups, indicating that the interaction between SOGG and LGG is the strongest, resulting in a stronger adhesion to LGG. This suggests that the introduction of thiol groups increases the adhesion between GG and LGG. The other three membranes also show adhesion to LGG, possibly because the hydroxyl or carboxyl groups on the polysaccharide structure form hydrogen bonds with the groups on LGG. Furthermore, after immersing the four different membranes in water for the same period, the SA / SOGG membrane remained relatively intact, indicating that disulfide bonds enhance the membrane's mechanical strength. SOGG can act as a bridge in the intestinal mucus through dual adhesion with intestinal mucus and LGG, ultimately adhering and colonizing LGG in the intestinal mucosa.

[0214] 4.3.5.2 Intestinal Adhesion

[0215] The adhesion of microspheres to the intestine was determined using the everted intestinal sac method. The adhesion rates calculated from the experimental results showed that the adhesion rates of colonic mucus to SOGG / SA microspheres and GG / SA microspheres were 77.67% and 46.17%, respectively. The results indicate that the colon exhibits a certain degree of mucosal adhesion to both SOGG / SA and GG / SA microspheres, primarily due to the abundance of villi on the colonic mucosa. These villi facilitate better adhesion of the microspheres to the colonic mucosa and epithelial cells. Simultaneously, the colonic region has a high concentration of goblet cells and a high level of mucin in the colonic mucosa, further increasing the adhesion of the microspheres in the colon. This may be due to the formation of hydrogen bonds or ionic bonds between the hydroxyl or amino groups on the polysaccharide chains and the groups on the mucus glycoproteins. The adhesion of mouse colonic mucosa to SOGG / SA microspheres was significantly higher than that to GG / SA microspheres. This is mainly because the free thiol groups on the surface of SOGG / SA microspheres react with the thiol groups on the mucus glycoproteins secreted by the colonic mucosa to form disulfide bonds, which further increases the mucosal adhesion of the microspheres.

[0216] 4.3.6 In vivo safety evaluation

[0217] like Figure 11 As shown, after 7 days of continuous gavage administration of LGG microspheres, the weight of healthy mice maintained a stable and normal growth. Compared with the control group, the mental state and activity level of the experimental group mice were almost identical.

[0218] The mice were then euthanized, and their hearts, livers, spleens, lungs, and kidneys were collected for HE staining and pathological analysis. Figure 12 As shown, the internal organ structures of mice administered LGG microspheres via gavage were not significantly different from those of mice in the non-gavage group, and no obvious inflammatory responses were detected in any tissue. These results indicate that the in vivo toxicity of LGG microspheres is negligible, which is beneficial for their in vivo application.

[0219] Application Example 1: Lactobacillus rhamnosus microspheres prepared in Example 1 for the treatment of DSS-induced colitis in mice.

[0220] 1. Laboratory animals

[0221] SPF-grade male C57BL / 6J mice, aged approximately 4-6 weeks, were purchased from Jinan Vital River Pharmaceutical Co., Ltd.

[0222] 2. Experimental Methods

[0223] 2.1 Construction of a mouse model of colitis

[0224] Before establishing the colitis model, C57BL / 6J mice were acclimatized to their environment by feeding them for 7 days. Mice were allowed free access to 3% DSS solution for 7 days to establish the colitis model. The DSS solution was changed every 1 day, and treatment was observed simultaneously. Mice were administered DSS via gavage starting on the day of model establishment, once daily, with gavage performed at approximately the same time each time, using 0.2 mL of bacterial solution each time. The normal control group and the DSS model group were given corresponding volumes of physiological saline, respectively.

[0225] Preparation of DSS: Dissolve DSS in sterile distilled water to prepare a 3% (w / v) solution.

[0226] Establishment of experimental animal models: 48 mice were randomly divided into 4 groups: Control group, DSS group, free LGG group, and LGG microsphere group, with 12 mice in each group. The grouping is as follows:

[0227] Control group: 0.2 mL of sterile saline was administered by gavage only.

[0228] DSS group: DSS administered orally plus 0.2 mL of sterile saline via gavage.

[0229] Free LGG group: DSS administered orally + 0.2 mL by gavage 2×10 9 cfu / mL bacterial suspension

[0230] LGG microsphere group: DSS was administered orally followed by 0.2 mL of LGG microsphere suspension (the best-performing microspheres prepared in Example 1) via gavage.

[0231] 2.2 Evaluation of DAI in mice

[0232] During the experiment, the mice's mental state, diet and water intake, activity, defecation, coat color, weight changes, and mortality were observed and recorded. As shown in Table 6, the average of the three scores—percentage of weight loss, fecal characteristics, and occult blood test (o-toluidine method)—was used as the Disease Activity Index (DAI). The efficacy of the drug was initially judged based on the mice's condition and DAI score.

[0233] DAI = (Weight Loss Score + Stool Characteristics Score + Severity of Rectal Bleeding Score) / 3

[0234] Table 6. Mouse DAI Scoring Criteria

[0235]

[0236] 2.3 Collection, length measurement, and HE staining of mouse colon tissue

[0237] Mice were observed multiple times daily. When more than 80% of the DSS group mice showed gross hematoma, they were euthanized and their abdominal cavities were dissected. The portion from the anus to the cecum was the colon, and its length was measured. Residual feces and blood were rinsed with pre-cooled PBS solution, and the moisture was blotted dry with filter paper. The colon was divided into two parts. One part was used for HE staining and immunohistochemical staining. A section of the colon 1 cm above the anus was immersed in paraformaldehyde and sent for testing. Histological scoring was performed based on the HE staining results. The other part was used for myeloperoxidase assay. The specimens were quickly transferred to a -80°C freezer for subsequent experiments to avoid repeated freeze-thaw cycles.

[0238] Table 7 Colonic Histological Scoring

[0239]

[0240]

[0241] 2.4 Changes in the spleen of mice

[0242] After euthanizing and dissecting the mice by cervical dislocation, the spleen was removed, weighed, and the spleen coefficient was calculated according to the spleen coefficient formula.

[0243] Spleen coefficient (%) = (Spleen weight / Mouse body weight) * 100%

[0244] 2.5 Detection of myeloperoxidase in colon tissue

[0245] The colonic tissue and internal feces were rinsed with PBS buffer solution. The colonic tissue was then minced and thoroughly homogenized using a tissue homogenizer to create a 5% homogenate, avoiding large pieces of tissue. The experimental procedures were performed according to the kit instructions from Nanjing Jiancheng Biotechnology Institute. The activity of the MPO enzyme was calculated by measuring the absorbance at 460 nm using the formula, with units of U / g wet weight.

[0246] MPO vitality = (A 测定 -A 对照 ) / (11.3×W)

[0247] In the formula: 11.3 is the reciprocal of the slope; A is the absorbance value at 460nm; W is the sample amount.

[0248] 2.6 Immunohistochemical analysis of mouse colon

[0249] The experimental procedure is the same as in 2.3, and colon tissue samples are delivered.

[0250] 3. Results and Discussion

[0251] 3.1 General observation of mice

[0252] Mice were induced to develop colitis by freely ingesting 3% DSS for 7 days. The changes in vital signs of mice in each group were observed during the experiment. The results for each group are as follows:

[0253] (1) Control group: One mouse lost weight rapidly, possibly due to esophageal damage during gavage. The remaining 11 mice had normal diet, water intake, and normal fecal color and shape, with smooth fur and active behavior.

[0254] (2) Model Group (DSS Group): Throughout the experiment, none of the 12 mice died. On days 3-4, the mice exhibited varying degrees of huddling together, rough fur, and mild diarrhea. On days 5-7, the mice drank less water and ate less. Starting on day 5, 20% of the mice showed symptoms such as lethargy, sluggishness, piloerection, arched back, and loose stools. As the experiment progressed, the diarrhea worsened, blood was visible at the anus, and the above symptoms intensified. The next step of the experiment could be carried out when more than 80% of the mice in the DSS group showed visible blood in their stools.

[0255] (3) Free LGG group (DSS+LGG group): From day 1 to day 5 of the experiment, all signs of the mice were normal. From day 5 to day 7, there were slight signs of anorexia and lethargy, and 30% of the mice had slightly looser stools, but there was no large-scale diarrhea or bloody stools. The mice's mental state and condition were better than those of the normal group.

[0256] (4) LGG microsphere group (DSS+Microsphere group): No mice died during the experiment. There was no significant difference between the mice and the normal group from day 1 to day 6. On day 6, 10% of the mice had slightly looser stools, but there was no large-scale diarrhea or bloody stools. The mice's mental state and condition were better than those of the normal group and the free LGG group.

[0257] 3.2 Changes in mouse body weight

[0258] To investigate the effects of free LGG and encapsulated LGG microspheres on mice with colitis, the body weight changes of each group of mice were recorded daily during the experiment. Results are as follows: Figure 13 As shown, the body weight of mice in the blank control group showed a gradual increasing trend, while the body weight of mice in the DSS group, DSS+LGG group, and DSS+LGG microsphere group all showed a decreasing trend. The body weight of mice in the DSS group decreased significantly from day 5 onwards, with a statistically significant difference (P<0.001). Compared with the DSS group, the body weight of mice in the DSS+LGG group and the DSS+LGG microsphere group decreased relatively more slowly. Meanwhile, the LGG microsphere group showed a relatively better therapeutic effect, with a reduction in the amount of loose stool, with a statistically significant difference (P<0.01). The results indicate that LGG microspheres have a certain therapeutic effect on mice with colitis.

[0259] 3.3 Evaluation of DAI in mice

[0260] During the experiment, the percentage of body weight loss, stool characteristics, and occult blood test results of mice in each group were recorded daily, and DAI scores were used for evaluation. Figure 14 As shown, the DAI score of mice in the DSS group continuously increased with the extension of experimental time. On day 5, mice in the DSS group exhibited visibly loose and bloody stools, lethargy, and other symptoms. Compared with the DSS+LGG group and the DSS+microsphere group, the DAI score of mice in the DSS group significantly improved from day 5 onwards. The results indicate that both LGG and LGG microspheres can effectively reduce the DAI score in mice with colitis, with the encapsulated probiotics showing a better effect, suggesting that LGG microspheres are more effective than free LGG in treating colitis.

[0261] 3.4 Evaluation of colon length and pathological damage

[0262] like Figure 15 As shown in Figure a, the colon length in the DSS group was significantly shorter, while the colon length in mice treated with LGG and LGG microspheres was significantly longer, indicating that both treatments have some efficacy in treating colitis. Meanwhile, there was a significant difference in colon length between the DSS+LGG group and the DSS+LGG microsphere group. Figure 15 As shown in b, after H&E staining, the colon of mice in the blank control group was in good condition, containing a large number of goblet cells, with intact and orderly intestinal epithelial and crypt structures, and no inflammatory cell infiltration. In the DSS group, mice had extensive necrosis and erosion of the colonic mucosal epithelial cells, with abundant inflammatory cell infiltration in the mucosal and submucosa layers. The number of goblet cells was significantly reduced, almost nonexistent, and the intestinal crypt structure was damaged. The colonic tissue damage score in the DSS group was significantly higher than that in the blank control group. After intervention with LGG and LGG microspheres, colonic mucosal ulcers and erosions were significantly reduced or disappeared, and the goblet cell arrangement returned to normal, both significantly reducing the colonic pathological score. Furthermore, compared with the LGG group, the LGG microsphere group significantly improved colonic mucosal damage in terms of histological score. All these results indicate that LGG microspheres have a better therapeutic effect on colitis than unembedded LGG.

[0263] 3.5 Changes in the spleen of mice

[0264] Depend on Figure 16 It was found that the spleen coefficient increased significantly in the DSS group, while it decreased with treatment with both free LGG and LGG microspheres. This indicates that both free and encapsulated probiotics have a certain therapeutic effect on colitis. Mice treated with LGG microspheres showed almost no significant difference compared to the control group, suggesting that LGG microspheres can alleviate the DSS-induced increase in spleen coefficient and have a certain therapeutic effect on colitis.

[0265] 3.6 Detection of myeloperoxidase in colon tissue

[0266] Depend on Figure 17 It was found that the MPO enzyme activity in the colon tissue of mice in the DSS group was significantly higher than that in the normal group. After the intervention of LGG and LGG microspheres, there was no significant difference between the mice and the normal group, indicating that both have a certain therapeutic effect on colitis. However, there was a significant difference between the LGG microsphere group and the LGG group (P<0.05), and the LGG microsphere group had a better therapeutic effect on colitis.

[0267] 3.7 Immunohistochemical analysis of mouse colon

[0268] Depend on Figure 18 The yellow-brown color indicates a positive protein. Compared to the control group, the IL-10 level was significantly downregulated after DSS stimulation, indicating a large infiltration of neutrophils and severe inflammation in the colon of mice in the DSS group. After treatment, both LGG and LGG microspheres significantly upregulated the colonic IL-10 level, which was reduced by DSS stimulation. This shows that LGG and LGG microspheres can alleviate colonic inflammation by upregulating IL-10 levels, with the LGG microsphere group showing the most significant upregulation effect.

[0269] The levels of IL-6 and TNF-α in the mouse colon increased from Figure 18 It was found that the levels of IL-6 and TNF-α in the colon of control mice were low, while the levels of IL-6 and TNF-α increased significantly after DSS stimulation. Treatment with LGG and LGG microspheres both downregulated the levels of IL-6 and TNF-α, which had increased after DSS stimulation, to varying degrees, and the differences were significant compared to the DSS group. Figure 18 It can be seen that both LGG and LGG microspheres can reduce intestinal inflammation by downregulating IL-6 and TNF-α levels, and the improvement effect of the LGG microsphere group is the most significant.

[0270] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A process for the preparation of sulfhydrylated oxidized guar gum / sodium alginate microspheres, characterized in that, Includes the following steps: S1. Oxidized guar gum is obtained by TEMPO oxidation of guar gum, and L-cysteine ​​is grafted onto the molecular chain of oxidized guar gum by amidation to generate thiolized oxidized guar gum. The TEMPO oxidation reaction includes the following steps: Guar gum was dissolved in deionized water, and sodium bromide and 2,2,6,6-tetramethylpiperidine oxide (TEMPO) were added. After dissolution, the solution was placed in an ice-water bath. The pH was adjusted to 10-10.5, and sodium hypochlorite solution was added dropwise over 10 minutes, maintaining the pH at 10-10.5, until all sodium hypochlorite was used up and the pH of the reaction no longer changed. The oxidation reaction was then terminated. The precipitate was precipitated with alcohol, centrifuged, and washed. The separated precipitate was redissolved in water, dialyzed, and freeze-dried to obtain oxidized guar gum (OGG). The amount of TEMPO added is 18-20 mg / g, guar gum; the available chlorine content in the sodium hypochlorite solution is 10%; the amount of sodium hypochlorite used is 20-25 mmol / g, guar gum; S2. Prepare a mixture containing thiolated oxidized guar gum, sodium alginate, and calcium carbonate. Add the bacterial suspension to the mixture and stir until homogeneous to obtain the aqueous phase. Prepare a liquid paraffin solution containing Span 80. Dissolve Span 80 completely to obtain the oil phase. Slowly add the aqueous phase to the oil phase, emulsify, and stir to form W / O droplets. Add glacial acetic acid and continue stirring to solidify. Demulsify, allow to stand, centrifuge, remove the oil phase, and collect the microspheres. The volume ratio of the aqueous phase to the oil phase is 1:2-4. The concentration of thiolated oxidized guar gum is 0.5%-0.75% by mass; in the aqueous phase, the mass ratio of sodium alginate to calcium carbonate is 1:4-5; and the amount of glacial acetic acid added is 0.25-0.35 mL. The microspheres are composed of sodium alginate and Ca. 2+ The ionic cross-linking network and the disulfide bonds formed by the self-oxidation of SOGG thiol groups cross-link to form a double network structure.

2. A process for the preparation of thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 1, wherein, The bacteria are Lactobacillus rhamnosus CICC 6141 Lactobacillus rhamnosus ).

3. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 1, wherein, The molecular weight of the guar gum is 200-250 kDa.

4. The process for preparing thiolated oxidized guar gum / sodium alginate microspheres according to claim 1, characterized in that, The ratio of guar gum to deionized water is 0.2-0.8 g: 190-210 mL.

5. The method of preparing thiolated oxidized guar gum / sodium alginate microspheres according to claim 1, wherein, Sodium bromide was added at a rate of 0.25-0.27 g / g, along with guar gum.

6. The method for preparing thiolated oxidized guar gum / sodium alginate microspheres as described in claim 1, characterized in that, The amidation reaction includes the following steps: Prepare an aqueous solution of oxidized guar gum with a mass fraction of 0.15-0.25%, add EDC to activate the carboxyl groups, and add NHS to fix the carboxyl groups after 15-25 min. Stir the reaction at room temperature in the dark for 30-50 min. Add L-cysteine ​​to the reaction solution, adjust the pH, and stir continuously at room temperature in the dark for 24 h. After the reaction is completed, dialyze to remove unreacted reagents. Freeze-dry the dialyzed liquid to obtain thiolized oxidized guar gum.

7. A process for the preparation of thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 6, wherein, The pH of the amidation reaction is 5.0-6.

0.

8. A process for the preparation of thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 7, wherein, The pH of the amidation reaction is 5.

5.

9. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 6, wherein, EDC:NHS = 4-5:1 mass ratio.

10. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 9, wherein, EDC:NHS = 5:1 mass ratio.

11. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 6, wherein, The mass ratio of oxidized guar gum to L-cysteine ​​is 1:5-10.

12. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 11, wherein, The mass ratio of oxidized guar gum to L-cysteine ​​is 1:

7.

13. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 6, wherein, The ratio of oxidized guar gum to EDC is 1:

5.

14. The method of preparing thiolated oxidized guar gum / sodium alginate microspheres according to claim 1, wherein, The concentration of the bacterial suspension was 2 x 10 9 cfu / mL; the volume ratio of the bacterial suspension to the mixed solution was 1:5; The volume fraction of Span 80 in the liquid paraffin solution is 1.5%-2.5%.

15. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 14, wherein, The volume fraction of Span 80 in the liquid paraffin solution is 2%.

16. The method for preparing thiolated oxidized guar gum / sodium alginate microspheres as described in claim 1, characterized in that, The volume ratio of the aqueous phase to the oil phase is 1:

3.

17. A process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 1, wherein, The amount of glacial acetic acid added is 0.3 mL.

18. The process for preparing thiolated oxidized guar gum / sodium alginate microspheres as claimed in claim 1, wherein, In the aqueous phase, the concentration of thiolated oxidized guar gum is 0.5%, the mass ratio of sodium alginate to calcium carbonate is 1:3, the volume ratio of the aqueous phase to the oil phase is 1:3, and the amount of glacial acetic acid added is 0.3 mL.

19. A sulfhydrylated oxidized guar gum / sodium alginate microsphere, characterized in that, The thiolated oxidized guar gum / sodium alginate microspheres are prepared by any of the preparation methods described in claims 1-18.

20. The sulfhydrylated oxidized guar gum / sodium alginate microsphere of claim 19, wherein, The thiolated oxidized guar gum / sodium alginate microspheres have a smooth, regular spherical structure with an average particle size of 260-350 μm.

21. The application of the thiolated oxidized guar gum / sodium alginate microspheres as described in claim 19 or 20 in the preparation of biomedical materials.

22. The use of the thiolated oxidized guar gum / sodium alginate microspheres as described in claim 19 or 20 in the preparation of drugs for treating colitis.

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