CN116725958A - Sulfhydrylation oxidation guar gum/sodium alginate microsphere and preparation method and application thereof - Google Patents
Sulfhydrylation oxidation guar gum/sodium alginate microsphere and preparation method and application thereof Download PDFInfo
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- CN116725958A CN116725958A CN202310640431.3A CN202310640431A CN116725958A CN 116725958 A CN116725958 A CN 116725958A CN 202310640431 A CN202310640431 A CN 202310640431A CN 116725958 A CN116725958 A CN 116725958A
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Classifications
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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
Technical Field
The invention belongs to the field of biomedical materials, and in particular relates to a sulfhydryl oxidized guar gum/sodium alginate microsphere, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Inflammatory bowel disease (Inflammatory bowel diseases, IBD) is a chronic disease of the intestinal tract with symptoms such as abdominal pain, diarrhea, hematochezia, and weight loss, which severely threatens human health. IBD has become a global public health problem. Although the pathogenesis of the disease is not yet clear, there is a potential correlation between the intestinal microbiota and the inflammatory profile. Probiotics can play a probiotic role in the body by modulating immune function, producing organic acids and antimicrobial compounds. However, since probiotics are susceptible to adverse factors such as low pH of gastric acid and bile after entering the digestive tract through oral administration, the survival rate of the probiotics reaching the intestinal tract is reduced, and it is difficult for a sufficient amount of probiotics to reach intestinal colonization, thereby exerting the probiotic effect. At present, microspheres are one of the most effective techniques for embedding probiotics. The microsphere can obviously enhance the tolerance of probiotics to severe environments, thereby increasing the number of live bacteria of the probiotics reaching the intestinal tract. Although probiotics can improve survival rate by embedding the microspheres in simulated gastric fluid, they have poor adhesion in the intestinal tract. Therefore, development of a novel drug delivery system with gastric acid resistance, intestinal targeting and intestinal adhesion fixation functions is urgently needed.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide the sulfhydrylation oxidation guar gum/sodium alginate microsphere, and the preparation method and the application thereof.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in a first aspect of the invention, a preparation method of sulfhydryl oxidized guar gum/sodium alginate microspheres is provided, comprising the following steps:
s1, carrying out TEMPO oxidation reaction on GG to obtain OGG, and grafting L-cys onto an OGG molecular chain through amidation reaction to generate SOGG;
s2, preparing a mixed solution containing SOGG, SA and calcium carbonate, adding a 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, centrifuging, removing the oil phase, and collecting the sulfhydryl guar gum/sodium alginate microspheres, namely LGG microspheres.
In a second aspect of the present invention, there is provided a thiolated guar gum/sodium alginate microsphere prepared by the above preparation method.
In a third aspect, the present invention provides an application of the above-mentioned thiolated oxidized guar gum/sodium alginate microsphere in the field of biomedical materials.
The beneficial effects of the invention are as follows:
few studies on intestinal targeting of modified GG-embedded probiotics have been reported. GG is a natural high molecular polysaccharide, low in cost, good in biocompatibility and nontoxic, and SOGG is prepared through amidation reaction, so that a new idea is provided for GG application.
According to the invention, the SOGG with mercapto is prepared by carrying out TEMPO system oxidation on natural polymer polysaccharide GG and then grafting and modifying L-cys through an amide bond. And (3) constructing the LGG microsphere by adopting an emulsion gel method and utilizing SOGG and SA to crosslink the loaded LGG. The LGG microsphere has good biocompatibility. The invention utilizes SOGG mercapto to form disulfide bond and SA and Ca 2+ And (3) preparing the double-network crosslinked microsphere by virtue of the characteristic of ion gel formation. The microsphere has intestinal adhesion and gastric acid resistance, and can improve survival rate of LGG in stomach; the sulfhydryl group on the microsphere plays a role in connecting probiotics and intestinal mucus, increases the field planting adhesion of the probiotics in the intestinal tract, and is beneficial to the probiotics to play a role in the intestinal tract. The invention provides a theoretical basis for the application of probiotics in the milk products, beverages and medicine industries.
The in vitro simulated gastrointestinal fluid experiment proves that the LGG microsphere prepared by the invention improves the survival rate of the LGG in gastric acid and the adhesion of intestinal tracts, and has a protective effect on the LGG.
Through in vivo simulation tests, the colitis mouse model constructed by 3% DSS induction proves that compared with free LGG for treating colitis, the LGG microsphere treatment can remarkably relieve colon shortening, mouse weight loss, disease activity index increase and spleen coefficient increase caused by DSS, and reduce colonitis cell infiltration, so that the LGG microsphere can effectively relieve colitis and has a good treatment effect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram showing the mechanism and action of the LGG microsphere prepared in example 1 of the present invention;
FIG. 2 is a graph of sodium hypochlorite usage versus GG, where a is the effect of NaClO usage on GG oxidation; b is the light transmittance of GG and OGG;
FIG. 3 is an infrared spectrum of SOGG characterization; wherein a is the infrared spectrogram of GG, OGG and the products of the OGG after acidification; b is the infrared spectrograms of GG, OGG and SOGG;
FIG. 4 is a diagram of GG, OGG, SOGG and L-cys 1 HNMR diagram;
FIG. 5 is a screen diagram of thiol graft ratio conditions; wherein a is the influence of EDC/NHS on the mercapto content; b is the influence of different reaction pH on the mercapto content; c is the influence of different feeding ratios on the mercapto content;
FIG. 6 is a screen diagram of conditions for preparing LGG microspheres; wherein a is the influence of SOGG concentration on the embedding rate; b is SA, caCO 3 The influence of the ratio of (2) on the embedding rate; c is the influence of the water-oil volume ratio on the embedding rate; d is the influence of the consumption of glacial acetic acid on the embedding rate;
FIG. 7 is an external appearance of LGG microspheres; wherein a is the freeze-dried LGG microsphere, and b is a morphological diagram of the microsphere under a common optical microscope; c. d is a scanning electron microscope image of the LGG microsphere at different magnification;
FIG. 8 is a graph showing the tolerance of LGG microspheres to simulated gastrointestinal fluids; wherein a is tolerance of free LGG to simulated gastrointestinal fluids; b is the tolerance of the LGG microsphere to simulated gastrointestinal fluids;
FIG. 9 is a graph showing the tolerance of LGG microspheres in simulating continuous gastrointestinal;
FIG. 10 shows LGG and film adhesion test results; wherein a is a film of four different polysaccharides; b is the adhesion of LGG to different films;
FIG. 11 is a graph showing the effect of LGG microspheres on mouse body weight;
FIG. 12 is an H & E stained pathology image of mice heart, liver, spleen, lung, kidney
FIG. 13 shows the change in body weight of mice during the experiment;
FIG. 14 is a DAI score of mice during the course of the experiment;
FIG. 15 shows colon length for each group of mice (a); (b) HE staining and histological scoring (200×); and (b) pouring a: compared to the blank, # # P <0.0001; b, injecting: comparing with the model group, P < 0.005, P <0.0001;
FIG. 16 is the spleen factor of mice; and (b) pouring a: compared to the blank, # # P <0.0001; b, injecting: comparing P <0.0001;
FIG. 17 shows MPO enzyme activity; and (b) pouring a: compared to the blank, # # P <0.0001; b, injecting: comparing P <0.0001;
FIG. 18 shows immunohistochemistry of the colon (a) IL-10, (b) TNF- α and (c) IL-6 of mice (200X); and (b) pouring a: p <0.05 compared to the blank; b, injecting: compared to the model group, #p <0.01, #p <0.05, #p <0.001.
Detailed Description
In view of the problems of low survival rate of probiotics in gastric juice and poor adhesiveness in intestinal tracts, the invention provides a sulfhydryl guar gum/sodium alginate microsphere and a preparation method and application thereof.
The invention provides a preparation method of sulfhydryl oxidized guar gum/sodium alginate microspheres, which comprises the following steps:
S1, carrying out TEMPO oxidation reaction on GG to obtain OGG, and grafting L-cys onto an OGG molecular chain through amidation reaction to generate SOGG;
s2, preparing a mixed solution containing SOGG, SA and calcium carbonate, adding a bacterial suspension into the mixed solution, and stirring uniformly to obtain a water phase; preparing a liquid paraffin solution containing Span80, wherein the Span80 is fully dissolved 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.
The invention uses SOGG and SA to pass through ionsCrosslinking and disulfide bond crosslinking to prepare the microsphere loaded with probiotics. The free sulfhydryl in SOGG has adhesion and can solve the problem of adhesion and colonization of probiotics in intestinal tracts. The sulfhydryl groups in SOGG can be self-oxidized to form disulfide bonds, and the disulfide bonds can be crosslinked to prepare the microsphere. SA and Ca 2+ The chelated ions are not cracked in the stomach, and calcium hydroxide is formed in the alkaline environment of the intestinal tract for cracking, so that the effect of protecting probiotics in the stomach is achieved. However, a single SA microsphere has many pores and poor mechanical properties, and the protective effect against probiotics is poor. Therefore, SA and SOGG are compounded and crosslinked to form a double-network hydrogel, so that the porous structure of the SA hydrogel is reduced. Meanwhile, GG can be degraded by colonic bacteria to achieve the effect of targeting release of probiotics by colon. Intestinal mucus is composed of many cysteine glycoproteins-mucins, with abundant sulfhydryl groups. The thiolated polymer may form disulfide bonds with cysteines on the probiotic surface protein. The sulfur polymer may also form disulfide bonds with the mucus gel layer by either sulfhydryl or disulfide bond exchange reactions. Therefore, the sulfur polymer can be used as a bridge for connecting probiotics and mucus, so that the probiotics and the mucus can be adhered for a long time. According to the invention, SOGG and SA are compounded to form the double-network microsphere with ion chelation and disulfide bond, so that the effects of protecting probiotics and adhering and planting intestinal tracts are realized.
In some examples of this embodiment, the bacterium is lactobacillus rhamnosus cic 6141 (Lactobacillus rhamnosus), purchased from the chinese industrial microbiological bacterial collection center.
In some examples of this embodiment, the TEMPO oxidation reaction includes the steps of:
dissolving GG in deionized water, adding NaBr and TEMPO, and placing the solution in an ice-water bath after dissolving; regulating pH to 10-10.5, dropwise adding NaClO solution into the solution, controlling pH to 10-10.5 within 10min, and stopping oxidation reaction until NaClO is completely consumed and 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 OGG.
The synthetic route of OGG is shown below:
in some examples of this embodiment, the GG has a molecular weight of 200-250kDa and the aqueous solution is mostly hazy. The existence of a large number of hydroxyl groups in GG molecular chains, and intramolecular hydrogen bond self-crosslinking is a main cause of poor GG solubility.
In some examples of this embodiment, the GG to deionized water dosage ratio is 0.2-0.8g:190-210mL.
In some examples of this embodiment, naBr is added in an amount of 0.25 to 0.27g/g, GG.
In some examples of this embodiment, TEMPO is added in an amount of 18-22mg/g, GG.
In some examples of this embodiment, the effective chlorine content of the NaClO solution is 10%; the amount of NaClO is 20-25mmol/g, GG. In a TEMPO oxidation system, naClO is used as an oxidant, and the amount of NaClO used determines the degree of GG oxidation. The TEMPO oxidation system can oxidize the hydroxyl at the C6 position of GG into carboxyl, and the TEMPO oxidation system can oxidize the hydroxyl at the C6 position of GG to more than 90% under the dosage of 20-25mmol/g of GG. When the amount of NaClO is increased again, the oxidation degree is not changed basically.
In some examples of this embodiment, the amidation reaction comprises the steps of:
preparing OGG water solution with 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 for reaction for 30-50min in dark place; adding L-cys into the reaction solution, adjusting the pH, and continuously stirring at room temperature for 24 hours in a dark environment; after the reaction is completed, dialyzing to remove unreacted reagents; and freeze-drying the dialyzed liquid to obtain SOGG.
The synthetic route of SOGG is as follows:
in some examples of this embodiment, the pH is 5.0 to 6.0. The reaction pH affects the thiol content in SOGG. When the reaction pH increases, the thiol content in SOGG increases and decreases. The thiol content reached a maximum at ph=5.5.
In some examples of this embodiment, EDC: nhs=4-5:1 mass ratio. The EDC/NHS ratio also affects the thiol content in SOGG. EDC acts to activate carboxyl groups and NHS acts to fix carboxyl groups. When EDC/nhs=5:1, the mercapto content reached the highest. This is probably because NHS has a competitive relationship with L-cys by binding the immobilized carboxyl group to the activated carboxyl group.
In some examples of this embodiment, the mass ratio of OGG to L-cys is 1:5-10. The mass ratio of OGG/L-cys also influences the thiol content in SOGG. As the input of L-cys increases, the mercapto content in SOGG increases and then decreases. When the feeding ratio is 1:7, the free mercapto content is the highest.
In some examples of this embodiment, the dosage ratio between OGG and EDC is 1:5.
In some examples of this embodiment, the concentration of the bacterial suspension is 2X 10 9 cfu/mL; the volume ratio of the bacterial suspension to the mixed solution is 1:5.
In some examples of this embodiment, the liquid paraffin solution has a Span80 volume fraction of 1.5% to 2.5%, and more preferably 2%.
In some examples of this embodiment, the concentration of SOGG in the aqueous phase is 0.25% to 0.75% by mass. The concentration of SOGG affects the entrapment rate of probiotics in the microspheres. The embedding rate of the microspheres increases with the concentration of SOGG, and tends to increase and decrease. The entrapment rate of the microspheres was maximized at a SOGG concentration of 0.5%.
In some examples of this embodiment, SA and CaCO are present in the aqueous phase 3 The mass ratio of (3) is 1:3-5. Encapsulation effect of microspheres and SA and CaCO 3 Is closely related to the mass ratio of (c). Thus SA/CaCO 3 The embedding effect of the microspheres can be influenced by the mass ratio. At a ratio of 1:4, the embedding effect of the microspheres is better.
In some examples of this embodiment, the volume ratio of the aqueous phase to the oil phase is 1:2-4. In the preparation process of the microsphere, emulsification plays a key role. The volume ratio of water to oil can influence the particle size of the microsphere and thus the quality of the microsphere. When the volume of the oil phase gradually increases, the embedding rate of the microspheres tends to rise and then fall. The quality of the prepared microsphere is good at the volume ratio of 1:3.
In some examples of this embodiment, the glacial acetic acid is added in an amount of 0.25 to 0.35mL, more preferably 0.3mL. Glacial acetic acid reacts with calcium carbonate to release Ca 2+ Plays a key role in SA gel formation. The glacial acetic acid is too little to be used, is not beneficial to SA gel formation, and is not easy to prepare microspheres; too much pH is too acidic and can cause death of the probiotic. The quality of the obtained microsphere is better under the dosage of 0.25-0.35mL, and the quantity of the live bacteria after embedding is better.
In some examples of this embodiment, SOGG concentration, SA/CaCO are considered in combination 3 The optimal preparation process conditions of the LGG microsphere obtained by orthogonal experiments are as follows: the concentration of SOGG in the water phase 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 0.3mL.
In another exemplary embodiment of the present invention, a thiol-modified guar gum/sodium alginate microsphere is provided, which is prepared by the above preparation method.
In some examples of this embodiment, the thiolated guar/sodium alginate microspheres have a regular spherical structure with a smooth surface and an average particle size of 260-350 μm.
Another exemplary embodiment of the present invention provides an application of the above-mentioned sulfhydryl oxidized guar gum/sodium alginate microsphere in the field of biomedical materials.
In some examples of this embodiment, the use is in the manufacture of a medicament for the treatment of colitis.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
1. Experimental materials
1.1 principal symbols
The symbols used in the experiments are shown in Table 1.
TABLE 1 Primary symbol Table
1.2 major reagents
The main materials and reagents used in the experiment are shown in Table 2
TABLE 2 Main materials and reagents
And (3) strain: lactobacillus rhamnosus cic 6141 (Lactobacillus rhamnosus) is purchased from the chinese industrial microbiological bacterial collection center.
And (3) cells: macrophage RAW264.7 was cultured by the present laboratory.
1.3 major instrumentation
The main instruments used in the experiment are shown in Table 3
TABLE 3 Main instruments
2. Test method
2.1 Synthesis of OGG
(1) 0.5g GG was weighed out and stirred in 200mL deionized water until completely dissolved, 0.13g NaBr (0.26 g/g, GG.) and 10mg TEMPO (20 mg/g, GG.) were added, and stirring was continued until the TEMPO and NaBr were completely dissolved.
(2) The solution was placed in an ice-water bath at 0℃and the pH was adjusted to 10.3 (+ -0.02), naClO solution was added dropwise, and after completion of the dropwise addition within 10min, TEMPO-mediated oxidation was initiated and the pH of the reaction was maintained at 10.3 (+ -0.02) with 0.5M NaOH. The consumption of NaOH solution was recorded every 10min until the pH of the reaction was no longer changed.
(3) 20mL of 95% ethanol was added, the pH of the solution was adjusted to 7, and the reaction was stopped.
(4) 3 volumes of ethanol are added to separate out OGG, the precipitate is washed 3 times with 75% ethanol solution, dissolved again in water, the solution is transferred to a dialysis bag and dialyzed in ultrapure water for 72h, and unreacted reagent is removed.
(5) The dialyzed OGG was placed in a-80 ℃ refrigerator and cooled overnight, and then freeze-dried for 48h to give a cotton-like solid.
(6) Acidification of OGG (hopg): 1g/L OGG was dispersed in 99% acetic acid solution, 10% HCl (37%, v/v) was added, mixed for 20min, centrifuged at 1000g for 20min, dried and excess acid was removed. The carboxylate on OGG was converted to the acidic form (carboxylic acid) for subsequent characterization.
2.2 Synthesis of SOGG
(1) OGG 0.094g was weighed and stirred in distilled water overnight to prepare a 0.2% solution, EDC was added to activate carboxyl groups, after 20min, NHS was added to fix carboxyl groups, and the reaction was stirred at room temperature in the absence of light for 40min.
(2) L-cys with different mass ratios (1:1, 1:3, 1:5, 1:7 and 1:10) with OGG/L-cys is added into the reaction solution, the reaction pH is regulated (4.0, 4.5, 5.0, 5.5 and 6.0), the reaction is protected from light, and the reaction solution is continuously stirred for 24 hours at room temperature.
(3) After the reaction was completed, the reaction mixture was dialyzed for 3d against a dialysis bag having an interception molecular weight of 7500 to remove unreacted reagents. The dialysis medium used: deionized water at 1d, ph=5; 2d, deionized water with ph=5 containing 1% NaCl; 3d, deionized water at ph=5. The water was changed every 12h to remove unreacted reagents.
(4) The dialyzed liquid was transferred and placed in a-80 ℃ refrigerator to be cooled overnight, and then freeze-dried for 48 hours to obtain a cotton-like solid.
2.3 preparation of LGG microspheres
2.3.1 preparation of LGG suspensions
LGG strain was inoculated into 10mL of MRS broth medium at an inoculum size of 2% in an ultra clean bench, and cultured in a constant temperature incubator at 37 ℃ for 24 hours. The activated LGG strain was then passaged at 2% inoculum size. Inoculated in a volume of 200mL of broth at an inoculum size of 2%, and cultured in a constant temperature incubator at 37℃for 14 hours. In this step, passaging was performed once more. The cultured bacterial liquid was centrifuged at 4000rpm for 10min, and the supernatant was removed. The bacterial sludge was washed with sterilized 0.85% NaCl solution and repeated three times until the supernatant became colorless. Finally, the bacterial mud is resuspended by using 0.85 percent physiological saline, and the concentration of the prepared bacterial solution is 2 multiplied by 10 9 cfu/mL is placed in a refrigerator at 4 ℃ for standby.
Preparation of 2.3.2LGG microspheres
SOGG and SA were weighed separately and dissolved in deionized water and stirred overnight for complete dissolution.
Aqueous phase: preparing a mixture containing SOGG, SA and CaCO 3 Fully stirring the mixed solution of the components to ensure that the mixed solution is uniformly mixed; and (3) uniformly stirring and mixing the bacterial suspension in the ratio of 2.3.1 and the mixed solution by vortex according to the volume ratio of 1:5.
An oil phase: liquid paraffin containing Span80 with a volume fraction of 2% was stirred at 45℃to dissolve Span80 sufficiently.
Slowly adding 5mL of water phase into 20mL of oil phase, coarse emulsifying with a magnetic stirrer, fine emulsifying with a shearing homogenizer at 5000rpm for 10min, mechanically stirring to form W/O liquid drops, adding 200 μL of glacial acetic acid, continuously stirring for 40min, and fully solidifying; adding 4 times of sterilized buffer solution to demulsify, standing for 2h, settling microsphere at the bottom of the beaker due to gravity, and floating oil phase on the water upper layer. Centrifuging (4000 rpm,10 min), removing oil phase and surfactant, repeating for three times, and collecting microsphere; and storing the collected probiotic microspheres in a refrigerator at the temperature of 4 ℃. FIG. 1 is a schematic diagram showing the mechanism of LGG microsphere formation and its function.
3. Optimization and characterization
3.1 characterization of OGG
3.1.1 Effect of sodium hypochlorite usage on OGG Oxidation degree
In a TEMPO oxidation system, naClO is used as an oxidant, and the amount of NaClO used determines the degree of GG oxidation. During the synthesis of OGG, naClO solution at ph=10.3 was added to 0.25% GG polysaccharide solution, the pH of the polysaccharide solution started to decrease, and 0.5m noh solution was added to maintain the pH of the GG polysaccharide solution at 10.3. When the pH of the solution is no longer decreasing, the addition of NaClO solution is continued and neutralization with 0.5M NaOH solution is continued, and the process is cycled until the NaClO solution is added dropwise again, with the polysaccharide solution pH being substantially unchanged. The change in consumption of NaClO solution versus the degree of oxidation OD was recorded.
3.1.2FTIR
GG. The mass ratio of the sample subjected to OGG freeze-drying to the dried potassium bromide is 1:100, the sample and the potassium bromide are fully and uniformly mixed by an agate mortar, a tablet press is used for tabletting, and infrared scanning is respectively carried out on GG and OGG by a Fourier infrared spectrometer.
3.2 characterization of SOGG
3.2.1FTIR
The same as in 3.1.2.
3.2.2 1 HNMR
Dissolving 10mg GG and SOGG in 1mL D 2 O, use is made of 1 HNMR resolved its chemical structure on a bruker am500 spectrometer.
3.2.3Ellman method for measuring thiol content
The basic principle of the quantitative detection of free mercapto groups by the Ellman reagent is as follows:
DTNB has no ultraviolet spectral absorption at 412nm and generates 2-nitro-5-mercaptobenzoic acid (TNB) after reaction with thiol-containing sample cross-linking 2- )，TNB 2- The reaction has high specificity and strong ultraviolet absorption at 412 nm. This experiment is led toAnd measuring a standard curve of the standard L-cys sample by using an ultraviolet spectrophotometry, and measuring the mercapto content in the SOGG by using 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 by single factor assay; SA CaCO 3 Mass ratio (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); effect of glacial acetic acid (0.2 mL, 0.3mL, 0.4mL, 0.5mL, 0.6 mL) on microsphere embedding effect. The embedding rate of probiotics is used as an index, and an orthogonal test L is adopted 9 (3 4 ) Design, the influence of 4 factors on the microsphere embedding rate is studied. SOGG-SA microsphere formula L 9 (3 4 ) The orthogonal test designs are shown in table 4.
TABLE 4 level of orthogonal experimental factors
3.3.1 determination of microsphere entrapment Rate
0.5g of microspheres was added to 9mL of the lysis solution for microsphere lysis, and the mixture was shaken up and down in a shaker at 37℃and 230r/min for 1 hour, sampled and counted by plate counting. The number of viable bacteria initially added was counted in the same manner.
The probiotic entrapment rate can be expressed as: embedding rate (%) = (m) 1 /m 0 )*100％
Wherein: m is m 0 Initiating the number of the added LGG viable bacteria, cfu/mL; m is m 1 LGG viable bacteria count, cfu/mL embedded in the microspheres.
3.3.2 microsphere appearance morphology and particle size characterization
Dispersing the microspheres in water, observing with a fluorescent inverted microscope, measuring the particle size of the microspheres, counting 100, and taking an average value. And observing the microstructure of the bacteria-carrying microsphere after freeze drying by a scanning electron microscope.
3.3.3LGG microsphere tolerance to simulated gastrointestinal fluids
Preparation of artificial Simulated Gastric Fluid (SGF): 1g of pepsin is added with 100mL of deionized water, the mixture is placed in a water bath kettle with the temperature of 37 ℃ for uniform stirring, the pH value is adjusted to 1.2 by 4M hydrochloric acid, and the sterile microporous filter membrane with the pH value of 0.2 mu M is used for sterilization, so that the preparation is ready for use.
Preparation of artificial Simulated Intestinal Fluid (SIF): 1g trypsin is added with 100mL deionized water, then 0.65g potassium dihydrogen phosphate is added, the pH value is regulated to 7.4 by sodium hydroxide solution, the solution is stirred uniformly, and then a sterile microporous filter membrane with the thickness of 0.2 mu m is used for filtration.
Collecting LGG in logarithmic growth phase, centrifuging, washing with physiological saline, and re-suspending to adjust final concentration to about 2×10 9 cfu/mL。
Tolerance to simulated gastric fluid: the free LGG bacterial suspension and the LGG microsphere sample are respectively taken into a test tube filled with 10mL simulated gastric fluid at 37 ℃ and treated for 120min at 80 r/min. And adding a vesicle solution into the probiotic microsphere, and performing pyrolysis at 37 ℃ and 230r/min for 1h, so that the probiotics are completely released. Viable bacteria were counted by plate counting, counted after 48 hours of incubation, and averaged 3 times.
Tolerance to simulated intestinal fluid: the free LGG bacterial suspension and the LGG microsphere sample are respectively taken into a 10mL simulated intestinal fluid test tube preheated at 37 ℃, and treated for 4 hours at 37 ℃ and 80r/min, and the rest experimental steps are the same as simulated gastric fluid.
Control group: free LGG was added to 0.85% physiological saline.
Tolerance to continuous gastrointestinal fluids (GIF): free LGG and LGG microspheres were transferred to pre-heated tubes containing 10mL simulated gastric fluid at 37℃and treated for 120min at 37℃on a shaker at 80 r/min. The simulated gastric fluid was removed by centrifugation 4000rpm for 10min and washed 3 times with physiological saline. 10mL of simulated intestinal fluid was added to the free LGG bacterial suspension and the tube of LGG microspheres, respectively, and the mixture was treated at 37℃for 4 hours at 80 r/min. Centrifuging, removing simulated intestinal fluid, washing with physiological saline, adding cyst lysis solution, lysing for 1h, counting plates, repeating for 3 times, and taking average value.
3.3.4 adhesion experiments
3.3.4.1LGG adhesion to film
(1) Preparation of SA film, SA+GG film, SA+OGG film, SA+SOGG film
The preparation method comprises the following steps of: 1.8% SA,1.8% SA+1.0% GG,1.8% SA+1.0% OGG,1.8% SA+1.0% SOGG, and the mixture was stirred at room temperature for 6 hours to dissolve the components sufficiently. Subsequently 1mL of glycerol was added to the membrane solution and sonicated for 20min to remove air bubbles. The membrane solutions (30 mL) were poured separately into plastic petri dishes (d=9 cm) and dried in an oven at 60 ℃ for 24h. Taking out the dried film, placing the film in a dryer with 50% humidity and 25 ℃ for 48 hours, taking out the film and uncovering the film for later use.
(2) Determination of adhesion of composite film to LGG
Cutting four composite films into 1cm 2 Adhere to the slide and irradiate under ultra-clean bench uv lamp for 30min. LGG bacterial suspension (0.2 mL, 2X 10) 8 cfu/mL) and a film (1 cm 2 ) Combining, incubating for 30min at 37 ℃, sterilizing, flushing with normal saline for three times, collecting flushing liquid, carrying out gradient dilution, and measuring the number of unattached probiotics bacteria by adopting a plate counting method. The adhesion rate was calculated according to the following adhesion rate calculation formula.
Adhesion (%) = ((a-B)/a) ×100%
A is the total LGG quantity, cfu/mL; b is the number of non-adhered LGGs, cfu/mL
3.3.4.2 in vitro simulation of adhesion of probiotic microspheres to intestinal tracts
According to the everting encapsulation method, the mucosa adhesion of the composite particles is determined, and the experimental steps are as follows:
male C57BL/6J mice were fasted for 24h before taking the intestinal tract, during which time water was free. The next day the mice were sacrificed, opened, the colon was removed, the intestinal tract was rinsed with normal saline and the intestinal contents removed, 6cm of the colon was cut off and stored in PBS buffer. After the colonic tract fragments are turned into capsules by a glass rod, the capsules are placed in 10mL PBS solution containing 10mg microspheres, slowly vibrated in a shaking table at 37 ℃ for 30min, the intestinal capsules are taken out, centrifuged, the microspheres are separated, the freeze-dried by a freeze dryer for 48h, the weight is measured, and the mucosa adhesiveness is calculated according to the following mucosa adhesiveness calculation formula.
Mucoadhesive (%) = ((C-D)/C) 100%
C is the initial microsphere weight, mg; d is the weight of the microspheres that did not adhere to the colon, mg.
3.3.5 biocompatibility of microspheres
3.3.5.1 cytotoxicity assay
The in vitro toxicity of SOGG-SA microspheres to RAW264.7 cells was evaluated separately using MTT colorimetric method. 200mg of blank microspheres were placed in 1mL of medium and extracted for 24 hours, and the extract was collected by centrifugation. RAW264.7 cells were counted using a cell counting plate to 5X 10 4 Individual cells/wells were seeded into 96-well plates and incubated in an incubator for 24h. The extract was diluted with medium to different concentrations (200, 100, 50, 25, 12.5, 6.25 mg/mL), respectively. The supernatants were discarded and replaced with different concentrations of extract, 100 μl per well, and 6 replicates per group were incubated for 24h. 10 mu L of MTT solution (5 mg/mL) is added into each hole, after incubation is carried out in an incubator for 4 hours, the supernatant is sucked, then 150 mu L of LDMSO is added into each hole, the light is prevented from shaking uniformly, the formazan is fully dissolved, untreated cells are used as a control, absorbance is detected at 570nm wavelength by an enzyme-labeled instrument, and the cell survival rate is calculated.
Survival (%) = ((a) s -A 0 )/(A c -A 0 ))*100％
In which A s : absorbance value of the sample; a is that c : absorbance of untreated cell groups; a is that 0 : absorbance values for blank wells.
3.3.5.2 hemolysis experiment
Blood from rats was centrifuged at 1000rpm for 10min at 4℃and the supernatant was removed, and red blood cells were obtained by repeated washing with PBS and diluted to 5% (V/V). 200mg of the blank microspheres were placed in 1mL of medium for 24 hours, and the extract was collected by centrifugation and diluted. The various diluted 0.5mL of extract was mixed with 0.5mL of red blood cells, placed in a shaking table at 37℃and 100rpm for 1h, and centrifuged. Positive control: 0.5mL of distilled water and 0.5mL of red blood cells; blank group: the hemolysis rate was calculated by measuring the absorbance at 540nm with a microplate reader using 0.5mL PBS buffer and 0.5mL red blood cells.
Hemolysis ratio (%) = ((a) s -A n )/(A p -A n ))*100
In which A s : absorbance value of the sample; a is that n : absorbance of the blank control group; a is that p : absorbance values for distilled water groups.
3.3.5.3 toxicity test in vivo
Mice were kept in animal houses for one week to acclimatize. To investigate whether the prepared lactobacillus rhamnosus delivery system was systemically toxic, mice were perfused with 0.2mL microspheres daily for 7d and periodically tested for changes in body weight. The control group was an equivalent amount of physiological saline. After the experiment, the mice were sacrificed, organs such as heart, liver, spleen, lung and kidney were washed with physiological saline, and the blood spots were fixed with 4% formaldehyde and transferred, and observed with an optical microscope.
4. Results and discussion
4.1 characterization of OGG
4.1.1 Effect of NaClO usage on GG
GG has a molecular weight of about 220kDa, and the aqueous solution is mostly turbid. The existence of a large number of hydroxyl groups in GG molecular chains, and intramolecular hydrogen bond self-crosslinking is a main cause of poor GG solubility. Hydrophilic groups carboxyl and sulfhydryl are introduced on GG through chemical modification, so that the water solubility of GG is improved. The solubility of GG aqueous solutions can be reflected in transparency, the higher the transparency, indicating better solubility. TEMPO oxidation systems are capable of oxidizing the hydroxyl group at the C6 position of GG to a carboxyl group. FIG. 2 shows that GG solution gradually changed from cloudy to clear as the amount of NaClO was increased; at the same time, the oxidation degree of GG is also gradually increased. The TEMPO oxidation system can oxidize the hydroxyl group at the C6 position of GG to more than 90% by calculation of the oxidation degree. When the amount of NaClO is increased again, the oxidation degree is not changed basically. The light transmittance of the GG solution after oxidation also increases. The result shows that the introduction of carboxyl can improve the solubility of GG, because the hydrophilicity of carboxyl accelerates the infiltration process of water molecules on GG.
4.1.2FTIR
From the infrared spectrogram analysis of fig. 3a, the main absorption peaks of the products after acidification of GG, OGG and OGG are similar, which indicates 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 occurs mainly at 1614cm -1 It is attributable to the asymmetric carboxylate (COO-) groups and C=O stretching vibrations of the symmetric COO-. Shift the OGG acidified c=o peak to 1734cm -1 Indicating that the-COOH is generated, the-COOH group is successfully introduced into the GG macromolecular structure, and the OGG is successfully prepared.
4.2 characterization of SOGG
4.2.1FTIR
SOGG is formed by amide bond formation between amine groups of L-cysteine hydrochloride and carboxylate groups of OGG. SOGG at 2696cm was observed from FIG. 3b -1 the-SH peak appeared at 1654cm -1 And 1541cm -1 Where amide peaks occur, c=o bonds and-N-H bonds of the amide groups, respectively. The amide reaction is proved to occur, and grafting is successful; at 1608cm -1 The presence of a carboxyl peak indicates that carboxyl and thiol coexist in SOGG.
4.2.2 1 HNMR
As shown in FIG. 4, the SOGG spectra showed distinct characteristic peaks at 3.34ppm and 2.85ppm, and the 3.35ppm peak was-COCH in cysteine derivatives, compared to the spectra of native GG and OGG 3 -part, 2.85ppm and-CH 2 The absorption sites of the SH methylene protons are similar, which indicates that the thiol units have been successfully grafted onto the sugar chains of GG, confirming successful synthesis of SOGG.
4.2.3Ellman method for measuring mercapto content
To accurately determine the degree of thiol grafting of SOGG, a standard curve of free thiol was first determined by the Ellman method. The linear regression equation for free thiol is: y= 12.857x-0.0071, r 2 =0.9993, the linear relationship between absorbance and thiol concentration was good. The free sulfhydryl group plays an adhesive role, and the disulfide bond is a crosslinking role in the preparation process of the microsphere.
The thiol content after amidation reaction is 47.67 mu mol/g at the beginning, and the thiol group content is relatively low, which may be caused by long molecular chains of OGG and low molecular mobility, and L-cys is not easily connected to the OGG main chain. The higher the sulfur group content, the stronger the adhesion of the resulting SOGG, so that the reaction conditions for synthesizing SOGG are simply optimized with the content of mercapto groups as an index. In order to increase the mercapto content, three factors which have a great influence on the amidation reaction are further screened out by literature investigation and preliminary experiments: the EDC/NHS ratio, reaction pH and L-cys addition were optimized for the three influencing factors.
4.2.3.1 mercapto graft ratio condition screening
4.2.3.1.1 Effect of different EDC/NHS ratios on thiol content
The pH=5.5 of the reaction is controlled, the feeding ratio is 1:7, the EDC/NHS ratio is changed, EDC plays a role in activating carboxyl, and NHS plays a role in fixing carboxyl. As can be seen from FIG. 5a, the mercapto content increased from 191.3. Mu. Mol/g to 332.18. Mu. Mol/g as the EDC/NHS ratio increased. When EDC/nhs=5:1, the mercapto content reached the highest. This is probably because NHS has a competitive relationship with L-cys by binding the immobilized carboxyl group to the activated carboxyl group. The experimental trial did not add NHS and the result was high thiol content without EDC/nhs=5:1. The amidation reaction is carried out when EDC/nhs=5:1 is selected according to the mercapto content.
4.2.3.1.2 Effect of different pH on thiol content
The pH of the amidation reaction was changed and the other conditions remained unchanged. As can be seen from FIG. 5b, when the reaction pH was increased from 4.0 to 6.0, the thiol content was increased and then decreased. At ph=5.5, the thiol content reached a maximum of 204.4 μmol/g, indicating that this experiment favors the amidation reaction at pH 5.5.
Effects of different feed ratios of 4.2.3.1.3 on thiol content
Different feeding ratios are the mass ratio of OGG/L-cys, the feeding amount of the L-cys is changed, and other reaction conditions are unchanged. As is clear from the results of FIG. 5c, the mercapto group content increased and then decreased with the increase in the amount of L-cys added. At a feed ratio of 1:7, the free mercapto content was up to 252.09. Mu. Mol/g, which may be related to the collision between L-cys and OGG. When the added amount of the L-cys is increased, the concentration of the solution is increased, the collision opportunity between molecules is increased, and the generated product is increased; as the amount of L-cys increases again, the progress of the reaction is inhibited, resulting in a decrease in the amount of the product produced.
In summary, the single factor reaction condition screening is performed by using the mercapto content of SOGG as an index, and finally EDC/nhs=5:1, ph=5.5 and the feed ratio of 1:7 are selected as the optimal reaction conditions. The free thiol content was calculated by the Ellman method: 332.18. Mu. Mol/g, total mercapto content: 618.128. Mu. Mol/g, 46.26% of the mercapto groups undergo autoxidation to disulfide bonds. The free sulfhydryl group has an adhesive effect and can play a role of a bridge between the LGG and intestinal mucus. Meanwhile, disulfide bonds can be formed by autoxidation, and can play a role in crosslinking in the preparation process of the microsphere.
4.3LGG microspheres
Preparation condition screening of 4.3.1LGG microspheres
Effects of 4.3.1.1SOGG concentration on the embedding Rate
From fig. 6a, it can be seen that the entrapment rate of the microspheres tends to increase and then decrease with the concentration of SOGG. When the SOGG concentration is 0.5%, the embedding rate of the microsphere is 57.67% at maximum. As SOGG concentration increases, the entrapment rate decreases. This is mainly due to the fact that as the SOGG concentration becomes greater, the viscosity of the polysaccharide solution increases, resulting in LGG not being easily uniformly dispersed in the solution. When the SOGG concentration is reduced, the particle size of the formed microsphere is small, the mechanical strength is low, and the probiotics can not be effectively embedded, so that the embedding effect is affected. When the SOGG concentration is increased from 0% to 2%, the particle size of the microsphere is gradually increased, mainly due to the increase of the viscosity of the polysaccharide solution, which is unfavorable for emulsification in the process of preparing the microsphere, and the particle size of the microsphere is increased. In summary, the optimal SOGG concentration for preparing LGG microspheres was 0.5%.
4.3.1.2SA/CaCO 3 Effect of the ratio on the embedding rate
Encapsulation effect of microspheres and SA and CaCO 3 Is closely related to the mass ratio of (c). At H + CaCO in the presence of 3 Ca is generated 2+ The Ca produced 2+ Chelating with SA to form a gel, thus SA/CaCO 3 The embedding effect of the microspheres can be influenced by the mass ratio. As can be seen from fig. 6b, the embedding rate of the microspheres increases and then decreases with increasing mass ratio. When the mass ratio is 1:1, caCO 3 At a lower concentration, with residual SA not being equal to Ca 2+ Crosslinking to form glue. At the moment, the microsphere is difficult to form, has larger particle size, is sticky, has thin microsphere wall and small mechanical strength, and therefore has poor effect of protecting probiotics in the stomach. As the mass ratio increases, ca in the reaction 2+ The embedded rate of the probiotics is gradually increased, and meanwhile, the particle size of the formed microspheres is gradually reduced, so that the probiotics are in a spherical shape, compact in structure and free of adhesion, and the protection of the probiotics can be enhanced. When the mass ratio is 1:4, the probiotics areThe embedding rate of the microbial microspheres reaches 43.38 percent at the highest. However, as the mass ratio continues to increase, ca 2+ Excess CaCO 3 Cannot be all combined with H + The reaction starts to increase the particle size of the microspheres, the spherical shape is poor, and the embedding rate starts to decrease. In summary, the best SA/CaCO for preparing LGG microspheres 3 The ratio is 1:4.
4.3.1.3 Effect of Water-oil ratio on embedding Rate
In the preparation process of the microsphere, emulsification plays a key role. The volume ratio of water to oil can influence the particle size of the microsphere and thus the quality of the microsphere. As can be seen from fig. 6c, the embedding rate of the microspheres tends to increase and decrease when the volume of the oil phase increases gradually. When the volume ratio of water to oil is 1:1, larger dispersed liquid drops are generated in the oil phase, and the prepared microspheres have large particle size, thinner microsphere walls and low mechanical strength and are easy to break, so that the embedding rate is lower. This is probably due to the relatively high content of microspheres formed due to the low content of emulsifier, which are extruded by collision with each other during stirring, making it difficult to sphere the microspheres. When the volume ratio of water to oil is 1:3, the embedding rate is 34.41% at the highest, and the microsphere is good in balling and free of adhesion. With the increase of the oil phase, the dispersion space of the microspheres is loose, so that the water phase can be fully emulsified, the particle size of the microspheres is gradually reduced, and the embedding rate of the microspheres is improved. The microsphere particle size is too small, so that probiotics are exposed outside the microsphere, and the microsphere is easy to break under the mechanical actions of stirring, centrifugation and the like, and finally the embedding rate is low. In summary, the optimal water to oil volume ratio for LGG microsphere preparation was 1:3.
Influence of the amount of glacial acetic acid 4.3.1.4 on the entrapment Rate
Glacial acetic acid reacts with calcium carbonate to release Ca 2+ Plays a key role in SA gel formation. The glacial acetic acid is too little to be used, is not beneficial to SA gel formation, and is not easy to prepare microspheres; too much use and too strong pH acidity can cause death of the probiotic. It is therefore important to screen the amount of glacial acetic acid that can be used to form the ball without causing the death of the probiotics. As shown in fig. 6d, the embedding rate tends to increase and decrease with increasing glacial acetic acid. When the addition amount of glacial acetic acid is 0.2mL, the small amount of glacial acetic acid results in residual calcium carbonate, so that the particle size of the microsphere becomes smallLarge, mechanically weak, so the embedding rate is low. When the addition amount of glacial acetic acid is 0.3mL, the embedding rate reaches a maximum of 81.56 percent, and at the moment, the microsphere particle size is uniform, the sphericity is good, and the mechanical strength is high. As the amount of glacial acetic acid increases, the embedding rate begins to decrease, i.e., the number of viable bacteria after embedding decreases, because too much glacial acetic acid that does not react with calcium carbonate decreases the pH of the overall system, causing the death of most probiotics. In summary, the optimal addition of glacial acetic acid to prepare the LGG microspheres was 0.3mL.
4.3.1.5 orthogonal test
The orthogonal experiment is carried out on the basis of the four single factor experiments, and SOGG concentration and SA/CaCO are selected 3 And (3) taking the factors with larger influence on the embedding rate of the LGG microspheres from the mass ratio, the water-oil volume ratio and the glacial acetic acid consumption as independent variables, measuring the embedding rate of the microspheres, and obtaining the optimal preparation process condition of the LGG microspheres through orthogonal experiments. Taking the embedding rate as an index, the larger the extremely difference is, the larger the influence of the factor on the embedding rate is. As can be seen from table 5, the impact of the four factors on the embedding rate was as follows: d (glacial acetic acid usage) > C (water-oil volume ratio) > A (SOGG concentration) > B (SA/CaCO) 3 Mass ratio of (c). Thus A is 1 B 1 C 2 D 2 For the best formulation, further validation is required as the formulation is not present in the orthogonal experimental list.
TABLE 5 microsphere formulation screening L 9 (3 4 ) Results of the orthogonal test
| 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 | D 2 | 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 | D 2 | 78.61 |
| 7 | A 3 | B 1 | C 3 | D 2 | 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 | |
| Preferred level | A 1 | B 1 | C 2 | D 2 |
Verification experiments according to the optimal formulation show that A 1 B 1 C 2 D 2 The embedding rate of the combination is higher and reaches (88.67+/-6.8)%, and A is tested 1 B 1 C 2 D 2 Is significantly higher than other groups (P<0.05). And the microsphere has good sphericity and smaller particle size. Thus, select A 1 B 1 C 2 D 2 The combined microspheres were subjected to the next experiment. With the embedding rate as an index, the mass fraction of SOGG is 0.5%, SA/CaCO 3 The mass ratio of the (B) is 1:3, the volume ratio of water to oil is 1:3, the using amount of glacial acetic acid is 0.3mL, and the (B) is used as the optimal formula of the LGG microsphere for verification of a subsequent experiment.
4.3.2LGG microsphere appearance
The wet microspheres and the freeze-dried microsphere morphology were observed by a common optical microscope and a scanning electron microscope, and the results are shown in fig. 7. As can be seen from fig. 7a, the LGG microspheres after freeze-drying were visually observed as white powder. As can be seen from the observation of the ordinary optical microscope in FIG. 7b, the LGG microsphere has a regular spherical structure with a smooth surface, and the average particle diameter is 294.46.+ -. 31.8. Mu.m.
As can be seen from the scanning electron microscope observations in fig. 7c and 7d, after the microspheres are freeze-dried, the surfaces of the spheres are wrinkled and concave, and the spheres are in a fusiform shape, which is a common phenomenon of freeze-drying. The micro-spheres are further observed in a local amplifying way, and the micro-bulge of some probiotics is obviously observed to be embedded in the spheres through red arrows, and some probiotics are adhered to the surfaces of the micro-spheres, so that few probiotics are shown to be on the surfaces of the micro-spheres.
4.3.3LGG microsphere tolerance to simulated gastrointestinal fluids
Free LGG and LGG microspheres were investigated for their viability under simulated gastrointestinal conditions. As the results in fig. 8a show, after incubation of free LGG in simulated gastric fluid for 2h, the number of viable bacteria was significantly reduced, and the colony count was reduced by approximately 7 Log. After 4h incubation in simulated intestinal fluid, the colony count was reduced by nearly 0.2 Log and negligible. Numerous studies have shown that most probiotics lose their viability significantly when they enter the gastrointestinal tract of humans. Subsequently, the tolerance of the LGG microspheres in simulated gastrointestinal fluids was studied, and as shown in FIG. 8b, after the LGG microspheres were respectively subjected to incubation of 2 hours of simulated gastric fluid and 4 hours of simulated intestinal fluid, the number of the embedded LGG viable bacteria was slightly reduced by approximately 0.7 Log and 0.2 Log, and the simulated gastrointestinal fluids showed stronger tolerance. The microsphere has a certain protection effect on probiotics. Before exploring the tolerance of LGG microspheres to simulated gastrointestinal fluids, it was investigated whether the activity of the probiotics was changed before and after embedding in order to avoid the effect of the embedding process on the activity of the probiotics themselves. The growth curve of the LGG before and after embedding is obtained by drawing the growth curve of the LGG after embedding, and the growth curves of the LGG before and after embedding have no obvious difference. Indicating that the embedding process of the microspheres has little effect on the LGG activity.
4.3.4LGG microsphere is used for simulating tolerance of continuous gastrointestinal fluid
The effect of continuous gastrointestinal fluids on free LGG and LGG microspheres was further simulated, and the protective effect of microspheres on LGG is further demonstrated in fig. 9. Free LGG was almost free of viable bacteria in continuous simulated gastrointestinal fluids (P < 0.0001) compared to saline group. In contrast, LGG microsphere-embedded probiotics were incubated in gastrointestinal fluids for 6 hours continuously. The reduction in Log was only 0.5 compared to the control group. These results demonstrate that the microspheres have a very important protective effect on LGG in gastrointestinal fluids, minimizing the damage to probiotics caused by gastric acid.
4.3.5 adhesion experiments
4.3.5.1LGG adhesion to film
To examine SA/CaCO 3 The adhesion of the microsphere to the LGG is verified indirectly by preparing SA, GG, OGG and SOGG to form a composite film. The results of the adhesion experiments of LGG to four different films are shown in fig. 10. As can be seen from fig. 10a, the mechanical properties of a single SA film are inferior to the other three groups. Among them, the SA/SOGG film has the best mechanical properties, which may enhance the mechanical strength of the film with disulfide bonds formed by autoxidation of disulfide bonds in SOGG. As can be seen from fig. 10b, the SA, SA/GG, SA/OGG and SA/SOGG films all had some adhesion to LGG, wherein the SA/SOGG films had higher adhesion to LGG than the other three groups, indicating that the SOGG interaction with LGG was the strongest and the LGG adhesion was stronger. It was demonstrated that the introduction of thiol groups increased the adhesion between GG and LGG. The other three films also have adhesion to LGG, probably because hydroxyl or carboxyl groups on the polysaccharide structure form hydrogen bonds with groups on LGG. In addition, the SA/SOGG films remained relatively intact after soaking the four different films in water for the same period of time, indicating that disulfide bonds enhance the mechanical strength of the films. SOGG can play a bridge role in intestinal mucus by double adhesion with intestinal mucus and LGG, and finally LGG is adhered and planted in intestinal mucosa.
4.3.5.2 intestinal adhesion
The adhesion of the microspheres to the intestinal tract is measured by adopting an eversion intestinal capsule method, and the adhesion rate of colon mucous to SOGG/SA microspheres and GG/SA microspheres is 77.67 percent and 46.17 percent respectively. From the results, the colon showed a certain mucoadhesion to both SOGG/SA microspheres and GG/SA microspheres, mainly due to the presence of a large amount of villi on the colonic mucosa, which helps the microspheres adhere better to colonic mucosa and epithelial cells. Meanwhile, the content of goblet cells in the colon part is more, the mucin level of the colon mucosa is higher, and the adhesiveness of the microsphere in the colon part is further increased. It may be due to the formation of hydrogen or ionic bonds between hydroxyl or amino groups on the polysaccharide chain and groups on the mucus glycoprotein. The adhesion of the mouse colon mucosa to the SOGG/SA microsphere is obviously higher than that of the mouse colon mucosa to the GG/SA microsphere, and the main reason is probably that the free sulfhydryl on the surface of the SOGG/SA microsphere reacts with the sulfhydryl on the mucous glycoprotein secreted by the colon mucosa to form disulfide bonds, so that the mucosa adhesion of the microsphere is further increased.
4.3.6 in vivo safety evaluation
As shown in fig. 11, healthy mice maintained stable body weight for normal growth after continuous gastric lavage of LGG microspheres for 7 d. There was little difference in mental state and activity state of the mice in the experimental group compared to the blank group.
Mice were then sacrificed and their hearts, livers, spleens, lungs, kidneys were collected for HE staining pathology. As shown in fig. 12, the visceral tissue structure of mice after the LGG microsphere gastric lavage was not significantly different from that of mice in the group without gastric lavage, and no obvious inflammatory reaction phenomenon was detected in each tissue. The above results indicate that LGG microspheres have negligible in vivo toxicity, which is advantageous for LGG microspheres for in vivo applications.
Application example 1 lactobacillus rhamnosus microsphere prepared in example 1 treatment of DSS-induced colitis mice
1. Experimental animal
SPF-grade C57BL/6J male mice of about 4-6 weeks of age were purchased from Jinan Weitong Lihua.
2. Experimental method
2.1 construction of the mouse model for colitis
C57BL/6J mice were acclimatized in animals for 7d prior to animal molding. Mice were free to drink 3% DSS solution for 7d, a colitis model was established, DSS solution was changed every 1d, and treatment observations were made. Mice were gavaged from DSS on the day of molding, once daily, and were gavaged at the same time as much as possible, with 0.2mL of bacteria solution each time. Normal group and DSS model group were respectively given corresponding volumes of physiological saline.
Preparation of DSS: DSS was dissolved in sterile distilled water to make a 3% (w/v) solution.
Establishing an experimental animal model: the 48 mice were randomly divided into 4 groups: control group, DSS group, free LGG group, LGG microsphere group, 12 per group, grouped as follows:
control group: only lavage 0.2mL sterilized physiological saline
DSS group: 0.2mL of sterilized normal saline with DSS and gastric lavage functions is drunk
Free LGG group: DSS+lavage 0.2mL2X10 is drunk 9 cfu/mL bacterial suspension
LGG microsphere set: drinking DSS+lavage 0.2mL LGG microsphere suspension (microsphere with optimal performance prepared in example 1)
2.2 DAI evaluation in mice
During the experiment, the mental state, diet drinking, activity, defecation condition, hair color, weight change, death condition and the like of the mice were observed and recorded. As shown in table 6, the average of the three scores of the weight loss percentage, fecal property and occult blood test (o-tolidine method) of the mice was used as a Disease Activity Index (DAI), and the administration effect was preliminarily determined based on the status of the mice and the DAI score.
Dai= (weight reduction score + stool trait score + hematochezia degree score)/3
TABLE 6 mouse DAI scoring criteria
2.3 colon tissue collection, length measurement and HE staining of mice
The state of the mice is observed several times daily, when 80% or more of mice in the DSS group have macroscopic hematochezia, the mice are sacrificed to open the abdominal cavity, the part between the anus and the cecum is the colon, and the length is measured. The residual feces and blood were rinsed with pre-chilled PBS solution and the filter paper was blotted dry. Dividing colon into two parts, wherein one part is used for HE staining and immunohistochemical staining, taking colon 1cm above anus, soaking in paraformaldehyde, and delivering sample; histological scoring was performed by HE staining results; and part of the sample is used for measuring myeloperoxidase, and the sample is rapidly transferred to a refrigerator at-80 ℃ to be stored for subsequent experiments, so that repeated freezing and thawing are avoided.
TABLE 7 colonography scoring
2.4 spleen changes in mice
After the mice are sacrificed and dissected by cervical dislocation, spleens are taken, the weights of the spleens are weighed, and spleen coefficients are calculated according to spleen coefficient formulas.
Spleen factor (%) = (spleen weight/mouse weight) ×100%
2.5 detection of myeloperoxidase in colon tissue
Washing colon tissue and internal feces with PBS buffer solution, shearing colon tissue, fully grinding with a tissue grinder, homogenizing the tissue as uniformly as possible without existence of large tissues, preparing 5% tissue homogenate, and performing experimental steps according to the instruction of kit of Nanjing's institute of biological engineering. The activity of MPO enzyme was calculated according to the formula by measuring the absorbance at 460nm in U/g wet weight.
MPO activity= (a Measurement -A Control )/(11.3×W)
Wherein: 11.3 is the inverse of the slope; a is the absorbance at 460 nm; w is the sample sampling amount.
2.6 colon immunohistochemical analysis of mice
The experimental operation steps are the same as 2.3, and colon tissue sample delivery is carried out.
3. Results and discussion
3.1 general observations of mice
The 3% DSS was freely introduced over 7 days to induce colitis in mice. The mice of each group were observed for signs changes during the course of the experiment, and the following results were obtained for each group:
(1) Normal group (Control group): 1 mouse lost weight more rapidly, probably due to esophageal damage during gastric lavage. The other 11 mice have normal diet, drinking water, feces color and shape, smooth hair and active spirit.
(2) Model group (DSS group): in the whole experiment process, 12 mice are not dead, and the mice have different degrees of binding aggregation and rough hair and slight diarrhea after being subjected to experiments for 3-4 days; the mice have reduced drinking water and diet after 5-7 days of the experiment, 20% of mice begin to have symptoms of listlessness, inflexible reaction, vertical hair, dorsum of arch, loose stool and the like; with the increase of the experimental time, diarrhea of the mice is aggravated, blood stains are visible at the anus, and the symptoms are aggravated. When 80% of mice in the DSS group have naked eye hematochezia, the next experiment can be carried out.
(3) Free LGG group (dss+lgg group): the mice were normal for each sign 1-5 days from the start of the experiment. On days 5-7, there was a slight anorexia and lazy, 30% of the faeces were slightly diluted, but no large area diarrhea occurred, and no hematochezia occurred. The mental state and the state of the mice are better than those of the normal group.
(4) LGG Microsphere group (dss+microsphere group): mice did not die during the experiment, there was no obvious difference between the experiment 1-6 days and the normal group, 10% of the mouse feces on the 6 th day were slightly diluted, but no large-area diarrhea occurred, and no hematochezia occurred. The mental state and condition of the mice were superior to those of the normal group and the free LGG group.
3.2 weight changes in mice
To investigate the effect of free LGG and embedded LGG microspheres on mice on the mice with colitis, changes in body weight of each group of mice were recorded daily during the experiment. Results as shown in fig. 13, the body weight of the mice in the blank group showed a gradual increase trend, and the DSS group, the dss+lgg group, and the dss+lgg microsphere group showed a decrease trend. DSS group mice showed a significant decrease in body weight from day 5d, with differences statistically significant (P < 0.001). The body weight of mice in the dss+lgg group and those in the dss+lgg microsphere group decreased relatively slowly, and at the same time, the LGG microsphere group had a relatively good therapeutic effect, the number of loose stool was reduced, and the difference was statistically significant (P < 0.01). The results show that the LGG microsphere has a certain therapeutic effect on colon inflammatory mice.
3.3 DAI evaluation in mice
During the experiment, the weight reduction percentage, stool characters and occult blood examination results of each group of mice are recorded every day, and DAI scoring evaluation is carried out. As shown in fig. 14, DSS group mice DAI increased continuously with the time of the experiment. The mice in the DSS group of the 5d part can visually see the symptoms of loose stool, hematochezia, listlessness and the like; the rise in mouse DAI was significantly improved from day 5d compared to dss+lgg, dss+microsphere groups. The results show that both the LGG and the LGG microspheres can effectively reduce the DAI score of the colonitis mice, wherein the embedded probiotics have better effect, which indicates that the LGG microspheres have better curative effect on colonitis than the free LGG.
3.4 evaluation of colon Length and pathological injury
As shown in fig. 15a, the colon length of DSS group was significantly shortened, while the colon length of mice treated with LGG and LGG microspheres was significantly lengthened, indicating that both had a certain efficacy in the treatment of colitis. At the same time, there was a significant difference in colon length between the dss+lgg group and the dss+lgg microsphere group. As shown in FIG. 15b, after H & E staining, the colon status of the mice in the blank group was good, and the mice contained a large number of goblet cells, and the intestinal epithelium and crypt structures were complete and ordered, and no inflammatory cell infiltration was observed. The colon of the DSS group mice is necrotic and erosive with a large amount of mucosal epithelial cells, the mucous layer and the lower layer can be infiltrated with a large amount of inflammatory cells, the number of goblet cells is obviously reduced and almost no goblet cells are damaged, and the colon tissue damage score of the DSS group is obviously higher than that of the blank group; LGG and LGG microspheres are used for prognosis, so that the ulcer and erosion of the colonic mucosa are obviously reduced or eliminated, the arrangement of goblet cells is restored to be normal, and the pathological score of colon is obviously reduced. In addition, the LGG microsphere group significantly improved colonic mucosal lesions compared to the histological scores of the LGG group, and the above results all demonstrate that the LGG microsphere has better therapeutic effect on colitis than non-embedded LGG.
3.5 spleen changes in mice
As can be seen from fig. 16, the increase in spleen factor was evident in the DSS group, and the spleen factor decreased with both free LGG and LGG microsphere treatment. The free probiotics and the embedded probiotics have certain therapeutic effects on enteritis. The mice treated by the LGG microsphere have almost no obvious difference from blank groups, which shows that the LGG microsphere can relieve the spleen factor enlargement caused by DSS induction and has a certain treatment effect on the enteritis.
3.6 detection of myeloperoxidase in colon tissue
As can be seen from fig. 17, the colon tissue MPO enzyme activity was significantly higher in DSS group mice than in normal group. The intervention of the LGG and the LGG microsphere treatment shows that the mice have no significant difference from the normal group, and the mice have certain therapeutic effect on the enteritis. However, the LGG microsphere group has significant difference (P is less than 0.05) from the LGG group, and the LGG microsphere group has good curative effect on enteritis.
3.7 colon immunohistochemical analysis of mice
As shown in fig. 18, the yellow-brown color is positive protein, and the content of IL-10 is significantly reduced when induced by DSS stimulation compared with the blank group, which indicates that the colon of the DSS group mice contains a large amount of neutrophil infiltration, and the inflammation is serious. Both LGG and LGG microspheres significantly up-regulate the reduced levels of colonic IL-10 upon DSS stimulation after treatment. It can be seen that LGG and LGG microspheres can alleviate colonic inflammation by up-regulating IL-10 content, with LGG microsphere sets having the most pronounced up-regulating effect.
As can be seen from FIG. 18, the IL-6 and TNF- α content in the colon of the mice was lower in the colon of the control mice, and the IL-6 and TNF- α content increased significantly when stimulated with DSS. The increased levels of IL-6 and TNF- α were secreted by DSS stimulation when treated with both LGG and LGG microspheres were down-regulated to varying degrees and were significantly different compared to the DSS group. As can be seen from fig. 18, both LGG and LGG microspheres reduced intestinal inflammation by down-regulating IL-6 and TNF- α content, and the improvement effect of LGG microsphere set was most remarkable.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should 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.
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