CN116725958A - A kind of thiolated oxidized guar gum/sodium alginate microsphere and its preparation method and application - Google Patents

Abstract

The invention discloses a sulfhydryl guar gum/sodium alginate microsphere and a preparation method and application thereof, belonging to the field of biomedical materials. According to the invention, the sulfhydryl oxidized guar gum and the sodium alginate base material are used for constructing the microsphere with pH response, colon targeting and intestinal adhesiveness by an emulsion gel method, and the microsphere technology is used for embedding probiotics, so that the survival rate of the probiotics in gastric juice is improved. Through in vitro verification, the microsphere embedding method successfully prevents the probiotics from being in direct contact with severe environment, improves the capability of resisting strong acid of the probiotics in the gastrointestinal tract, and enhances the adhesion and implantation effect of the probiotics in the intestinal tract. Animal experiments are carried out by simulating colonitis, and the LGG microsphere has the best curative effect on colonitis. Compared with a plurality of side effects brought by antibiotic therapy, the treatment method treats inflammatory bowel disease by a means with biological safety, and has a certain clinical application prospect.

Description

A kind of thiolated oxidized guar gum/sodium alginate microsphere and its preparation method and application

Technical field

The invention belongs to the field of biomedical materials, and specifically relates to a thiolated oxidized guar gum/sodium alginate microsphere and its preparation method and application.

Background technique

The information in this Background section is disclosed solely for the purpose of increasing understanding of the general background of the invention and is not necessarily considered to be an admission or in any way implying that the information constitutes prior art that is already known to a person of ordinary skill in the art.

Inflammatory bowel diseases (IBD) are chronic intestinal diseases accompanied by symptoms such as abdominal pain, diarrhea, bloody stools, and weight loss, which seriously threaten human health. IBD has become a global public health problem. Although the pathogenesis of the disease remains unclear, there is a potential association between the gut microbiota and inflammatory signatures. Probiotics can play a beneficial role in the body by regulating immune function and producing organic acids and antibacterial compounds. However, since probiotics are easily affected by adverse factors such as low pH of gastric acid and bile after entering the digestive tract through oral administration, the survival rate of probiotics reaching the intestinal tract is reduced, and it is difficult for a sufficient amount of probiotics to reach the intestinal colon and colonize. exert a probiotic effect. 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 viable bacteria that reach the intestinal tract. Although encapsulation of probiotics in simulated gastric juice through microspheres can improve the survival rate, their adhesion in the intestine is poor. Therefore, there is an urgent need to develop new drug delivery systems with gastric acid resistance, intestinal targeting, and intestinal adhesive colonization functions.

Contents of the invention

In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a thiolated oxidized guar gum/sodium alginate microsphere and its preparation method and application. The present invention uses thiolated oxidized guar gum and sodium alginate. As the base material, microspheres with gastric acid resistance, colon targeting and intestinal adhesion were prepared and constructed through the emulsification gel method. Microsphere technology was used to encapsulate probiotics and improve the survival rate of probiotics in gastric juice and intestinal tract. Adhesion.

In order to achieve the above objects, the technical solution of the present invention is:

A first aspect of the invention provides a method for preparing thiolated oxidized guar gum/sodium alginate microspheres, which includes the following steps:

S1. OGG is obtained by performing TEMPO oxidation reaction on GG, and L-cys is grafted onto the OGG molecular chain through amidation reaction to generate SOGG;

S2. Prepare a mixed solution containing SOGG, SA and calcium carbonate, add bacterial suspension to the mixed solution and stir well to obtain a water phase; prepare a liquid paraffin solution containing Span 80, and fully dissolve Span 80 to obtain an oil phase; Slowly add the water phase into the oil phase, emulsify, stir to form W/O droplets, add glacial acetic acid and continue stirring to solidify; break the emulsification, let stand, centrifuge, remove the oil phase, and collect to obtain thiolated oxidized guar gum. /Sodium alginate microspheres, referred to as LGG microspheres.

A second aspect of the present invention provides a thiolated oxidized guar gum/sodium alginate microsphere, which is prepared by the above preparation method.

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.

The beneficial effects of the present invention are:

At present, few studies on intestinal targeting of modified GG-encapsulated probiotics have been reported. GG is a natural polymer polysaccharide with low price, good biocompatibility and non-toxicity. The present invention prepares SOGG through amidation reaction, which provides new ideas for the application of GG.

The present invention prepares SOGG with thiol groups by oxidizing the natural polymer polysaccharide GG in a TEMPO system and then grafting and modifying L-cys through an amide bond. LGG microspheres were constructed using the emulsification gel method, using SOGG and SA to cross-link and load LGG. The LGG microspheres have good biocompatibility. The present invention utilizes the characteristics that the mercapto group of SOGG can auto-oxidize to form a disulfide bond and SA gels with Ca ²⁺ ions to prepare double network cross-linked microspheres. The microspheres have intestinal adhesion and gastric acid resistance, which can improve the survival rate of LGG in the stomach; the sulfhydryl groups on the microspheres play a role in connecting probiotics and intestinal mucus, increasing the colonization and adhesion of probiotics in the intestines , which helps probiotics play a probiotic role in the intestines. The invention provides a theoretical basis for the application of probiotics in dairy products, beverages and pharmaceutical industries.

It has been verified by in vitro simulated gastrointestinal fluid experiments that the LGG microspheres prepared in the present invention improve the survival rate and intestinal adhesion of LGG in gastric acid, and have a protective effect on LGG.

Through in vivo simulation tests, the colitis mouse model induced by 3% DSS proved that compared with the treatment of colitis with free LGG, the LGG microsphere treatment of the present invention can significantly alleviate the colon shortening, mouse weight loss, and disease caused by DSS. The activity index increased, the spleen coefficient became larger, and the infiltration of colonic inflammatory cells was reduced, indicating that LGG microspheres can effectively relieve colitis and have good therapeutic effects.

Description of drawings

The description and drawings that constitute a part of the present invention are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

Figure 1 is a schematic diagram of the formation mechanism and function of LGG microspheres prepared in Example 1 of the present invention;

Figure 2 is a graph showing the effect of the dosage of sodium hypochlorite on GG, where a is the effect of the dosage of NaClO on the oxidation degree of GG; b is the transmittance of GG and OGG;

Figure 3 shows the infrared spectrum of SOGG characterization; a is the infrared spectrum of the acidified products of GG, OGG and OGG; b is the infrared spectrum of GG, OGG and SOGG;

Figure 4 shows the ¹ HNMR images of GG, OGG, SOGG and L-cys;

Figure 5 is a condition screening chart for thiol grafting rate; where a is the effect of EDC/NHS on thiol content; b is the effect of different reaction pH on thiol content; c is the effect of different feeding ratios on thiol content;

Figure 6 is a screening diagram of the preparation conditions of LGG microspheres; where a is the effect of SOGG concentration on the embedding rate; b is the effect of the ratio of SA:CaCO ₃ on the embedding rate; c is the water-oil volume ratio on the embedding rate The influence of; d is the influence of the dosage of glacial acetic acid on the embedding rate;

Figure 7 shows the appearance and morphology of LGG microspheres; a is the freeze-dried LGG microsphere, b is the morphology of the microspheres under an ordinary optical microscope; c and d are scanning electron microscope images of LGG microspheres under different magnification ratios. ;

Figure 8 is a graph showing the tolerance of LGG microspheres to simulated gastrointestinal fluid; where a is the tolerance of free LGG to simulated gastrointestinal fluid; b is the tolerance of LGG microspheres to simulated gastrointestinal fluid;

Figure 9 shows the tolerance of LGG microspheres in simulated continuous gastrointestinal tract;

Figure 10 shows the test results of adhesion between LGG and film; where a is the film of four different polysaccharides; b is the adhesion between LGG and different films;

Figure 11 shows the effect of LGG microspheres on mouse body weight;

Figure 12 shows pathological images of mouse heart, liver, spleen, lung, and kidney after H&E staining.

Figure 13 shows the changes in mouse body weight during the experiment;

Figure 14 shows the DAI score of mice during the experiment;

Figure 15 shows (a) colon length of mice in each group; (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;

Figure 16 shows 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;

Figure 17 shows the MPO enzyme activity; Note a: compared with the blank group, ###P<0.0001; Note b: compared with the model group, ****P<0.0001;

Figure 18 shows the immunohistochemistry (200×) of (a) IL-10, (b) TNF-α and (c) IL-6 in mouse colon; 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 ways

In view of the problems of low survival rate of probiotics in gastric juice and poor adhesion in the intestinal tract, the present invention proposes a thiolated oxidized guar gum/sodium alginate microsphere and its preparation method and application.

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

S1. OGG is obtained by performing TEMPO oxidation reaction on GG, and L-cys is grafted onto the OGG molecular chain through amidation reaction to generate SOGG;

S2. Prepare a mixed solution containing SOGG, SA and calcium carbonate, add bacterial suspension to the mixed solution and stir well to obtain a water phase; prepare a liquid paraffin solution containing Span80, fully dissolve Span80 to obtain an oil phase; slowly add the water phase Add to the oil phase, emulsify, stir to form W/O droplets, add glacial acetic acid and continue stirring to solidify; break the emulsification, let stand, centrifuge, remove the oil phase, and collect microspheres.

In the present invention, SOGG and SA are used to prepare probiotic-loaded microspheres through ionic cross-linking and disulfide bond cross-linking. The free sulfhydryl groups in SOGG are adhesive and can solve the problem of adhesion and colonization of probiotics in the intestine. The sulfhydryl groups in SOGG auto-oxidize to form disulfide bonds, which can be cross-linked to prepare microspheres. The ions formed by the chelation of SA and Ca ²⁺ do not decompose in the stomach, but form calcium hydroxide in the alkaline intestinal environment and decompose, which plays a role in protecting probiotics in the stomach. However, a single SA microsphere has many pores and poor mechanical properties, and has poor protective effect on probiotics. Therefore, SA and SOGG were compounded and cross-linked to form a double network hydrogel, thereby reducing the porous structure of the SA hydrogel. At the same time, GG can be degraded by colon bacteria to achieve colon-targeted release of probiotics. Intestinal mucus is composed of glycoprotein mucins with many cysteine residues and is rich in sulfhydryl groups. Thiolated polymers can form disulfide bonds with cysteine residues on probiotic surface proteins. Thiopolymers can also form disulfide bonds with the mucus gel layer through exchange reactions of sulfhydryl groups or disulfide bonds. Therefore, sulfopolymers can serve as a bridge between probiotics and mucus, enabling long-term adhesion of probiotics to mucus. The present invention combines SOGG and SA to form microspheres with a double network of ion chelation and disulfide bonds, thereby achieving the effects of protecting probiotics and adhering to intestinal colonization.

In some examples of this embodiment, the bacterium is Lactobacillus rhamnosus CICC 6141 (Lactobacillus rhamnosus), purchased from the China Industrial Microbial Culture Collection Center.

In some examples of this embodiment, the TEMPO oxidation reaction includes the following steps:

Dissolve GG in deionized water, add NaBr and TEMPO, put the solution in an ice water bath after dissolution; adjust the pH to 10-10.5, add NaClO solution dropwise to the solution, finish dripping within 10 minutes, and control the pH to 10-10.5. The oxidation reaction is terminated until all NaClO is consumed and the pH of the reaction no longer changes; alcohol is precipitated, and the precipitate is centrifuged and washed; the separated precipitate is redissolved in water, dialyzed, and freeze-dried to obtain OGG.

Among them, the synthesis route of OGG is as follows:

In some examples of this embodiment, the molecular weight of GG is 200-250 kDa, and the aqueous solution is mostly turbid. There are a large number of hydroxyl groups in the GG molecular chain, and intramolecular hydrogen bond self-crosslinking is the main reason for the poor solubility of GG.

In some examples of this embodiment, the dosage ratio of GG to deionized water is 0.2-0.8g:190-210mL.

In some examples of this embodiment, the amount of NaBr added is 0.25-0.27g/g, GG.

In some examples of this embodiment, the added amount of TEMPO is 18-22 mg/g, GG.

In some examples of this embodiment, the available chlorine content in the NaClO solution is 10%; the dosage of NaClO is 20-25 mmol/g, GG. In the TEMPO oxidation system, NaClO is used as an oxidant, and its dosage determines the degree of GG oxidation. The TEMPO oxidation system can oxidize the hydroxyl group at the C6 position of GG to a carboxyl group. At a dosage of 20-25mmol/g of GG, the TEMPO oxidation system can oxidize the hydroxyl group at the C6 position of GG to more than 90%. When the amount of NaClO is increased, the degree of oxidation remains basically unchanged.

In some examples of this embodiment, the amidation reaction includes the following steps:

Prepare an OGG aqueous solution with a mass fraction of 0.15-0.25%, add EDC to activate the carboxyl group, add NHS to fix the carboxyl group after 15-25 minutes, stir the reaction at room temperature in the dark for 30-50 minutes; add L-cys to the reaction solution, adjust the pH, and avoid light Stir continuously at room temperature for 24 hours; after the reaction is completed, dialyze to remove unreacted reagents; freeze-dry the dialyzed liquid to obtain SOGG.

Among them, the synthesis route of SOGG is as follows:

In some examples of this embodiment, the pH is 5.0-6.0. The reaction pH affects the thiol content in SOGG. When the reaction pH increases, the thiol content in SOGG first increases and then decreases. When pH=5.5, the sulfhydryl content reaches the highest level.

In some examples of this implementation, EDC:NHS=4-5:1 mass ratio. The ratio of EDC/NHS also affects the sulfhydryl content in SOGG. EDC plays the role of activating carboxyl groups, and NHS plays the role of fixing carboxyl groups. When EDC/NHS=5:1, the sulfhydryl content reaches the highest level. This may be because the NHS fixed carboxyl group combines with the activated carboxyl group and competes with L-cys.

In some examples of this implementation, the mass ratio of OGG to L-cys is 1:5-10. The mass ratio of OGG/L-cys also affects the sulfhydryl content in SOGG. As the input amount of L-cys increases, the thiol content in SOGG first increases and then decreases. When the feed ratio is 1:7, the free thiol content is the highest.

In some examples of this implementation, the dosage ratio between OGG and EDC is 1:5.

In some examples of this embodiment, the concentration of the bacterial suspension is 2×10 ⁹ cfu/mL; the volume ratio of the bacterial suspension and the mixed liquid is 1:5.

In some examples of this embodiment, the volume fraction of Span80 in the liquid paraffin solution is 1.5%-2.5%, more preferably 2%.

In some examples of this embodiment, the concentration of SOGG in the aqueous phase is 0.25%-0.75%, mass percentage. The concentration of SOGG affects the encapsulation rate of probiotic bacteria in microspheres. The embedding rate of microspheres increases first and then decreases as the concentration of SOGG increases. When the SOGG concentration is 0.5%, the embedding rate of microspheres is maximum.

In some examples of this embodiment, the mass ratio of SA to CaCO ₃ in the aqueous phase is 1:3-5. The encapsulation effect of microspheres is closely related to the mass ratio of SA to _CaCO3 . Therefore, the mass ratio of SA/CaCO ₃ will affect the embedding effect of microspheres. At the ratio of 1:4, the embedding effect of microspheres is better.

In some examples of this embodiment, the volume ratio of the water phase to the oil phase is 1:2-4. In the preparation process of microspheres, emulsification plays a key role. The water-oil volume ratio can affect the particle size of microspheres and thus the quality of microspheres. When the volume of the oil phase gradually increases, the embedding rate of microspheres first increases and then decreases. At a volume ratio of 1:3, the quality of the microspheres produced was good.

In some examples of this embodiment, the added amount of glacial acetic acid is 0.25-0.35 mL, and more preferably 0.3 mL. Glacial acetic acid reacts with calcium carbonate to release Ca ²⁺ , which plays a key role in SA gel formation. If too little glacial acetic acid is used, it is not conducive to SA gel formation and it is difficult to prepare microspheres; if too much glacial acetic acid is used, the pH will be too acidic, which will cause the death of probiotics. At the dosage of 0.25-0.35mL, the quality of the microspheres obtained and the number of viable bacteria after embedding were both good.

In some examples of this embodiment, the optimal preparation of LGG microspheres was obtained through orthogonal experiments by comprehensively considering the effects of SOGG concentration, SA/CaCO ₃ mass ratio, water-to-oil volume ratio, and glacial acetic acid dosage on the encapsulation rate of LGG microspheres. The process conditions are: in the water phase, the concentration of SOGG is 0.5%, the mass ratio of SA to calcium carbonate is 1:3, the volume ratio of the water phase to the oil phase is 1:3, and the addition amount of glacial acetic acid is 0.3 mL.

Another typical embodiment of the present invention provides a thiolated oxidized guar gum/sodium alginate microsphere, which is prepared by the above preparation method.

In some examples of this embodiment, the thiolated oxidized guar gum/sodium alginate microspheres have a regular spherical structure with a smooth surface and an average particle size of 260-350 μm.

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.

In some examples of this embodiment, the application is in the preparation of medicaments related to the treatment of colitis.

In order to enable those skilled in the art to understand the technical solution of the present invention more clearly, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

Example 1

1. Experimental materials

1.1 Main symbols

The symbols used in the experiments are shown in Table 1.

Table 1 Main symbol table

1.2 Main reagents

The main materials and reagents used in the experiment are shown in Table 2

Table 2 Main materials and reagents

Strain: Lactobacillus rhamnosus CICC 6141 (Lactobacillus rhamnosus) was purchased from the China Industrial Microbial Culture Collection Center.

Cells: Macrophages RAW264.7 were cultured in our laboratory.

1.3 Main instruments

The main instruments used in the experiment are shown in Table 3

Table 3 Main instruments

2. Test method

2.1Synthesis of OGG

(1) Weigh 0.5g GG and stir in 200mL of deionized water until completely dissolved. Add 0.13g NaBr (0.26g/g, calculated as GG), 10mg TEMPO (20mg/g, calculated as GG), and continue stirring until TEMPO and NaBr completely dissolved.

(2) Place the solution in an ice water bath at 0°C, adjust the pH to 10.3 (±0.02), add NaClO solution drop by drop, finish dropping within 10 minutes, start the TEMPO-mediated oxidation process, and use 0.5M NaOH to maintain the pH of the reaction at 10.3(±0.02). Record the amount of NaOH solution consumed every 10 minutes until the pH of the reaction no longer changes.

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

(4) Add 3 times the volume of ethanol to precipitate OGG. The precipitate is washed three times with 75% ethanol solution and redissolved in water. The solution is transferred to a dialysis bag and dialyzed in ultrapure water for 72 hours to remove unreacted reagents.

(5) The dialyzed OGG was frozen in a -80°C refrigerator overnight, and then freeze-dried for 48 hours to obtain a cotton-like solid.

(6) Acidification of OGG (HOGG): Disperse 1g/L OGG in 99% acetic acid solution, add 10% HCl (37%, v/v), mix for 20min, centrifuge at 1000g for 20min, dry, and remove excess acid . The carboxylate on OGG was converted to the acidic form (carboxylic acid) for subsequent characterization.

2.2Synthesis of SOGG

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

(2) Add L-cys with different mass ratios to OGG/L-cys (1:1, 1:3, 1:5, 1:7 and 1:10) into the reaction solution, and adjust the reaction pH (4.0, 4.5 , 5.0, 5.5 and 6.0), protected from light, and stirred continuously at room temperature for 24 hours.

(3) After the reaction, dialyze for 3 days using a dialysis bag with a molecular weight intercept of 7500 to remove unreacted reagents. The dialysis medium used was: deionized water with pH=5 on day 1; deionized water with pH=5 containing 1% NaCl on day 2; deionized water with pH=5 on day 3. Change the water every 12 hours and remove unreacted reagents.

(4) Transfer the dialyzed liquid and freeze it in a -80°C refrigerator overnight, and then freeze-dry it for 48 hours to obtain a cotton-like solid.

2.3 Preparation of LGG microspheres

2.3.1 Preparation of LGG bacterial suspension

The LGG strain was inoculated into 10 mL MRS broth culture medium at an inoculum volume of 2% in a clean workbench, and cultured in a 37°C constant temperature incubator for 24 h. The activated LGG strain was then passaged at an inoculum volume of 2%. Inoculate 2% of the inoculum into a 200 mL broth medium and culture it in a 37°C constant-temperature incubator for 14 h. Perform this step again for passage. Centrifuge the cultured bacterial solution at 4000 rpm for 10 minutes and remove the supernatant. Wash the bacterial slurry with sterile 0.85% NaCl solution three times until the supernatant becomes colorless. Finally, resuspend the bacterial sludge with 0.85% physiological saline, and prepare the bacterial liquid with a concentration of 2×10 ⁹ cfu/mL and store it in a 4°C refrigerator for later use.

2.3.2 Preparation of LGG microspheres

Weigh SOGG and SA respectively, dissolve them in deionized water, and stir overnight to fully dissolve them.

Water phase: Prepare a mixed solution containing SOGG, SA and CaCO ₃ , stir thoroughly to mix evenly; vortex and mix the bacterial suspension and the mixed solution in 2.3.1 according to a volume ratio of 1:5.

Oil phase: liquid paraffin containing 2% Span80 by volume, stir at 45°C to fully dissolve Span80.

Slowly add 5 mL of water phase into 20 mL of oil phase, use a magnetic stirrer to coarsely emulsify, and then use a shear homogenizer at 5000 rpm to finely emulsify for 10 min. After mechanical stirring to form W/O droplets, add 200 μL of glacial acetic acid and continue stirring for 40 min. It is fully solidified; add 4 times the volume of sterilized buffer solution to break the emulsification, and let it stand for 2 hours. The microspheres will settle at the bottom of the beaker due to gravity, and the oil phase will float on the water layer. Centrifuge (4000 rpm, 10 min) to remove the oil phase and surfactant, repeat three times, and collect the microspheres; store the collected probiotic microspheres in a 4°C refrigerator. Figure 1 is a schematic diagram of the formation mechanism and function of LGG microspheres.

3. Optimization and characterization

3.1 Characterization of OGG

3.1.1 Effect of sodium hypochlorite dosage on OGG oxidation degree

In the TEMPO oxidation system, NaClO is used as an oxidant, and its dosage determines the degree of GG oxidation. During the synthesis of OGG, a NaClO solution with pH = 10.3 was added to the 0.25% GG polysaccharide solution. The pH of the polysaccharide solution began to decrease. A 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 decreases, continue to add NaClO solution, and continue to neutralize with 0.5M NaOH solution. This process continues until NaClO solution is added dropwise, and the pH of the polysaccharide solution remains basically unchanged. Record the relationship between the consumption of NaClO solution and the oxidation degree OD.

3.1.2FTIR

The mass ratio of the freeze-dried samples of GG and OGG to dried potassium bromide is 1:100. Use an agate mortar to mix the sample and potassium bromide thoroughly, press the tablets with a tablet press, and measure GG and OGG with a Fourier transform infrared spectrometer respectively. Infrared scanning.

3.2 Characterization of SOGG

3.2.1FTIR

Same as the experimental method in 3.1.2.

3.2.2 ¹ HNMR

Take 10 mg of GG and SOGG respectively and fully dissolve them in 1 mL of D ₂ O, and use ¹ H NMR to analyze their chemical structures on a Bruker AM500 spectrometer.

3.2.3 Determination of sulfhydryl content by Ellman method

The basic principle of Ellman's reagent for quantitative detection of free sulfhydryl groups is as follows:

DTNB has no UV spectrum absorption at 412nm. When cross-linked with samples containing thiol groups, it generates 2-nitro-5-mercaptobenzoic acid (TNB ^2- ). TNB ^2- has strong UV absorption at 412nm. , this reaction has high specificity. In this experiment, UV spectrophotometry was used to determine the standard curve of the standard L-cys sample, and then the sulfhydryl content in SOGG was determined through the standard curve.

3.3 Optimization of preparation process of LGG microspheres

Different SOGG mass fractions (0, 0.5%, 1.0%, 1.5%, 2.0%) were determined through single factor experiments; SA:CaCO ₃ mass ratios (1:1, 1:2, 1:3, 1:4, 1: 5); water to oil volume ratio (1:1, 1:2, 1:3, 1:4, 1:5); dosage of glacial acetic acid (0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL). Effect of microsphere embedding effect. Taking the encapsulation rate of probiotics as an indicator, an orthogonal experiment L ₉ (3 ⁴ ) design was used to study the influence of four factors on the entrapment rate of microspheres. The orthogonal experimental design of SOGG-SA microsphere formula L ₉ (3 ⁴ ) is shown in Table 4.

Table 4 Orthogonal Experiment Factor Level Table

3.3.1 Determination of microsphere embedding rate

Add 0.5 g of microspheres to 9 mL of lysis solution for microsphere lysis, shake up and down in a shaker at 37°C and 230 r/min for 1 hour, take samples, and count by plate counting. The number of viable bacteria added initially was counted using the same method.

The encapsulation rate of probiotics can be expressed as: entrapment rate (%) = (m ₁ /m ₀ )*100%

In the formula: m ₀ , the number of viable LGG bacteria initially added, cfu/mL; m ₁ , the number of viable LGG bacteria embedded in the microspheres, cfu/mL.

3.3.2 Characterization of appearance, morphology and particle size of microspheres

Disperse the microspheres in water, observe with a fluorescent inverted microscope, measure the particle size of the microspheres, count 100, and take the average value. The microstructure of the freeze-dried bacteria-loaded microspheres was then observed using a scanning electron microscope.

3.3.3 Tolerance of LGG microspheres to simulated gastrointestinal fluid

Preparation of artificial simulated gastric juice (SGF): add 1g pepsin to 100mL deionized water, add water and place in a 37°C water bath and stir evenly, adjust the pH to 1.2 with 4M hydrochloric acid, and remove with a 0.2μm sterile microporous filter membrane Bacteria, ready for use.

Preparation of artificial simulated intestinal fluid (SIF): add 1g trypsin to 100mL deionized water, then add 0.65g potassium dihydrogen phosphate, adjust the pH to 7.4 with sodium hydroxide solution, stir the above solution evenly, and then use 0.2μm sterile microfiber hole filter membrane for filtration.

Collect LGG in the logarithmic growth phase and centrifuge, wash and resuspend with physiological saline, and adjust the final concentration to approximately 2×10 ⁹ cfu/mL.

Tolerance to simulated gastric juice: Take free LGG bacterial suspension and LGG microsphere samples into 10mL simulated gastric juice test tubes at 37°C, and process at 80r/min for 120min. Add the encapsulation solution to the probiotic microspheres and lyse them at 37°C and 230r/min for 1 hour to completely release the probiotics. The viable bacteria were counted by the plate counting method. After 48 h of incubation, the counts were repeated three times and the average value was taken.

Tolerance to simulated intestinal fluid: Take free LGG bacterial suspension and LGG microsphere samples into 10mL simulated intestinal fluid test tubes preheated at 37°C, and process them at 37°C and 80r/min for 4 hours. The remaining experimental steps are the same as those for simulated gastric juice.

Control group: free LGG was added to 0.85% physiological saline.

Tolerance to continuous gastrointestinal fluid (GIF): Free LGG and LGG microspheres were transferred into test tubes containing 10 mL of simulated gastric fluid preheated at 37°C, and treated with a shaker at 80r/min at 37°C for 120 minutes. Centrifuge at 4000 rpm for 10 min to remove simulated gastric juice, and wash three times with physiological saline. Add 10 mL of simulated intestinal fluid to the test tubes of free LGG bacterial suspension and LGG microspheres respectively, and treat at 37°C and 80 r/min for 4 hours. Centrifuge, remove simulated intestinal fluid, wash with physiological saline, add cyst solution, lyse for 1 hour, plate count, repeat 3 times and take the average.

3.3.4 Adhesion test

3.3.4.1 Adhesion between LGG and membrane

(1) Preparation of SA membrane, SA+GG membrane, SA+OGG membrane, SA+SOGG membrane

Prepare 30 mL of film solution respectively: 1.8% SA, 1.8% SA+1.0% GG, 1.8% SA+1.0% OGG, 1.8% SA+1.0% SOGG, stir at room temperature for 6 hours, and fully dissolve. Subsequently, 1 mL of glycerol was added to the membrane liquid and ultrasonicated for 20 min to remove air bubbles. The film liquid (30 mL) was poured into plastic petri dishes (D = 9 cm) and dried in an oven at 60°C for 24 h. Take out the dried film, place it in a desiccator at 50% humidity and 25°C for 48 hours, then take it out and peel it off for later use.

(2) Determination of the adhesion of composite films to LGG

The four composite films were cut into 1cm ² , adhered to the glass slide, and irradiated under ultraviolet light on a clean workbench for 30 minutes. Combine the LGG bacterial suspension (0.2mL, 2×10 ⁸ cfu/mL) and the film (1cm ² ), incubate at 37°C for 30 minutes, rinse with sterile physiological saline three times, collect the rinse fluid and dilute it gradiently, and remove the unadhered probiotics. The number of bacteria was determined by plate counting method. Calculate the adhesion rate according to the following adhesion rate calculation formula.

Adhesion rate (%) = ((A-B)/A)*100%

A is the number of total LGGs, cfu/mL; B is the number of unadhered LGGs, cfu/mL.

3.3.4.2 In vitro simulation of probiotic microspheres and intestinal adhesion

According to the eversion encapsulation method, the mucosal adhesion of composite particles is measured. The experimental steps are as follows:

Male C57BL/6J mice were fasted for 24 h before intestinal removal, with free access to water during this period. The mice were sacrificed the next day, their abdomen was opened, the colon was removed, the intestine was flushed with physiological saline and the intestinal contents were removed, and 6 cm of the colon was cut and stored in PBS buffer. After everting the colon tract segment into a capsule with a glass rod, place it in 10 mL of PBS solution containing 10 mg of microspheres, shake slowly in a shaker at 37°C for 30 minutes, remove the intestinal capsule, centrifuge, separate the microspheres, and freeze in a freeze dryer Dry for 48 hours, weigh, and calculate mucoadhesion according to the following mucoadhesion calculation formula.

Mucoadhesion (%) = ((C-D)/C)*100%

C is the weight of the initial microspheres, mg; D is the weight of the microspheres that have not adhered to the colon, mg.

3.3.5 Biocompatibility of microspheres

3.3.5.1 Cytotoxicity experiment

The MTT colorimetric method was used to evaluate the in vitro toxicity of SOGG-SA microspheres to RAW264.7 cells. Take 200 mg of blank microspheres and place them in 1 mL of culture medium for extraction for 24 hours, and centrifuge to collect the extract. RAW264.7 cells were counted using a cell counting plate, seeded into a 96-well plate at 5×10 ⁴ cells/well, and incubated in an incubator for 24 h. The extract liquid was diluted with culture medium to different concentrations (200, 100, 50, 25, 12.5, 6.25 mg/mL). Discard the supernatant and replace with extract solutions of different concentrations, 100 μL per well, and culture 6 replicate wells in each group for 24 hours. Add 10 μL MTT solution (5 mg/mL) to each well, incubate for 4 hours in the incubator, aspirate the supernatant, then add 150 μL DMSO to each well, and shake well in the dark to fully dissolve the formazan. Use untreated cells as a control. , use a microplate reader to detect the absorbance at a wavelength of 570 nm, and calculate the cell survival rate.

Survival rate (%)=((A _s -A ₀ )/(A _c -A ₀ ))*100%

In the formula, _As : the absorbance value of the sample; _Ac : the absorbance of the untreated cell group; _A0 : the absorbance value of the blank well.

3.3.5.2 Hemolysis experiment

Take the blood of rats, centrifuge at 1000 rpm, 10 min, 4°C, remove the supernatant, wash repeatedly with PBS to obtain red blood cells and dilute to 5% (V/V). Place 200 mg of blank microspheres in 1 mL of culture medium and extract for 24 hours, collect the extract by centrifugation, and dilute the extract. Mix 0.5 mL of different diluted extracts and 0.5 mL of red blood cells, place on a shaker at 37°C, 100 rpm, for 1 hour, and centrifuge. Positive control: 0.5mL distilled water and 0.5mL red blood cells; blank group: 0.5mL PBS buffer solution and 0.5mL red blood cells. Use a microplate reader to measure the absorbance at 540nm and calculate the hemolysis rate.

Hemolysis rate (%) = ((A _s -A _n )/(A _p -A _n ))*100

In the formula, _As : the absorbance value of the sample; A _n : the absorbance value of the blank control group; A _p : the absorbance value of the distilled water group.

3.3.5.3 In vivo toxicity test

Mice were kept in the animal room for one week to acclimate. In order to study whether the prepared Lactobacillus rhamnosus delivery system has systemic toxicity, mice were administered 0.2 mL microspheres daily for 7 days, and changes in the mice's body weight were regularly detected. The control group was equal volume of normal saline. After the experiment, the mice were sacrificed, and the blood stains of the heart, liver, spleen, lungs, kidneys and other organs were rinsed with physiological saline, fixed with 4% formaldehyde and sent to the samples for observation with an optical microscope.

4. Results and discussion

4.1 Characterization of OGG

4.1.1 Effect of NaClO dosage on GG

The molecular weight of GG is about 220kDa, and the aqueous solution is mostly turbid. There are a large number of hydroxyl groups in the GG molecular chain, and intramolecular hydrogen bond self-crosslinking is the main reason for the poor solubility of GG. Hydrophilic groups carboxyl and sulfhydryl groups were introduced on GG through chemical modification to improve its water solubility. The solubility of GG aqueous solution can be reflected by the transparency. The higher the transparency, the better the solubility. The TEMPO oxidation system can oxidize the C6 hydroxyl group of GG to a carboxyl group. Figure 2 shows that as the amount of NaClO increases, the GG solution gradually changes from turbid to clear; at the same time, the oxidation degree of GG also gradually increases. Calculated by the degree of oxidation, the TEMPO oxidation system can oxidize the hydroxyl group at the C6 position of GG to more than 90%. When the amount of NaClO is increased, the degree of oxidation remains basically unchanged. The light transmittance of the GG solution also continued to increase after oxidation. The results show that the introduction of carboxyl groups can improve the solubility of GG, because the hydrophilicity of the carboxyl groups accelerates the infiltration process of water molecules into GG.

4.1.2FTIR

From the infrared spectrum analysis in Figure 3a, it can be seen that the main absorption peaks of the acidified products of GG, OGG and OGG are similar, indicating that the main structures of GG, OGG and HOGG are consistent, and the TEMPO oxidation system does not destroy the main structure. The C=O absorption peak in the carboxylic acid group after GG oxidation mainly appears at 1614 cm ^-1 , which can be attributed to the C=O stretching vibration of the asymmetric carboxylate (COO-) group and the symmetric COO-. After acidifying OGG, the C=O peak moved to 1734cm ^-1 , indicating that -COOH was generated, the -COOH group was successfully introduced into the GG macromolecular structure, and OGG was successfully prepared.

4.2 Characterization of SOGG

4.2.1FTIR

SOGG is formed through the formation of an amide bond between the amine group of L-cysteine hydrochloride and the carboxylate group of OGG. From Figure 3b, it is observed that SOGG has a -SH peak at 2696cm ^-1 , and amide peaks at 1654cm ^-1 and 1541cm ^-1 , which are generated by the C=O bond and -NH bond of the amide group respectively. It proves that the amide reaction occurred and the grafting was successful; there is a carboxyl peak at 1608cm ^-1 , indicating that carboxyl and sulfhydryl groups coexist in SOGG.

4.2.2 ¹ HNMR

As shown in Figure 4, compared with the spectra of natural GG and OGG, the spectrum of SOGG has obvious characteristic peaks at 3.34ppm and 2.85ppm. The 3.35ppm peak is the -COCH ₃ - moiety in cysteine derivatives. , 2.85ppm is close to the absorption site of -CH ₂ -SH methylene proton. This result shows that the thiol unit has been successfully grafted to the sugar chain of GG, confirming the successful synthesis of SOGG.

4.2.3 Determination of sulfhydryl content by Ellman method

In order to accurately determine the degree of thiol grafting of SOGG, the standard curve of free thiol groups was first determined by the Ellman method. The linear regression equation of free thiol groups is: y=12.857x-0.0071, R ² =0.9993, and the linear relationship between absorbance and thiol concentration is good. Free sulfhydryl groups play an adhesion role, and disulfide bonds serve as cross-linking effects during the preparation of microspheres.

The initial thiol content after amidation reaction was 47.67 μmol/g, and the thiol group content was relatively low. This may be due to the long molecular chain of OGG and low molecular mobility, making it difficult for L-cys to connect to the OGG main chain. Caused. Due to the high sulfur group content, the resulting SOGG has stronger adhesion, so the reaction conditions for synthesizing SOGG were simply optimized using the sulfhydryl group content as an indicator. In order to increase the sulfhydryl content, three factors that have a greater impact on the amidation reaction were further screened out through literature surveys and preliminary experiments: the ratio of EDC/NHS, reaction pH and the amount of L-cys added, and conditions were determined for these three influencing factors. optimization.

4.2.3.1 Screening of conditions for thiol grafting rate

4.2.3.1.1 Effect of different EDC/NHS ratios on sulfhydryl content

Control the pH of the reaction = 5.5, the feed ratio is 1:7, and change the ratio of EDC/NHS. EDC acts to activate the carboxyl group, and NHS acts to fix the carboxyl group. It can be seen from Figure 5a that as the ratio of EDC/NHS increases, the thiol content increases from 191.3 μmol/g to 332.18 μmol/g. When EDC/NHS=5:1, the sulfhydryl content reaches the highest level. This may be because the NHS fixed carboxyl group combines with the activated carboxyl group and competes with L-cys. The experiment tried not to add NHS, but the result was that the sulfhydryl content was not as high as when EDC/NHS=5:1. Therefore, EDC/NHS=5:1 is selected according to the sulfhydryl content for amidation reaction.

4.2.3.1.2 Effect of different pH on sulfhydryl content

Change the pH of the amidation reaction while keeping other conditions unchanged. It can be seen from Figure 5b that when the reaction pH increases from 4.0 to 6.0, the sulfhydryl content first increases and then decreases. When pH=5.5, the thiol content reached the highest level of 204.4 μmol/g, indicating that this experiment is conducive to the amidation reaction under the condition of pH 5.5.

4.2.3.1.3 Effect of different feeding ratios on sulfhydryl content

Different input ratios are the mass ratio of OGG/L-cys. When the input amount of L-cys is changed, other reaction conditions remain unchanged. It can be seen from the results in Figure 5c that as the input amount of L-cys increases, the thiol content first increases and then decreases. When the feeding ratio was 1:7, the free thiol content was the highest at 252.09 μmol/g, which may be related to the collision between L-cys and OGG. When the amount of L-cys added increases, the solution concentration becomes larger, the chance of collision between molecules increases, and more products are generated; as the amount of L-cys gradually increases, the process of the reaction will be inhibited, resulting in the generation of products. reduce.

In summary, the single-factor reaction conditions were screened using the sulfhydryl content of SOGG as an indicator, and finally EDC/NHS = 5:1, pH = 5.5, and a feed ratio of 1:7 were selected as the optimal reaction conditions. The free sulfhydryl group content was calculated by Ellman method: 332.18 μmol/g, the total sulfhydryl group content: 618.128 μmol/g, and 46.26% of the sulfhydryl groups were self-oxidized into disulfide bonds. Free sulfhydryl groups have an adhesive effect and can serve as a bridge between LGG and intestinal mucus. At the same time, disulfide bonds can auto-oxidize to form disulfide bonds, which can play a cross-linking role during the preparation of microspheres.

4.3LGG microspheres

4.3.1 Screening of preparation conditions of LGG microspheres

4.3.1.1 Effect of SOGG concentration on embedding rate

It can be seen from Figure 6a that the embedding rate of microspheres increases first and then decreases as the concentration of SOGG increases. When the SOGG concentration is 0.5%, the embedding rate of microspheres is maximum 57.67%. When the SOGG concentration continues to increase, the entrapment rate decreases. This is mainly because when the concentration of SOGG increases, the viscosity of the polysaccharide solution increases, making it difficult for LGG to be evenly dispersed in the solution. When the concentration of SOGG decreases, the particle size of the formed microspheres becomes smaller and the mechanical strength is low, and the probiotics cannot be effectively embedded, affecting the embedding effect. When the SOGG concentration increased from 0% to 2%, the particle size of the microspheres gradually increased. This was mainly due to the increase in the viscosity of the polysaccharide solution, which was not conducive to emulsification during the preparation of microspheres, resulting in an increase in the particle size of the microspheres. In summary, the optimal SOGG concentration for preparing LGG microspheres is 0.5%.

4.3.1.2 Effect of SA/CaCO ₃ ratio on embedding rate

The encapsulation effect of microspheres is closely related to the mass ratio of SA to _CaCO3 . In the presence of H ⁺ , CaCO ₃ will produce Ca ²⁺ , and the generated Ca ²⁺ will chelate with SA to form a gel. Therefore, the mass ratio of SA/CaCO ₃ will affect the embedding effect of microspheres. It can be seen from Figure 6b that as the mass ratio continues to increase, the embedding of microspheres first increases and then decreases. When the mass ratio is 1:1, the CaCO ₃ concentration is low, and there is remaining SA that is not cross-linked with Ca ²⁺ to form a gel. At this time, the microspheres are not easy to form, have large particle sizes and are adherent, have thin walls, and have low mechanical strength, so the protective effect on probiotics in the stomach is poor. As the mass ratio increases, Ca ²⁺ increases in the reaction and can be more fully cross-linked with SA. Therefore, the entrapment rate of probiotics gradually increases, and the particle size of the formed microspheres gradually becomes smaller, taking on a spherical shape and structure. Tight and non-adhesive, it can enhance the protection of probiotics. When the mass ratio is 1:4, the encapsulation rate of probiotic microspheres reaches a maximum of 43.38%. However, as the mass ratio continues to increase, there is excess Ca2 ⁺ , _CaCO3 cannot all react with H ⁺ , the particle size of the microspheres begins to increase, the spherical shape is poor, and the embedding rate begins to decrease. In summary, the optimal SA/ _CaCO ratio for preparing LGG microspheres is 1:4.

4.3.1.3 Effect of water-oil ratio on embedding rate

In the preparation process of microspheres, emulsification plays a key role. The water-oil volume ratio can affect the particle size of microspheres and thus the quality of microspheres. As shown in Figure 6c, when the oil phase volume gradually increases, the embedding rate of microspheres shows a trend of first increasing and then decreasing. When the water-to-oil volume ratio is 1:1, larger dispersed droplets are produced in the oil phase. The prepared microspheres have large particle sizes, thin microsphere walls, low mechanical strength and are easy to break, so the embedding rate is low. This may be due to the low content of emulsifier and the relatively high content of microspheres formed. During the stirring process, the microspheres collide and squeeze each other, making it difficult for the microspheres to form into balls. When the volume ratio of water to oil is 1:3, the embedding rate is the highest at 34.41%. At this time, the microspheres have a good spherical shape and no adhesion. As the oil phase increases, the dispersion space of the microspheres becomes looser, allowing the water phase to be fully emulsified, and the particle size of the microspheres gradually becomes smaller, which is beneficial to improving the embedding rate of the microspheres. If the particle size of the microspheres is too small, the probiotics will be exposed outside the microspheres. At the same time, the microspheres will easily break under mechanical action such as stirring and centrifugation, resulting in a low encapsulation rate. In summary, the optimal water-to-oil volume ratio for LGG microsphere preparation is 1:3.

4.3.1.4 Effect of glacial acetic acid dosage on embedding rate

Glacial acetic acid reacts with calcium carbonate to release Ca ²⁺ , which plays a key role in SA gel formation. If too little glacial acetic acid is used, it is not conducive to SA gel formation and it is difficult to prepare microspheres; if too much is used, the pH will be too acidic, which will cause the death of probiotic bacteria. Therefore, it is very important to select the amount of glacial acetic acid that can form balls without causing the death of probiotics. As shown in Figure 6d, as the amount of glacial acetic acid increases, the embedding rate first increases and then decreases. When the added amount of glacial acetic acid is 0.2 mL, the small amount of glacial acetic acid results in residual calcium carbonate, which makes the particle size of the microspheres larger and the mechanical strength is weak, so the embedding rate is low. When the added amount of glacial acetic acid is 0.3 mL, the embedding rate reaches a maximum of 81.56%. At this time, the microspheres have uniform particle size, good spherical shape, and high mechanical strength. As the dosage of glacial acetic acid gradually increases, the encapsulation rate begins to decrease, that is, the number of viable bacteria after embedding decreases. This is because too much glacial acetic acid that has not reacted with calcium carbonate lowers the pH of the entire system, resulting in the loss of most probiotics. Bacteria die. In summary, the optimal amount of glacial acetic acid to prepare LGG microspheres is 0.3 mL.

4.3.1.5 Orthogonal test

This orthogonal experiment was conducted on the basis of the above four single-factor experiments, and the factors that had a greater impact on the embedding rate of LGG microspheres were selected: SOGG concentration, SA/CaCO ₃ mass ratio, water-to-oil volume ratio, and glacial acetic acid dosage. The independent variable is to measure the embedding rate of microspheres, and obtain the optimal preparation process conditions of LGG microspheres through orthogonal experiments. Taking the embedding rate as an indicator, the larger the range, the greater the impact of this factor on the embedding rate. It can be seen from Table 5 that the impact of the four factors on the embedding rate is as follows: D (amount of glacial acetic acid) > C (water to oil volume ratio) > A (concentration of SOGG) > B (mass ratio of SA/CaCO ₃ ) . Therefore, A ₁ B ₁ C ₂ D ₂ is the best formula. Since this formula does not appear in the orthogonal experiment list, further verification is required.

Table 5 L ₉ (3 ⁴ ) orthogonal test results of microsphere formula screening

Test number A B C D Embedding rate % 1 A ₁ B ₁ C ₁ D ₁ 79.59 2 A ₁ B ₂ C _{2 D2} 84.88 3 A ₁ B ₃ C ₃ D ₃ 69.61 4 A ₂ B ₁ C ₂ D ₃ 70.94 5 A ₂ B ₂ C ₃ D ₁ 77.83 6 A ₂ B ₃ C _{1 D2} 78.61 7 A ₃ B ₁ C _{3 D2} 84.36 8 A ₃ B ₂ C ₁ D ₃ 68.48 9 A ₃ B ₃ C ₂ D ₁ 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 k ₁ 78.03 78.30 75.56 79.26 k ₂ 75.79 77.06 78.73 82.62 k ₃ 77.74 76.20 77.27 69.68 R 2.23 2.10 3.17 12.94 better level A ₁ B ₁ C _{2 D2}

The verification test based on the optimal formula shows that the embedding rate of the A ₁ B ₁ C ₂ D ₂ combination is relatively high, reaching (88.67±6.8)%. After testing, the embedding rate of A ₁ B ₁ C ₂ D ₂ is significantly higher than other groups (P<0.05). Moreover, the microspheres have good sphericity and small particle size. Therefore, the microspheres with the combination of A ₁ B ₁ C ₂ D ₂ were selected for the next experiment. Taking the encapsulation rate as an indicator, the mass fraction of SOGG is 0.5%, the mass ratio of SA/ _CaCO3 is 1:3, the volume ratio of water to oil is 1:3, and the dosage of glacial acetic acid is 0.3mL. As the optimal amount of LGG microspheres, The optimal formula will be verified by subsequent tests.

4.3.2 Appearance and morphology of LGG microspheres

The morphology of wet microspheres and freeze-dried microspheres was observed by ordinary optical microscope and scanning electron microscope, and the results are shown in Figure 7. As can be seen from Figure 7a, the freeze-dried LGG microspheres are white powder when observed with the naked eye. It can be seen from the ordinary optical microscope observation in Figure 7b that LGG microspheres have a regular spherical structure with a smooth surface and an average particle size of 294.46±31.8 μm.

From the scanning electron microscope observation in Figure 7c and Figure 7d, it can be seen that after the microspheres are freeze-dried, wrinkles and depressions appear on the surface of the spheres, and the spheres become spindle-shaped, which is a common phenomenon in freeze-drying. When the microsphere is further magnified and observed, it can be clearly seen through the red arrow that some probiotics are slightly raised and embedded inside the sphere, and some probiotics adhere to the surface of the microsphere, indicating that there are a small number of probiotics on the surface of the microsphere.

4.3.3 Tolerance of LGG microspheres to simulated gastrointestinal fluid

To explore the viability of free LGG and LGG microspheres under simulated gastrointestinal conditions. As shown in Figure 8a, the number of viable bacteria decreased significantly after free LGG was incubated in simulated gastric juice for 2 hours, and the number of colonies decreased by nearly 7 Logs. After incubation in simulated intestinal fluid for 4 hours, the number of colonies decreased by nearly 0.2 Log, which was negligible. A large number of studies have shown that most probiotics will significantly lose their activity after entering the human gastrointestinal tract. The tolerance of LGG microspheres in simulated gastrointestinal fluid was then studied. As shown in Figure 8b, after incubation in simulated gastric fluid for 2 hours and simulated intestinal fluid for 4 hours, the number of viable LGG bacteria after encapsulation was slightly reduced by nearly 0.7. Log, 0.2 Log, showing strong tolerance to simulated gastrointestinal fluid. This shows that the microspheres have a certain protective effect on probiotics. Before exploring the tolerance of LGG microspheres to simulated gastrointestinal fluid, in order to avoid the impact of the encapsulation process on the activity of the probiotics themselves, we studied whether the activity of the probiotics changed before and after encapsulation. It was obtained by plotting the growth curve of LGG after embedding. There was no significant difference in the growth curve of LGG before and after embedding. This shows that the embedding process of microspheres has almost no effect on the activity of LGG.

4.3.4 Tolerance of LGG microspheres in simulated continuous gastrointestinal fluid

The effect of continuous gastrointestinal fluid on free LGG and LGG microspheres was further simulated, and Figure 9 further demonstrates the protective effect of microspheres on LGG. Compared with the normal saline group, free LGG had almost no viable bacteria in the continuous simulated gastrointestinal fluid (P<0.0001). In contrast, probiotics embedded in LGG microspheres were continuously incubated in gastrointestinal fluid for 6 h. Compared with the control group, it was only reduced by 0.5 Log. These results confirm that microspheres have a very important protective effect on LGG in gastrointestinal fluid and minimize the damage caused by gastric acid to probiotic bacteria.

4.3.5 Adhesion test

4.3.5.1 Adhesion between LGG and membrane

In order to examine the adhesion of SA/CaCO ₃ microspheres to LGG, SA was prepared with GG, OGG and SOGG to form composite films, which can indirectly verify the adhesion of SOGG to LGG. The experimental results of adhesion between LGG and four different films are shown in Figure 10. As can be seen from Figure 10a, the mechanical properties of the single SA film are worse than those of the other three groups. Among them, the SA/SOGG film has the best mechanical properties, which may be due to the fact that the disulfide bonds formed by the auto-oxidation of the disulfide bonds in SOGG enhance the mechanical strength of the film. It can be seen from Figure 10b that SA film, SA/GG film, SA/OGG film and SA/SOGG film all have a certain adhesion effect on LGG. Among them, the adhesion rate of SA/SOGG film on LGG is higher than that of the other three groups, indicating that SOGG and The interaction between LGG is the strongest, and it has a stronger adhesion effect on LGG. It shows that the introduction of thiol groups increases the adhesion between GG and LGG. The other three membranes are also adhesive to LGG, probably because the hydroxyl or carboxyl groups on the polysaccharide structure form hydrogen bonds with the groups on LGG. In addition, after the four different films were immersed in water for the same time, the SA/SOGG film remained relatively intact, indicating that the disulfide bonds enhanced the mechanical strength of the film. SOGG can play a bridging role in intestinal mucus through dual adhesion with intestinal mucus and LGG, and ultimately adhere and colonize LGG in the intestinal mucosa.

4.3.5.2 Intestinal adhesion

The adhesion between the microspheres and the intestine was measured using the everted intestinal sac method. Based on the adhesion rate calculation from the experimental results, the adhesion rates of colonic mucus to SOGG/SA microspheres and GG/SA microspheres were 77.67% and 46.17, respectively. %. It can be seen from the results that the colon shows a certain degree of mucosal adhesion to both SOGG/SA microspheres and GG/SA microspheres, mainly due to the presence of a large number of villi on the colon mucosa, which help the microspheres to better adhere to the colon mucosa. and epithelial cells. At the same time, there are more goblet cells in the colon and higher mucin levels in the colon mucosa, which further increases the adhesion of the microspheres in the colon. It may be due to the formation of hydrogen or ionic bonds between the hydroxyl or amino groups on the polysaccharide chain and the groups on the mucus glycoprotein. The adhesion of mouse colon mucosa to SOGG/SA microspheres is significantly higher than that to GG/SA microspheres. The main reason may be that the free sulfhydryl groups on the surface of SOGG/SA microspheres interact with the mucus glycoprotein secreted by the colon mucosa. The sulfhydryl reaction forms a disulfide bond, further increasing the mucosal adhesion of the microspheres.

4.3.6 In vivo safety evaluation

As shown in Figure 11, after 7 days of continuous intragastric administration of LGG microspheres to healthy mice, the weight of the mice remained stable and continued to grow normally. Compared with the blank group, there was almost no difference in the mental state and activity status of the mice in the experimental group.

The mice were then sacrificed, and their hearts, livers, spleens, lungs, and kidneys were collected for HE staining pathological analysis. As shown in Figure 12, the structure of the internal organs of the mice after intragastric administration of LGG microspheres was not significantly different from that of the mice in the non-administered group, and no obvious inflammatory reaction was detected in any tissue. The above results show that the in vivo toxicity of LGG microspheres is negligible, which is beneficial to the in vivo application of LGG microspheres.

Application of Lactobacillus rhamnosus microspheres prepared in Example 1 to treat DSS-induced colitis mice

1. Experimental animals

SPF grade C57BL/6J male mice about 4-6 weeks old were used and purchased from Jinan Vitong Lever.

2. Experimental methods

2.1 Construction of colitis mouse model

Before animal modeling, C57BL/6J mice were fed for 7 days to adapt to the environment. The mice drank 3% DSS solution freely for 7 days to establish a colitis model. The DSS solution was replaced every 1 day while treatment was observed. The mice were given DSS on the day of modeling, and the mice were given DSS once a day. Try to choose the same time point for the intragastric administration, with 0.2 mL of bacterial solution each time. The normal group and DSS model group were given corresponding volumes of physiological saline respectively.

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

Establishment of experimental animal model: 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 conditions are as follows:

Control group: only 0.2mL sterile saline was administered intragastrically

DSS group: Drinking DSS + intragastric administration of 0.2mL sterile saline

Free LGG group: Drinking DSS + intragastric administration of 0.2mL 2×10 ⁹ cfu/mL bacterial suspension

LGG microsphere group: drinking DSS + intragastric administration of 0.2 mL LGG microsphere suspension (microspheres with the best performance prepared in Example 1)

2.2 DAI evaluation of mice

During the experiment, the mice's mental state, food and water, activities, defecation, coat color, weight changes, and death were observed and recorded. As shown in Table 6, the average of the three scores of mouse weight loss percentage, fecal characteristics and occult blood detection (o-toluidine method) was used as the disease activity index (DAI), and the preliminary judgment was based on the mouse status and DAI score. Drug administration effect.

DAI = (weight loss score + stool character score + blood in stool score)/3

Table 6 Mouse DAI scoring criteria

2.3 Mouse colon tissue collection, length measurement and HE staining

The status of the mice was observed several times a day. When more than 80% of the mice in the DSS group showed gross blood in their stools, the mice were sacrificed and their abdominal cavities were opened. The colon was measured between the anal opening and the cecum. Wash the remaining feces and blood with pre-cooled PBS solution, and absorb the water with filter paper. The colon was divided into two parts. One part was used for HE staining and immunohistochemical staining. The colon 1cm above the anus was soaked in paraformaldehyde and sent for sample. The HE staining results were used for histological scoring. The other part was used for marrow. For peroxidase determination, the specimens were quickly transferred to a -80°C refrigerator and stored for subsequent experiments to avoid repeated freezing and thawing.

Table 7 Colon histological scores

2.4 Changes in mouse spleen

After the mice were sacrificed by cervical dislocation and dissected, the spleen was removed, the weight of the spleen was measured, and the spleen coefficient was calculated according to the spleen coefficient formula.

Spleen coefficient (%)=(spleen weight/mouse weight)*100%

2.5 Detection of myeloperoxidase in colon tissue

Rinse the colon tissue and the feces inside with PBS buffer solution. Cut the colon tissue into pieces and grind it fully with a tissue grinder. The tissue homogenate should be as uniform as possible without large pieces of tissue. Make a 5% tissue homogenate. The experimental steps are as follows: Kit instructions from Nanjing Jiancheng Bioengineering Institute. By measuring the absorbance value at 460nm, the activity of MPO enzyme is calculated according to the formula, and the unit is U/g wet weight.

MPO activity=(A _measurement -A _control )/(11.3×W)

In the formula: 11.3 is the reciprocal of the slope; A is the absorbance value at 460nm; W is the sample sampling volume.

2.6 Immunohistochemical analysis of mouse colon

The experimental steps are the same as 2.3, and colon tissue samples are sent.

3. Results and discussion

3.1 Observation of general conditions of mice

Colitis was induced in mice after 7 days of ad libitum administration of 3% DSS. Observe the physical changes of mice in each group during the experiment. The following are the results of each group:

(1) Normal group (Control group): One mouse lost weight rapidly, which may be due to damage to the esophagus during gastric administration, resulting in weight loss. The remaining 11 mice had normal diet, drinking water, and feces color and shape, with smooth coat color and active spirit.

(2) Model group (DSS group): During the entire experiment, none of the 12 mice died. On the 3-4th day of the experiment, the mice appeared in varying degrees of clustering, rough hair, and mild diarrhea; Experiment 5 On the -7th day, the mice drank less water and ate less. On the 5th day, 20% of the mice showed symptoms such as listlessness, inflexible reactions, piloerection, and loose stools. As the experimental time increased, the mice's diarrhea worsened. Blood stains can be seen in the anal opening, and the above symptoms are aggravated. When more than 80% of the mice in the DSS group show gross blood in their stools, the next experiment can be carried out.

(3) Free LGG group (DSS+LGG group): 1-5 days after the start of the experiment, all physical signs of the mice were normal. On the 5th to 7th day, there was slight anorexia and laziness, and 30% of the feces became slightly thinner, but there was no large-scale diarrhea or blood in the stool. The mental state and condition of the mice were better than those of the normal group.

(4) LGG microsphere group (DSS+Microsphere group): None of the mice died during the experiment. There was no significant difference between the 1-6 days of the experiment and the normal group. On the 6th day, 10% of the mice's feces became slightly thinner. However, there was no widespread diarrhea or blood in the stool. The mental state and condition of the mice were better than those of the normal group and the free LGG group.

3.2 Changes in mouse body weight

In order to study the effects of free LGG and embedded LGG microspheres on mice with colitis, the body weight changes of mice in each group were recorded every day during the experiment. The results are shown in Figure 13. The weight of the mice in the blank group showed a gradual increase trend, while the DSS group, DSS+LGG group and DSS+LGG microsphere group all showed a downward trend. The weight of mice in the DSS group decreased significantly from the 5th day, and the difference was statistically significant (P<0.001). Compared with the weight of mice in the DSS group, the weight of mice in the DSS+LGG group and DSS+LGG microsphere group decreased relatively slowly. At the same time, the treatment effect of the LGG microsphere group was relatively better, with a reduced number of loose stools. The difference was: Statistical significance (P<0.01). The results showed that LGG microspheres had a certain therapeutic effect on colitis mice.

3.3 DAI evaluation of mice

During the experiment, the weight loss percentage, stool characteristics, and occult blood test results of mice in each group were recorded every day, and the DAI score was evaluated. As shown in Figure 14, as the experimental time prolongs, the DAI of mice in the DSS group continues to increase. On the 5th day, the mice in the DSS group could see symptoms such as loose stools, blood in the stool, and lethargy. Compared with the DSS+LGG group and the DSS+microsphere group, the increase in DAI in mice was significantly improved from the 5th day. The results showed that both LGG and LGG microspheres could effectively reduce the DAI score of mice with colitis, and the effect of encapsulated probiotics was better, indicating that LGG microspheres were more effective in treating colitis than free LGG.

3.4 Colon length and pathological damage evaluation

As shown in Figure 15a, the length of the colon of the DSS group was significantly shorter, while the length of the colon of mice treated with LGG and LGG microspheres was significantly longer, indicating that both have certain effects on the treatment of colitis. At the same time, there was a significant difference in the length of the colon between the DSS+LGG group and the DSS+LGG microsphere group. As shown in Figure 15b, after H&E staining, the colon of the mice in the blank group was in good condition, containing a large number of goblet cells, the intestinal epithelium and crypt structures were intact and orderly, and there was no inflammatory cell infiltration. A large number of mucosal epithelial cells in the colon of mice in the DSS group were necrotic and eroded, a large number of inflammatory cells were infiltrated in the mucosal layer and lower layer, the number of goblet cells was significantly reduced and the intestinal crypt structure was damaged. The colon tissue damage score of the DSS group was significantly higher than that of the blank group; After the intervention of LGG and LGG microspheres, the ulcers and erosions of the colon mucosa were significantly reduced or disappeared, and the arrangement of goblet cells returned to normal, all of which significantly reduced the colon pathology score. In addition, compared with the histological score of the LGG group, the LGG microsphere group significantly improved the colon mucosal damage. The above results all show that the LGG microsphere has a better therapeutic effect on colitis than unembedded LGG.

3.5 Changes in mouse spleen

As can be seen from Figure 16, the spleen coefficient of the DSS group increased significantly, and with the treatment of free LGG and LGG microspheres, the spleen coefficient decreased. This shows that both free probiotics and embedded probiotics have certain therapeutic effects on colitis. There was almost no significant difference between mice treated with LGG microspheres and the blank group, indicating that LGG microspheres could alleviate the enlargement of the spleen coefficient induced by DSS and have a certain therapeutic effect on colitis.

3.6 Detection of myeloperoxidase in colon tissue

As shown in Figure 17, 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 microsphere treatment, 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). The LGG microsphere group had a better therapeutic effect on colitis.

3.7 Immunohistochemical analysis of mouse colon

As can be seen from Figure 18, yellow-brown color is a positive protein. Compared with the blank group, the IL-10 content is significantly down-regulated when stimulated by DSS. This indicates that the colon of mice in the DSS group contains a large number of neutrophils infiltrating and the inflammation is severe. After treatment, both LGG and LGG microspheres could significantly increase the content of colonic IL-10, which was stimulated by DSS and decreased secretion. It can be seen that LGG and LGG microspheres can alleviate colon inflammation by up-regulating IL-10 content, among which the LGG microsphere group has the most obvious up-regulation effect.

The contents of IL-6 and TNF-α in the colon of mice can be seen from Figure 18. The contents of IL-6 and TNF-α in the colon of mice in the control group were low. When stimulated with DSS, the contents of IL-6 and TNF-α increased significantly. . When treated with LGG and LGG microspheres, the levels of IL-6 and TNF-α secreted increased by DSS stimulation were reduced to varying degrees, and there were significant differences compared with the DSS group. As can be seen from Figure 18, both LGG and LGG microspheres can reduce intestinal inflammation by down-regulating the levels of IL-6 and TNF-α, and the improvement effect of the LGG microsphere group is the most significant.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. The preparation method of the sulfhydryl oxidized guar gum/sodium alginate microsphere is characterized by comprising the following steps:

s1, performing TEMPO oxidation reaction on guar gum to obtain oxidized guar gum, and grafting L-cysteine onto a molecular chain of the oxidized guar gum through amidation reaction to generate sulfhydrylated oxidized guar gum;

S2, preparing a mixed solution containing sulfhydrylation guar gum, sodium alginate and calcium carbonate, adding bacterial suspension into the mixed solution, and stirring uniformly to obtain a water phase; preparing a liquid paraffin solution containing span 80, and fully dissolving span 80 to obtain an oil phase; slowly adding the water phase into the oil phase, emulsifying, stirring to form W/O liquid drops, adding glacial acetic acid, and continuously stirring to solidify; demulsification, standing, centrifugation, removal of oil phase and collection to obtain the microsphere.

2. The method for preparing the thiolated guar gum/sodium alginate microspheres according to claim 1, wherein the bacteria is lactobacillus rhamnosus cic 6141 (Lactobacillus rhamnosus).

3. The method for preparing the sulfhydryl oxidized guar gum/sodium alginate microspheres according to claim 1, wherein the TEMPO oxidation reaction comprises the following steps:

dissolving guar gum in deionized water, adding sodium bromide and 2, 6-tetramethylpiperidine oxide TEMPO, and placing the solution in an ice water bath after dissolving; regulating pH to 10-10.5, dropwise adding sodium hypochlorite solution into the solution, controlling pH to 10-10.5 within 10min, and stopping oxidation reaction until sodium hypochlorite is completely consumed and the pH of the reaction is not changed; alcohol precipitation, and washing after centrifuging the precipitate; redissolving the separated precipitate in water, dialyzing, and freeze-drying to obtain the oxidized guar OGG;

Preferably, the guar gum has a molecular weight of 200-250kDa;

preferably, the dosage ratio of guar gum to deionized water is 0.2-0.8g:190-210mL;

preferably, the addition amount of sodium bromide is 0.25-0.27g/g, guar gum;

preferably, the addition amount of TEMPO is 18-22mg/g, guar gum;

preferably, the effective chlorine content in the sodium hypochlorite solution is 10%; the dosage of the sodium hypochlorite is 20-25mmol/g, and guar gum is used.

4. The method for preparing the thiolated guar gum/sodium alginate microspheres according to claim 1, wherein the amidation reaction comprises the following steps:

preparing an oxidized guar gum water solution with the mass fraction of 0.15-0.25%, adding EDC to activate carboxyl, adding NHS to fix carboxyl after 15-25min, and stirring at room temperature in a dark place for reaction for 30-50min; adding L-cysteine into the reaction solution, adjusting the pH value, and continuously stirring at room temperature for 24 hours in a light-resistant environment; after the reaction is completed, dialyzing to remove unreacted reagents; freeze-drying the dialyzed liquid to obtain sulfhydrylation oxidized guar gum;

preferably, the pH is 5.0 to 6.0, more preferably 5.5;

preferably, EDC: nhs=4-5:1 mass ratio, further preferably 5:1;

Preferably, the mass ratio of the oxidized guar gum to the L-cysteine is 1:5-10, and more preferably 1:7;

preferably, the dosage ratio of the oxidized guar gum to the EDC is 1:5.

5. The method for preparing the thiolated guar gum/sodium alginate microspheres according to claim 1, wherein the concentration of the bacterial suspension is 2 x 10 ⁹ cfu/mL; the volume ratio of the bacterial suspension to the mixed solution is 1:5;

preferably, in the liquid paraffin solution, the volume fraction of span 80 is 1.5% -2.5%, and more preferably 2%.

6. The method for preparing the thiolated guar gum/sodium alginate microspheres according to claim 1, wherein the concentration of the thiolated guar gum in the aqueous phase is 0.25% -0.75%, and more preferably 0.5%;

preferably, in the aqueous phase, the mass ratio of sodium alginate to calcium carbonate is 1:3-5, and more preferably 1:4;

preferably, the volume ratio of the aqueous phase to the oil phase is 1:2-4, more preferably 1:3;

preferably, the glacial acetic acid is added in an amount of 0.25 to 0.35mL, more preferably 0.3mL.

7. The method for preparing the thiolated guar gum/sodium alginate microspheres according to claim 6, wherein the concentration of the thiolated guar gum in the water phase is 0.5%, the mass ratio of sodium alginate to calcium carbonate is 1:3, the volume ratio of the water phase to the oil phase is 1:3, and the addition amount of glacial acetic acid is 0.3mL.

8. A thiolated oxidized guar gum/sodium alginate microsphere, characterized in that it is prepared by the preparation method of any one of claims 1-7.

9. The thiolated guar gum/sodium alginate microsphere according to claim 8, wherein the thiolated guar gum/sodium alginate microsphere has a regular spherical structure with a smooth surface and an average particle size of 260-350 μm.

10. Use of a thiolated guar gum/sodium alginate microsphere according to claim 8 or 9 in the field of biomedical materials;

preferably, the use is in the manufacture of a medicament for the treatment of colitis.