CN114057904B - Sulfhydryl radix Codonopsis polysaccharide and application thereof in preparation of colon targeting probiotic microcapsule - Google Patents
Sulfhydryl radix Codonopsis polysaccharide and application thereof in preparation of colon targeting probiotic microcapsule Download PDFInfo
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- CN114057904B CN114057904B CN202111292748.XA CN202111292748A CN114057904B CN 114057904 B CN114057904 B CN 114057904B CN 202111292748 A CN202111292748 A CN 202111292748A CN 114057904 B CN114057904 B CN 114057904B
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Abstract
The invention relates to the technical field of probiotic application, in particular to sulfhydryl radix codonopsis polysaccharide and application thereof in colon targeting probiotic microcapsule preparation. The sulfhydryl radix codonopsis polysaccharide can remarkably improve the adhesion effect of intestinal mucus on probiotics, and has remarkable effect. The probiotic microcapsule prepared by combining the sulfhydryl codonopsis pilosula polysaccharide and the probiotics can well protect the probiotics from the digestion action of the gastrointestinal tract, the probiotics are delivered to the colon in a targeting way, the complete release of the probiotics in the colon is realized, and the stay time of the probiotics in the colon can be remarkably prolonged, so that the effective colonization of the probiotics in the colon is promoted and the probiotics effect is exerted.
Description
Technical Field
The invention relates to the technical field of probiotic application, in particular to sulfhydryl radix codonopsis polysaccharide and application thereof in colon targeting probiotic microcapsule preparation.
Background
Probiotics are also called microecological regulator and live bacteria preparation, and can improve the health condition of organisms by regulating and controlling the balance of intestinal microecology. When intestinal microecology is unbalanced, exogenous probiotics are usually required to be supplemented to regulate the composition of intestinal flora, so that diseases are improved. However, probiotics only perform effectively when present in the intestine for a long period of time and up to a certain amount. But the fact is not as desirable. Exogenous probiotics are generally difficult to resist the digestion of the gastrointestinal tract, and researches show that the survival rate of bifidobacteria after passing through gastric juice environment is less than 2 per mill. Even the surviving probiotics are difficult to adapt to the intestinal environment, are repelled by the original resident flora of the intestinal tract, can not realize proliferation and long-term colonization in the intestinal tract, and finally become the external body of the 'passing bacteria' discharged along with the excrement. The colon is the main habitat of intestinal flora and is also the main place for probiotics to act, and the pH value of the colon is generally about 6.5-7.5, unlike the stomach and small intestine, and most of digestive enzymes are inactivated, so that the growth and the propagation of probiotics are facilitated. Therefore, how to protect the probiotics from digestion and to get them to the colon successfully is key to the function of the probiotics.
To solve this problem, researchers have conducted a number of experiments to screen digestive tract drug-resistant strains or to construct genetic strains to increase the resistance of probiotics to the extreme environment of the digestive tract. However, screening of probiotics is not only labor intensive, costly and time consuming, but also difficult to screen for adherent bacteria with specific functions. Although genetically engineered bacteria have good results, researchers and consumers have doubt about such bacteria, and the use of such genetically engineered products in foods is not allowed according to law. Delivery by microencapsulation is therefore an effective way of protecting probiotics from gastric acid and intestinal bile salts in the food industry.
On the other hand, after delivery to the intestine, probiotics require long-term colonization within the intestine to perform a long-term effective function. The intestinal adhesion effect of the probiotics is enhanced, and the intestinal colonization of the probiotics can be effectively promoted. The formation of strong interactions between intestinal mucus layers and probiotics is of great significance for adhesion and colonization of probiotics in the intestinal tract. Mucus binding proteins such as lipoteichoic acid and surface binding proteins on the surface of probiotics may interact with intestinal mucus through biological recognition of ligand receptors. However, ligand-receptor binding is relatively weak, different strains have different binding capacities, and the indigenous flora in the intestine competes with ingested bacteria. Thus, how to strengthen the interaction between intestinal mucus and probiotics is a hotspot of current research.
Disclosure of Invention
The invention provides a sulfhydryl pilose asiabell root polysaccharide for solving the problems in the prior art and provides an application of the sulfhydryl pilose asiabell root polysaccharide in preparation of probiotics microcapsules. The sulfhydryl radix codonopsis polysaccharide can obviously improve the adhesion effect of intestinal mucus on probiotics, so that the effective colonization of the probiotics in the colon is promoted, the probiotics effect is exerted, and the effect is obvious.
The invention relates to sulfhydryl radix codonopsis polysaccharide, which is prepared by the following method:
(1) Preparation of carboxymethyl codonopsis pilosula polysaccharide
Dissolving 1-2 g of codonopsis pilosula polysaccharide in 50-100mL of deionized water, adding 150-300 mL of 3mol/L NaOH solution after full dissolution, and stirring for 10min; adding 20-40 g of monochloroacetic acid, and reacting for 4 hours at 65 ℃; cooling to room temperature, adjusting pH to 7.0, and dialyzing; vacuum drying the dialyzed product to obtain carboxymethyl pilose asiabell root polysaccharide;
(2) Preparation of sulfhydryl pilose asiabell root polysaccharide
Dissolving 100-200 mg of carboxymethyl codonopsis pilosula polysaccharide in 20-40 mL of distilled water, and fully dissolving; then adding 100-500 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), activating carboxyl in the reaction system, and stirring for 1h at room temperature; adding N-acetyl-L-cysteine (NAC) under the protection of nitrogen, and stirring for reaction for 24 hours; removing EDC and NAC in the system by distilled water dialysis; and (3) freeze-drying the product obtained by dialysis to obtain the sulfhydryl pilosula polysaccharide.
The mercapto content in the mercapto-codonopsis pilosula polysaccharide is higher than 250 mu mol/g.
The invention relates to an application of the sulfhydryl radix codonopsis polysaccharide in the production of foods, health products or medicines.
The invention also relates to a probiotic product comprising probiotics and the sulfhydryl codonopsis pilosula polysaccharide.
The probiotics can be selected from any one or a combination of two or more of bifidobacterium, lactobacillus, streptococcus, lactococcus, leuconostoc, propionibacterium, saccharomycetes, pediococcus and staphylococcus.
The probiotics may be selected from any one or a combination of two or more of bifidobacterium longum, bifidobacterium adolescentis, bifidobacterium breve, bifidobacterium infantis, bifidobacterium animalis, bifidobacterium bifidum, lactobacillus acidophilus, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus reuteri, lactobacillus fermentum, lactobacillus bulgaricus, streptococcus thermophilus, kluyveromyces marxianus, pediococcus acidilactici, pediococcus pentosaceus, staphylococcus calf, staphylococcus xylosus, staphylococcus meat, leuconostoc mesenteroides, lactococcus lactis subspecies lactis, lactococcus lactis milk fat subspecies, lactococcus lactis diacetyl subspecies.
Further preferably, the probiotic preparation is a probiotic microcapsule.
Further preferably, the probiotic microcapsule is prepared by the following method:
(1) Preparing sodium alginate microcapsules containing probiotics and sulfhydryl radix codonopsis polysaccharide;
(2) Coating for the first time by chitosan;
(3) A second coating was performed using Eudragit S100.
Further preferably, the probiotic microcapsules are prepared by the following method:
(1) Preparation of sodium alginate microcapsule containing probiotics and sulfhydryl radix codonopsis polysaccharide
Adding sulfhydryl radix Codonopsis polysaccharide into probiotic bacterial suspension according to the proportion of 0.5-1.0% g/mL to prepare Cheng Yi raw bacteria/sulfhydryl radix Codonopsis polysaccharide suspension; mixing sodium alginate solution with the concentration of 3-5% g/mL with probiotic bacteria/sulfhydryl radix codonopsis polysaccharide suspension according to the weight ratio of 1: 1-5, and preparing into a bacterial gel mixture, wherein the viable count of the probiotics is 10 8 -10 9 CFU/mL; spraying 20-50 mL of fungus gel mixture into 1-2L of calcium chloride solution with the concentration of 2-4%g/mL by using an atomizer, and mechanically stirring at 300rpm for 30min for calcification; centrifuging the obtained solution at 4deg.C and 5000r/min for 10min, and washing with distilled water to obtain microcapsule A;
(2) First coating with chitosan
Adding the microcapsule A into chitosan solution with concentration of 4-10% g/mL according to the proportion of 0.5% mg/mL, stirring at 100rpm for 40min, centrifuging, collecting precipitate, and washing with distilled water to obtain microcapsule B;
(3) Secondary coating with Eudragit S100
And adding the microcapsule B into Eudragit S100 coating liquid with the concentration of 2-5% g/mL according to the proportion of 0.5% mg/mL, fully shaking for 4 hours for coating, centrifuging, collecting precipitate, washing with distilled water, and removing residual solution, thus obtaining the probiotic microcapsule.
The invention also relates to application of the probiotic microcapsule in preparing foods, health products or medicines.
The invention has the beneficial effects that:
the sulfhydryl content in the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 prepared by the invention is 279.50 +/-5.97 mu mol/g, and is in a sulfhydryl and carboxyl coexisting state. Compared with the pilose asiabell root polysaccharide CPP-2 and the carboxymethyl pilose asiabell root polysaccharide C-CPP-2, the sulfhydryl pilose asiabell root polysaccharide sC-CPP-2 prepared by the method has stronger interaction with intestinal mucus, stronger adhesive force and remarkable effect.
The sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 can remarkably enhance the adhesion effect of intestinal mucus on probiotics and improve the intestinal adhesiveness of the probiotics, thereby being beneficial to the field planting and proliferation of the probiotics in the intestinal tract. The results of the in vitro mucus adhesion test and the in vitro intestinal adhesion test show that the adhesion rate of intestinal mucus added with the sulfhydryl radix codonopsis polysaccharide to probiotics is obviously improved as high as 68.21 percent and 74.07 percent respectively. The scanning electron microscope observation result shows that the maximum amount of probiotics adhered to the colon of the rat injected with the sulfhydryl radix codonopsis polysaccharide simultaneously achieves unexpected technical effects.
The colon targeting probiotic microcapsule is prepared by combining probiotics and sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2. The probiotics microcapsule can better resist the extreme environment of the gastrointestinal tract, and the survival rate is respectively up to 95.2% and 69.15% after simulating the gastric and intestinal environments for 180 min. From the release profile of the probiotics, it is known that the probiotics in the microcapsules are hardly released during gastric digestion, but are slowly released in the small intestine environment and can be completely released after reaching the colon. Therefore, the probiotics microcapsule provided by the invention can well protect probiotics from the digestion effect of gastrointestinal tracts, target the probiotics to the colon and realize the complete release in the colon.
The experimental results of mice show that the probiotic microcapsule-fed mice can still observe the residual probiotic in the colon at 48 hours, while the probiotic is discharged from the body along with the feces at 48 hours in other groups of mice. Therefore, the probiotic microcapsule obviously prolongs the residence time of the probiotics in the colon and successfully realizes the targeted adhesion and field planting in the colon.
Aiming at a DSS-induced ulcerative colitis mouse, the probiotic microcapsule provided by the invention can effectively relieve the colonitis of the mouse and reduce the damage degree of colon, and the effect is obviously better than that of a microcapsule added with only probiotics or sulfhydryl codonopsis polysaccharide sC-CPP-2; the activity of myeloperoxidase in the colon of a mouse fed with the probiotic microcapsule is obviously reduced, the antioxidation effect is enhanced, the content of pro-inflammatory cytokines in the serum and colon of the mouse is also obviously reduced, the abundance of intestinal pathogenic bacteria is obviously reduced, the abundance of the probiotic is increased, and the content of short-chain fatty acid in the colon content is highest, especially the content of butyric acid is obviously improved. Therefore, the mercapto-codonopsis polysaccharide sC-CPP-2 provided by the invention is combined with probiotics, the anti-inflammatory effect is obviously better than that of the microcapsule added with the probiotics or the mercapto-codonopsis polysaccharide sC-CPP-2, the microcapsule has obvious relieving effect on acute colitis induced by DSS, and intestinal flora disorder caused by inflammation can be effectively repaired, and unexpected technical effects are achieved.
In addition, the probiotic microcapsule has good stability, and after 70d of storage, the survival rate of the probiotic is still up to 44.97%, which is obviously higher than that of free probiotic powder and commercial probiotic microcapsule.
The sulfhydryl radix codonopsis polysaccharide or the probiotic microcapsule provided by the invention can be widely applied to the production of foods, health products or medicines, and has wide application prospect.
Drawings
FIG. 1 is a zeta potential change analysis of Codonopsis pilosula polysaccharide before and after modification;
FIG. 2 shows the dynamic rheological properties and apparent viscosity analysis of intestinal mucus and CPP-2, C-CPP-2, sC-CPP-2 blends (A: mucus and CPP-2; B: mucus and C-CPP-2; C: mucus and sC-CPP-2;D: apparent viscosity);
FIG. 3 shows maximum peel force (MDF) of Codonopsis pilosula polysaccharide to intestinal mucosal surface (A) and Total Work of Adhesion (TWA) (B);
FIG. 4 is a graph showing the rate of adhesion of probiotic extracellular mucus;
FIG. 5 is a graph of the probiotic in vitro intestinal adhesion rate;
FIG. 6 is a laser confocal observation of intestinal adhesion of probiotics;
FIG. 7 is a scanning electron microscope view of the morphology of the microcapsules (A: LGG microcapsules; B: LGG/sC-CPP-2 microcapsules);
FIG. 8 is a particle size analysis (A: LGG microcapsules: B: LGG/sC-CPP-2 microcapsules);
FIG. 9 is a representation of the encapsulation status of LGG/sC-CPP-2 microcapsules for probiotics;
FIG. 10 shows the survival and release rates (A: survival; B: release rate) of probiotic microcapsules in the gastrointestinal tract;
FIG. 11 is a graph showing the storage stability of probiotic microcapsules at 4℃and 25 ℃;
FIG. 12 shows the intestinal distribution of probiotics in different groups of lavage;
FIG. 13 is a fluorescent quantitative PCR detection of intestinal colonization of probiotics;
FIG. 14 shows the mice weight change (A) and DAI score (B) during the experiment;
FIG. 15 is a comparison of colon length and spleen weight for different groups of mice;
FIG. 16 is a plot of colon tissue HE staining of mice (A: CD group; B: DSS group; C: A (Empty) group; D: A (sC-CPP-2) group; E: A (F.prausnitzii) group; F: A (F.prausnitzii/sC-CPP-2) group; G: free F.prausnitzii group);
FIG. 17 shows MPO activity in colon tissue of mice;
FIG. 18 is a graph of the antioxidant level of colon tissue of mice;
FIG. 19 shows TNF- α, IL-1β, IL-6 and IL-10 levels in mouse serum;
FIG. 20 shows TNF- α, IL-1β, IL-6 and IFN- γ levels in colon tissue of mice;
FIG. 21 is a differential analysis of intestinal flora based on genus level;
fig. 22 shows the short chain fatty acid content of the colon contents of mice.
Detailed Description
The following detailed description of specific embodiments of the invention is provided in connection with the accompanying drawings and examples in order to provide a better understanding of the invention, and its various aspects and advantages. However, the detailed description and examples set forth below are intended for purposes of illustration and are not intended to limit the invention.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 preparation method of mercapto Codonopsis polysaccharide (sC-CPP-2)
1. Preparation of carboxymethyl Codonopsis pilosula polysaccharide (C-CPP-2)
Dissolving 1-2g of codonopsis pilosula polysaccharide (CPP-2) in 50-100mL of deionized water, adding 150-300mL of NaOH solution (3 mol/L) after full dissolution, and stirring for 10min; adding 20-40g of monochloroacetic acid, and reacting for 4 hours at 65 ℃; cooling to room temperature, adjusting pH to 7.0, dialyzing until the residual reagent is eliminated; vacuum drying the dialyzed product to obtain carboxymethylated Codonopsis pilosula polysaccharide (C-CPP-2);
2. preparation of mercapto Codonopsis polysaccharide (sC-CPP-2)
Dissolving 100-200mg carboxymethylated Codonopsis pilosula polysaccharide C-CPP-2 in 20-40mL distilled water, and dissolving completely; then adding 100-500mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), activating carboxyl in the reaction system, and stirring for 1h at room temperature; adding N-acetyl-L-cysteine (NAC) under the protection of nitrogen, and stirring for reaction for 24 hours; dialyzing with distilled water for 24h, and removing EDC and NAC in the system; freeze-drying to obtain sulfhydryl radix Codonopsis polysaccharide (sC-CPP-2).
EXAMPLE 2 structural characterization of the mercapto-dangshen polysaccharide (sC-CPP-2)
1. Measurement of mercapto group content
The content of sulfhydryl groups in the sC-CPP-2 of the sulfhydryl codonopsis pilosula polysaccharide is determined by an Ellman's method. Preparing 12.5, 25, 50, 100 and 200 mu mol/L of L-cysteine solution; 1mL of L-cysteine solution was added to a certain amount of DTNB solution, and the mixture was allowed to stand for 10min, and the absorbance at 412nm was measured, and a standard curve was established.
50-100mg of sulfhydryl radix codonopsis polysaccharide sC-CPP-2 is taken and dissolved in 100-200ml of deionized water, the sample solution is detected according to the method, and the sulfhydryl content is calculated by using a standard curve.
The results show that: the sulfhydryl group content in the sulfhydryl radix codonopsis polysaccharide (sC-CPP-2) prepared by the method is 279.50 +/-5.97 mu mol/g.
2. Analysis of potential variation
The zeta-potentials of CPP-2, C-CPP-2 and sC-CPP-2 were determined by potentiometric proton titration with an automatic potentiometric titrator (Metrohm, switzerland), respectively.
As a result, as shown in FIG. 1, after the CPP-2 was carboxymethylated, the zeta potential was decreased from-8.8 mV to-67.5 mV (C-CPP-2), and the zeta potential of sC-CPP-2 was increased to-53.6 mV again, indicating that sC-CPP-2 was successfully modified with a thiol group and was in a state where the thiol group and the carboxyl group coexist.
EXAMPLE 3 interaction relationship of mercapto Codonopsis polysaccharide (sC-CPP-2) with intestinal mucus
The interaction relation between the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 prepared in the example 1 and intestinal mucus is analyzed by using a rheometer and a classical stretching experiment. The specific implementation mode is as follows:
1. measurement of CPP-2, C-CPP-2 and interaction of sC-CPP-2 with intestinal mucus by rheology
The interaction of intestinal mucus with CPP-2, C-CPP-2 and sC-CPP-2 is characterized by using rheological oscillation time scanning to analyze the viscoelasticity change of the intestinal mucus with CPP-2, C-CPP-2 and sC-CPP-2.
CPP-2, C-CPP-2 and sC-CPP-2 (5-10 mg/mL) were added to an equal volume of intestinal mucus, respectively; each mixture was loaded and measured on a 60mm turret of a rheometer (AR-1500, ta Inc.) at 37 ℃; scanning oscillation time in a linear viscoelasticity area at the frequency of 1Hz, and reading a point every 2min until the curve is stable; the above three mixtures were tested at 37℃for shear rates of 0.01-100s -1 Viscosity change under conditions. The results are shown in FIG. 2.
As can be seen from FIGS. 2 (A-C), at a frequency of 1Hz, the loss modulus G 'of the intestinal mucus and CPP-2, C-CPP-2 mixtures were all greater than the storage modulus G', indicating that the mixtures exhibited viscous dominant dilute solution properties, with typical viscous fluid characteristics. The mixture gradually approaches to an elastic dominant state in the detection time when the sC-CPP-2 is intersected with the G 'and G' of the intestinal mucus, which indicates that a gel network with viscoelasticity is formed between the sC-CPP-2 and the intestinal mucus through disulfide bonds, and the interaction between the sulfhydryl-modified codonopsis pilosula polysaccharide and the intestinal mucus is stronger.
As can be seen from FIG. 2 (D), the time is 0.01-100s -1 In the accelerated shear process, the apparent viscosity of the three mixtures is reduced along with the increase of the shear rate, and the apparent viscosity of the sC-CPP-2 mixture is higher than that of the mixture of C-CPP-2 and CPP-2, so that the interaction between the sC-CPP-2 and intestinal mucus is stronger than that between the mixture of C-CPP-2 and CPP-2.
2. Measurement of interaction of CPP-2, C-CPP-2 and sC-CPP-2 with intestinal mucus by tonicity
CPP-2, C-CPP-2 and sC-CPP-2 with certain concentrations are respectively and uniformly compressed in a plane test disc (d=10mm), the test disc is connected with a sliding block of a syringe pump by using a tensionless string, and the syringe pump (V2, guanjie, china) is fixed above a balance by using a bracket. Colon tissue of mice was fixed to the bottom of the beaker with instant glue and Phosphate Buffered Saline (PBS) was injected. The syringe pump slide block is adjusted to enable the test disc to descend to the surface of colon tissue, and incubation is carried out for 20min. The syringe pump slide was then raised at a constant speed of 2.83mm/min until the test disc separated from the colonic tissue. Balance data were recorded once per second, the maximum separation force (MDF) was calculated and the Total Work of Adhesion (TWA) was calculated according to the trapezoidal rule. The detection results are shown in FIG. 3.
As can be seen from FIG. 3, the maximum separation force obtained between sC-CPP-2 and mucus was 101.82.+ -. 5.78mN, which is approximately 1.5 times the maximum separation force obtained between C-CPP-2 and mucus (71.54.+ -. 4.60 mN), which was 2 times the maximum separation force obtained between CPP-2 mucus (49.53.+ -. 3.90 mN). The Total Work of Adhesion (TWA) of sC-CPP-2 is as high as 120.07 + -6.81 μJ, which is significantly higher than that of C-CPP-2 and CPP-2. Thus, the adhesion between the sulfhydryl modified pilose asiabell root polysaccharide and intestinal mucus is obviously enhanced.
In conclusion, compared with the codonopsis pilosula polysaccharide CPP-2 and the carboxymethyl codonopsis pilosula polysaccharide C-CPP-2, the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 prepared by the method has stronger interaction with intestinal mucus, stronger adhesive force and remarkable effect.
EXAMPLE 4 use of mercaptocodonopsis pilosula polysaccharide (sC-CPP-2) for enhancing intestinal adhesion of probiotics
In the invention, lactobacillus rhamnosus GG (LGG) is taken as an example, and the adhesion effect of intestinal mucus on lactobacillus rhamnosus GG (LGG) under the condition of adding sC-CPP-2 is analyzed through in-vitro adhesion test and laser confocal microscope observation. The specific implementation mode is as follows:
1. in vitro mucilage adhesion test
Adding intestinal mucus with regulated concentration into 96-well cell culture plate, and adding 100-200 μl physiological saline/LGG, CPP-2/LGG, C-CPP-2/LGG, and sC-CPP-2/LGG suspension (containing LGG viable bacteria number at 10) 8 -10 9 CFU/mL), the mixture was sealed and placed in a refrigerator at 4 ℃ overnight, and the buffer was aspirated and washed 2 times. Then adding 1-5% SDS-NaOH lysate to release the adhered bacteria. And counting live bacteria on the flat plate, and calculating the adhesion rate of intestinal mucus to probiotics according to the following formula.
Wherein: a is total bacterial count (CFU/mL);
b is the number of bacteria adhered (CFU/mL).
As shown in FIG. 4, the adhesion rate of the intestinal mucus from the sC-CPP-2 group to the probiotics was significantly improved by 68.21% as compared with the Normal Saline (NS), CPP-2 and C-CPP-2 groups. Therefore, the adhesion between the surface mercapto groups of the sC-CPP-2 and the probiotics and the intestinal mucus is formed, and the adhesion of the intestinal mucus to the probiotics can be remarkably enhanced.
2. In vitro intestinal adhesion test
In a sterile operation table, the intestinal tracts of male Sprague-Dawley rats are planed, 4 parts of 1.5cm×1.5cm×2mm intestinal tracts are spread on a sterile glass slide, and 10-100 μl of physiological saline/LGG, CPP-2/LGG, C-CPP-2/LGG, sC-CPP-2/LGG suspension are respectively dripped(the number of viable bacteria containing LGG was 10 8 -10 9 CFU/mL), and placing them in sterilized centrifuge tubes, placing the slide glass in the centrifuge tubes, slightly shaking for 2 hours, taking out, flushing with 100-500 μl of physiological saline, collecting the flushing liquid, performing gradient dilution, and counting viable bacteria, and calculating the adhesion rate according to the following formula.
Wherein: a is total bacterial count (CFU/mL);
b is the number of non-adherent bacteria (CFU/mL).
As shown in FIG. 5, the adhesion of the intestinal mucus of the sC-CPP-2 group to the probiotics is strongest, the adhesion rate reaches 74.07%, and the adhesion rate is remarkably higher than that of the Normal Saline (NS) group, the CPP-2 group and the C-CPP-2 group. Therefore, the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 can obviously enhance the adhesion effect of intestinal mucus to probiotics. 3. Laser confocal scanning electron microscope for observing adhesion performance of mucus of probiotics
The colon of male Sprague-Dawley rats was cut into small loops of 3-5cm in length and ligated on one side using a medical suturing procedure. Probiotic use 
BacLight (TM) bacterial Activity kit Syto 9 staining, 100-500. Mu.L of LGG bacterial suspension after staining (10 9 CFU/mL containing 0.5-1% CPP-2, C-CPP-2 or sC-CPP-2), slowly injecting into colon, fastening the other side with surgical thread, ligating, culturing in PBS solution at 37deg.C for 30min, making into intestinal slice, and scanning with laser confocal scanning electron microscope for 3D layer scan to observe the adsorption of probiotic in mucus. The results are shown in FIG. 6.
As can be seen from FIG. 6, the small amounts of probiotics in the CPP-2 and C-CPP-2 groups adhered to the intestinal tracts of rats, while the probiotics in the sC-CPP-2 group adhered most.
In conclusion, the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 prepared by the method can obviously enhance the adhesion effect of intestinal mucus on probiotics and improve the intestinal adhesiveness of the probiotics, thereby being beneficial to the field planting and proliferation of the probiotics in the intestinal tract and achieving unexpected technical effects.
Example 5 preparation method of colon targeting probiotic microcapsules
1. Sodium alginate microcapsule for preparing probiotics/sulfhydryl radix codonopsis polysaccharide by air atomization method
Adding sulfhydryl radix Codonopsis polysaccharide sC-CPP-2 into probiotic bacterial suspension according to the proportion of 0.5-1.0% g/mL to prepare Cheng Yi probiotic/sC-CPP-2 suspension; sodium alginate solution with concentration of 3-5% g/mL and probiotic bacteria/sC-CPP-2 suspension according to the ratio of 1:1-5, and preparing into a bacterial gel mixture, wherein the viable count of the probiotics is 10 8 -10 9 CFU/mL; spraying 20-50mL of the fungus gel mixture into 1-2L 2-4%g/mL of calcium chloride solution by using an atomizer, and mechanically stirring at 300rpm for 30min for calcification; centrifuging the obtained solution at 4deg.C and 5000r/min for 10min, collecting to obtain microcapsule A, and washing with distilled water for three times;
2. chitosan coating
Adding the microcapsule A into chitosan solution with concentration of 4-10% g/mL according to the proportion of 0.5% mg/mL, stirring at 100rpm for 40min, centrifuging to collect microcapsule B, and washing twice with distilled water;
3. eudragit S100 coating
Preparing 2-5% Eudragit S100 acetone solution, fully dissolving, adding distilled water containing 1-5mL Tween 80, fully mixing, evaporating acetone solvent in the solution by a reduced pressure evaporation method, adding microcapsule B into the prepared Eudragit S100 coating solution, and fully shaking for 4h for coating; centrifuging to collect precipitate, washing with distilled water, and removing residual solution to obtain probiotic microcapsule.
Example 6 characterization and Property analysis of colon targeted probiotic microcapsules
In this example, using commercially available Lactobacillus rhamnosus GG (LGG) as an example, microcapsules added with LGG and sC-CPP-2 and microcapsules added with only LGG were prepared by the method described in reference to example 5, respectively, and characterization and property analysis were performed on the prepared microcapsules.
1. Scanning electron microscope analysis
Microscopic observation of the prepared LGG/sC-CPP-2 microcapsule was performed by using a Scanning Electron Microscope (SEM), and the result is shown in FIG. 7.
As can be seen from fig. 7, no leakage of the probiotics was found on the surface of the LGG/sC-CPP-2 microcapsule prepared by the present invention, indicating that the probiotics were completely encapsulated inside the microcapsule. Moreover, compared with the microcapsule prepared by only adding the LGG, the microcapsule prepared by adding the LGG and the mercapto-dangshen polysaccharide sC-CPP-2 does not greatly change in size, so that the size and the form of the probiotic microcapsule are not influenced by the addition of the mercapto-dangshen polysaccharide sC-CPP-2.
2. Particle size analysis
The particle size of the colon targeted probiotic microcapsule prepared in example 4 was detected by a micrometer particle size analyzer, and the result is shown in fig. 8.
As can be seen from FIG. 8, the LGG/sC-CPP-2 microcapsule prepared by the present invention has a particle size of 164.07.+ -. 10.48. Mu.m, allowing encapsulation of probiotics (1-2 μm) during the crosslinking process.
3. Characterization of probiotic packaging conditions by laser confocal microscopy
Staining with Syto 9 prior to encapsulation of probiotics according to
The BacLight (TM) bacterial activity kit protocol was run and the encapsulation status of the probiotics was characterized using laser confocal, the results are shown in figure 9.
From FIG. 9, it can be seen that the probiotics in the LGG/sC-CPP-2 microcapsule were successfully embedded inside the microcapsule.
4. Survival rate and release rate analysis of probiotics in probiotic microcapsules in vitro simulating gastrointestinal tract environment
In the test, commercial free lactobacillus rhamnosus GG (LGG) bacterial powder is adopted for comparison, and the specific operation steps are as follows:
4 parts of each of 3 samples of free LGG powder, LGG microcapsule and LGG/sC-CPP-2 microcapsule were transferred to a pre-heated tube containing simulated gastric fluid, and treated at 37℃and 80rpm for 0, 30, 90 and 180min, respectively. Centrifuging to collect microcapsules and thalli, removing gastric juice, washing the microcapsules or thalli with PBS, fully releasing the microcapsules or thalli, performing gradient dilution, performing viable count on the LGG/sC-CPP-2 microcapsules, the LGG bacterial powder and the commercial LGG microcapsules, and calculating the survival rate of probiotics by using the following formula, wherein each treatment is repeated for 3 times.
Respectively taking 4 tubes of LGG/sC-CPP-2 microcapsule, free LGG bacterial powder and commercial LGG microcapsule after 180min of gastric juice digestion, centrifugally collecting the microcapsule and bacterial cells, removing gastric juice, flushing the microcapsule or bacterial cells with PBS, respectively transferring the microcapsule or bacterial cells into a test tube filled with 9.9mL of simulated intestinal juice, and treating for 0, 30, 90 and 180min at 37 ℃ and 80 rpm. Centrifuging, washing the microcapsule or thallus with PBS, removing intestinal juice, fully releasing the microcapsule, counting viable bacteria, and calculating the survival rate of probiotics according to the formula.
LGG/sC-CPP-2 microcapsule samples are transferred into simulated gastric fluid, incubated at 37 ℃ and 80rpm for 3 hours, then transferred into artificial intestinal fluid for 3 hours, then transferred into artificial colon fluid for 3 hours, 1 tube of samples are taken every 15 minutes, and the absorbance value of the release medium at 600nm is measured. And (3) drawing a release curve of probiotics, and detecting the release rate of the LGG/sC-CPP-2 microcapsule in the stomach, the small intestine and the colon.
The results are shown in FIG. 10.
As can be seen from fig. 10 (a), the survival rate of free probiotic LGG rapidly decreases in the acidic environment of the stomach, and after 180min, the survival rate is only 2.07%, indicating that the survival rate of free LGG is extremely low after passing through the extreme environment of the stomach, and hardly reaches the intestinal tract; commercial LGG microcapsules survive 180min in simulated gastric fluid environment, but survive 180min in simulated intestinal environment, and are undetectable; the survival rate of the LGG/sC-CPP-2 microcapsule still reaches 95.2% and 69.15% after simulating the gastric environment and the intestinal environment for 180min, so that the LGG/sC-CPP-2 microcapsule provided by the invention can better resist the extreme environment of the gastrointestinal tract and successfully deliver probiotics to the colon.
From fig. 10 (B) the probiotic release profile shows that the probiotic in LGG/sC-CPP-2 microcapsules is hardly released during gastric digestion, but is slowly released in the small intestine environment and is fully released after reaching the colon. The LGG/sC-CPP-2 microcapsule can well protect probiotics from being digested by gastrointestinal tracts, and the probiotics are targeted to realize complete release in colon.
5. Evaluation of storage stability of probiotic microcapsules
The amounts of LGG/sC-CPP-2 microcapsules, free LGG powder and commercially available LGG microcapsules were stored at 4℃and 25℃respectively and the survival rate of probiotics was examined at 0, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70d and the results are shown in FIG. 11.
As can be seen from FIG. 11, the variation in storage stability of LGG/sC-CPP-2 microcapsules, free LGG powder and commercially available LGG microcapsules was small under the condition of 4 ℃; however, the stability of the LGG/sC-CPP-2 microcapsules varies greatly under 25 ℃ storage conditions, wherein the survival rate of probiotics after 70d storage is as high as 44.97%, which is significantly higher than that of free LGG and commercial LGG microcapsules (p < 0.05).
6. Evaluation of the effect of the probiotic microcapsules on the proliferation and colonization of the probiotics
(1) Mouse feeding
8-10 week old BALB/c male mice (supplied by Fukang Biotechnology Co., ltd., beijing) were fed 3 animals per cage, were light/dark cycled for 12h, were fed water ad libitum, were acclimated for 1 week, and were fasted (not water forbidden) for at least 48h before the experiment.
(2) Analysis of probiotic distribution in the intestinal tract of mice by fluorescence imaging
Distribution of LGG/sC-CPP-2 microcapsules, free LGG powder and commercial LGG microcapsule post-intragastric probiotics in the intestinal tract was observed by in vivo fluorescence imaging (amihtx, spectroscopic imaging, USA). Use of probiotic prior to embedding 
BacLight (TM) bacterial activity kit Syto 9 staining, 63 mice were randomly divided into three groups (free LGG powder, commercially available LGG microcapsules, LGG/sC-CPP-2 microcapsules). Each mouse was fed with the free stain accordinglyLGG (300. Mu.L, LGG concentration 10) 9 CFU/mL), suspension containing LGG/sC-CPP-2 microcapsules or commercially available LGG microcapsules (300. Mu.L, each containing 10) 9 CFU/mL LGG). Each group was sacrificed at 1, 2, 4, 8, 12, 24 and 48 hours for fluorescence imaging by resecting stomach, small intestine and large intestine, respectively. The results are shown in FIG. 12.
From fig. 12, it can be seen that LGG/sC-CPP-2 microcapsules can significantly extend the residence time of probiotics in the intestinal tract relative to free LGG and commercial LGG microcapsules. Mice fed free LGG were able to observe a small amount of probiotics in their cecum and colon after 12h, but not at all after 24h and 48h, indicating that probiotics were completely expelled from the body with faeces and were unable to achieve colonisation in the gut; a small amount of probiotics can be observed at the tail end of cecum of the mice fed with the LGG microcapsule after 24 hours, but no probiotics can be observed at 48 hours, which indicates that the probiotics are discharged out of the body along with the feces at 48 hours; however, the residual probiotics can still be observed in the colon of the mice fed with the LGG/sC-CPP-2 microcapsule at 48 hours, which proves that the LGG/sC-CPP-2 microcapsule remarkably improves the residence time of the probiotics LGG in the colon and successfully realizes the targeted adhesion and colonization in the colon.
(3) Detection of LGG colonic colonisation by real-time fluorescent quantitative PCR (RT-PCR)
The mice were randomly divided into four groups, each group of mice was fed free LGG (300-500. Mu.L), LGG/sC-CPP-2 microcapsules or LGG microcapsule suspension (300-500. Mu.L, containing 10) 9 CFU/mL LGG) and PBS (300-500. Mu.L). The mice were sacrificed at 1, 6 and 9 days after the end of the gavage, respectively, and the colon contents of the mice were collected. LGG in the colon of mice was quantitatively analyzed by shanghai megi company. The results are shown in FIG. 13.
As can be seen from fig. 13, the colonization of the intestinal tract by the probiotic LGG was different in mice of different treatment groups. Wherein:
after 1d of gastric lavage, the LGG bacteria amount in the intestinal tracts of the mice of each treatment group reaches the highest;
after 6d of gavage, mice fed free LGG were almost leveled with the bacteria content of PBS group mice, while LGG bacteria content in LGG microcapsule fed mice was significantly higher than that in PBS group and free LGG group, but there was still a significant trend of decrease compared to 1d, but bacteria content in the intestinal tract of LGG/sC-CPP-2 microcapsule fed mice was still high;
after 9d of gastric lavage, the LGG bacteria amounts in the LGG microcapsule group and the free LGG group mice were leveled with the PBS group, indicating that little LGG added after gastric lavage was present in the colon; while the LGG/sC-CPP-2 microcapsule group mice had slightly decreased amounts of LGG bacteria, but were still significantly higher than the other treatment groups. Therefore, the LGG/sC-CPP-2 microcapsule provided by the invention can obviously promote adhesion and colonization of probiotics in colon.
Example 7 application of colon targeting probiotic microcapsules in relieving colitis in mice
Taking a dextran sodium sulfate (dextran sulfate sodium, DSS) induced ulcerative colitis mouse as an example, taking clostridium prausnitzii (F.prausnitzii) as an experimental probiotic, preparing F.prausnitzii/sC-CPP-2 microcapsules, co-delivering clostridium prausnitzii and mercapto codonopsis polysaccharide to the colon, increasing the adhesion and colonization and proliferation of the clostridium prausnitzii/sC-CPP-2 microcapsules in the colon, and further examining the effect of the F.prausnitzii/sC-CPP-2 microcapsules on relieving the enteritis and the effect of the F.prausnitzii/sC-CPP-2 microcapsules on regulating the intestinal flora of the mice with the enteritis. The specific implementation mode is as follows:
preparation of F.prausnitzii and sC-CPP-2 microcapsules
Prepared by the method described in example 4 of the present invention:
(1) Sodium alginate microcapsules added with F.prausnitzii and sC-CPP-2, namely A (F.prausnitzii/sC-CPP-2);
(2) Sodium alginate microcapsule with added sC-CPP-2 only, namely A (sC-CPP-2);
(3) Sodium alginate microcapsules with addition of only f.prausnitzii, i.e. a (f.prausnitzii);
(4) Empty sodium alginate microcapsules, i.e. a (Empty).
2. Grouping, modeling and administration of animals
Mice were divided into 7 groups of 6 mice each, acclimatized for 1 week, and group modeling and dosing began on day 8. In addition to the healthy group, the other groups had 3% (w/v) DSS added to the mice' drinking water, while the experimental group was gavaged 1 time daily and mice were sacrificed on day 8. The specific grouping and stomach filling doses are as follows:
(1) Health (CD) group: normal drinking water (without DSS), 100-500 μl of PBS was infused daily;
(2) Model (DSS) group: 100-500 mu L of PBS for stomach irrigation every day;
(3) Sodium alginate Empty sphere a (Empty) group: 100-500 mu L of sodium alginate microcapsule freeze-dried powder suspension for daily gastric lavage;
(4) Group A (sC-CPP-2): 100-500 mu L of microcapsule suspension of stomach A (sC-CPP-2) is infused every day;
(5) Group a (f.prausnitzii): 100-500 μl of microcapsule suspension of stomach a (f.prausnitzii) is infused daily;
(6) Group A (F.prausnitzii/sC-CPP-2): 100-500 mu L of microcapsule suspension of stomach A (F.prausnitzii/sC-CPP-2) is infused every day;
(7) Free f.prausnitzii group: and 100-500 mu L of the clostridium gastralgia bacteria powder suspension is infused every day.
3. Sample collection
(1) Fecal sample collection
Collecting mouse feces, and storing at-80deg.C.
(2) Serum collection
The abdominal aorta was bled and serum was isolated and stored at-80 ℃.
(3) Colon tissue and content harvesting
Mice were sacrificed after blood collection and colorectal was collected from each group of mice. The anus to ileocecal junction distance was measured and the mouse colorectal was fixed in 10-20% neutral formalin solution.
The other mice took out the colon content, rinsed the colon with sterile physiological saline, blotted dry with filter paper, and transferred the colon content and colon tissue rapidly into liquid nitrogen, and later transferred to-80 ℃ for preservation. 4. Body weight change and disease activity index calculation
During the test period, mice were monitored for water consumption and weight changes. In addition, the fecal status was observed, and at the same time, the fecal occult blood condition of the mice was detected, and the fecal blood condition was classified into five grades: the color is not developed and is negative; secondly, the yellow-green color is the positive (+) of occult blood; thirdly, the blue-green color is positive with occult blood (++); (IV) the dark blue-green color is positive in occult blood (++); fifth, blood is visible to the naked eye, and then hematochezia is visible to the naked eye. Finally, the disease activity index (Disease activity index, DAI) of the mice was calculated according to the scoring criteria of table 1.
TABLE 1 disease Activity index (Disease activity index, DAI) scoring criteria
As a result, as shown in FIG. 14, on the 3 rd day of the modeling, mice in each group, except for the CD group, exhibited various degrees of symptoms such as reduced drinking water, weight loss, darkened hair, listlessness, reduced activity, loose stool, and the like, and the DSS group exhibited symptoms such as loose stool, light hematochezia, and the like. On day 5 of modeling, mice in other groups except the CD group all showed different degrees of hematochezia, with the hematochezia in the DSS group being more pronounced and mice died. On day 7 of modeling, mice in the CD group were in good condition, and the hematochezia phenomenon of mice in the other groups was worse than the previous day, and the weight was significantly reduced. The weight change of each group of mice was shown in FIG. 14A, and the weight of the mice in the other groups was reduced except for the increase in the weight of the mice in the CD group on the 7 th day of the modeling, wherein the reduction in the weight of the DSS group was most remarkable and the reduction in the weight of the A (F.prausnitzii/sC-CPP-2) group was the lowest. As shown in fig. 14B, the DAI scores of the mice in each group, except the CD group, increased from day 3 of the modeling, wherein the DAI score on day 7 of the a (Empty) group was not significantly different from that of the DSS group, the DAI score of the a (f.prausitzii/sC-CPP-2) group increased most slowly, and the score on day 7 was lower, indicating that the a (f.prausitzii/sC-CPP-2) group had the best effect of alleviating colitis in the mice.
5. Colon length and spleen weight measurement in mice
The anus to the cecum end whole section of the colon of the mice in different groups were taken, the length was measured, and photographing and recording were performed. Spleen of the mice was taken for photographing and weighing recording.
As a result, as shown in FIG. 15, the difference in colon length of mice in different treatment groups was visually seen, and the colon length of mice in the CD group was longest, and the spleen size was normal without enlargement. Compared with the CD group, the colon of the DSS group is obviously shortened, and the splenomegaly phenomenon is obvious. Group a (Empty) is similar to DSS group, with significantly shortened colon and pronounced splenomegaly. The colonitis symptoms of the mice in the other groups are relieved to different degrees, particularly the colonic inflammation of the group A (F.prausnitzii/sC-CPP-2) has the longest colonic length and the lowest spleen weight compared with the other groups except the group CD, which indicates that the group A (F.prausnitzii/sC-CPP-2) has a certain relieving effect on colonic inflammation of colonic inflammatory mice.
6. Colon HE staining and tissue injury scoring
Colon paraffin sections were prepared and HE stained, and HE stained sections were scanned using a section scanner and scored for tissue damage. The scoring criteria are shown in table 2.
TABLE 2 tissue injury scoring Table
The results are shown in FIG. 16, in which the colonic mucosa of the CD group mice is intact and continuous, the glands are aligned, the crypts are normal, and no inflammatory cell infiltrates. The colon injury degree of the mice in the DSS group and the A (Empty) group is serious, bleeding and deep ulcers are visible, obvious acute inflammatory reaction is formed, and a large amount of inflammatory cells infiltrate the mucous membrane and submucosa. The A (sC-CPP-2), A (F. Prausnitzii) and Free F. Prausnitzii groups also exhibited different degrees of colon tissue damage, but the damage was lighter than in the DSS and A (Empty) groups. The group A (F.prausnitzii/sC-CPP-2) had the least colon lesions. Furthermore, as can be seen from the DAI score, the degree of damage to the colon tissue is in order from large to small: DSS group > a (Empty) group > Free f.prausnitzii group > a (sC-CPP-2) group > a (f.prausnitzii) > a (f.prausnitzii/sC-CPP-2) group > CD group.
The results show that the F.prausnitzii/sC-CPP-2 microcapsule provided by the invention can effectively relieve colonitis of mice, and the effect is obviously better than that of a microcapsule added with only F.prausnitzii or sC-CPP-2. Therefore, the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 provided by the invention can be used with probiotics in a combined way, so that the adhesion effect of intestinal mucus on the probiotics can be obviously enhanced, the colonisation of the probiotics in the colon is facilitated, the probiotics effect is exerted, the colonitis of mice is effectively relieved, and unexpected technical effects are achieved.
7. Myeloperoxidase (MPO) activity detection in colon tissue
10-20% colon tissue homogenate was prepared. The homogenate was used for protein concentration determination according to BCA protein assay kit (soribao) instructions. Homogenates were taken and assayed for MPO activity in colon tissue of mice according to the instructions of MPO (Myeloperoxidase) assay kit (Nanjing).
As shown in FIG. 17, the MPO levels of mice in the different groups were different, the MPO values were highest in the DSS group and the A (Empty) group, significantly higher than those in the CD group, and the colon MPO values were lower in the A (sC-CPP-2) group, the A (F.prausitzii) group and the Free F.prausitzii group, indicating that the symptoms of colitis were alleviated. While the colon MPO value of group A (F.prausnitzii/sC-CPP-2) was the lowest among the other groups except the CD group, significantly lower than that of the DSS group, indicating that the colitis inflammation was the least in group A (F.prausnitzii/sC-CPP-2). Therefore, the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 provided by the invention can be used with probiotics to effectively relieve the colonitis of mice, and unexpected technical effects are achieved.
8. Detection of antioxidant index in colon tissue
Colon tissue homogenate samples were prepared and tested for Nitric Oxide (NO), malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) content according to the kit (nanjing build) instructions.
As shown in figure 18, the levels of oxidative stress products NO and MDA in colon tissues were significantly increased (p < 0.05) compared to the CD group, and the levels of GSH and SOD for antioxidant effect were significantly reduced, indicating that the level of oxidative stress in DSS-induced acute colitis mice was significantly increased. MDA, NO, GSH and SOD content were not significantly different from DSS group in group a (Empty) compared to CD group. The NO and MDA content of group A (sC-CPP-2), group A (F.prausnitzii) and group Free F.prausnitzii were slightly reduced compared to the DSS group, while the GSH and SOD content was slightly increased. In addition, group A (F.prausnitzii/sC-CPP-2) exhibited lower NO and MDA levels and higher GSH and SOD levels. Therefore, the F.prausnitzii/sC-CPP-2 microcapsule provided by the invention can relieve damage of DSS-induced oxidative stress to the intestinal tract of a mouse through antioxidation.
9. ELISA determination of cytokine content in colon and serum
The content of TNF-alpha, IL-1 beta, IL-6 and IL-10 in the serum of the mice is determined according to the instruction of the kit. Taking colon tissue of a mouse at a distance of 1-2cm from cecum, placing the colon tissue into centrifugal tubes, placing 4 zirconium beads into each centrifugal tube, adding pre-cooled sterile PBS (phosphate buffer solution) with a volume of 9 times, placing the mixture into a tissue grinding homogenizer for grinding, centrifuging at 12000g and 4 ℃ for 15min, and collecting supernatant for cytokine detection. The levels of cytokines TNF- α, IL-1β, IL-6 and IFN- γ in the colon of the mice were determined according to the ELISA kit instructions.
The cytokine content in serum of mice is shown in FIG. 19, the TNF-alpha, IL-1 beta and IL-6 content of DSS group is obviously increased (p < 0.05), the IL-10 content is obviously reduced (p < 0.05), and compared with DSS group, the other groups can reduce the TNF-alpha, IL-1 beta and IL-6 content in serum to different degrees, and the IL-10 content is increased, wherein A (F.prausitzii/sC-CPP-2) group can obviously reduce the TNF-alpha, IL-1 beta and IL-6 content in serum (p < 0.05), and the IL-10 content is increased (p < 0.05).
Cytokine levels in the colon of mice are shown in FIG. 20, with significant increases in TNF- α, IL-1β, IL-6 and IFN- γ levels (p < 0.05) in the DSS group. Each of the other groups was able to reduce the levels of TNF- α, IL-1β, IL-6 and IFN- γ in serum to a different extent than the DSS group, wherein group A (F.prausnitzii/sC-CPP-2) was able to significantly reduce the levels of TNF- α, IL-1β, IL-6 and IFN- γ in serum (p < 0.05).
The results show that the F.prausnitzii/sC-CPP-2 microcapsule provided by the invention has remarkable anti-inflammatory effect and the effect is obviously better than that of a microcapsule added with only F.prausnitzii or sC-CPP-2. Therefore, the sulfhydryl codonopsis pilosula polysaccharide sC-CPP-2 provided by the invention is used in combination with probiotics, and has remarkable relieving effect on DSS-induced acute colitis.
10. Probiotic microcapsule has effect in regulating intestinal flora diversity and metabolite SCFAs of mice with colon inflammation
(1) Intestinal flora diversity analysis
Samples of colon contents of each group of mice were taken according to the following
Total bacterial DNA was extracted from the colon contents using the Soil DNA Kit (Omega Bio-Tek, USA) Kit instructions. DNA integrity was checked by agarose gel electrophoresis and the DNA concentration of each set of samples was checked using a NanoDrop 2000 (Thermo, USA). Then, using the upstream primer 338F: ACTCCTACGGGAGGCAGCA and downstream primer 806R: GGACTACHVGGGTWTCT AAT PCR amplification was performed on the V3 to V4 hypervariable regions of the 16S r RNA genes of each set of DNA samples. PCR products were identified and purified using agarose gel electrophoresis detection and AxyPrep DNA Gel Extraction Kit (Axygen, USA), using Quantus TM The Fluorometer (Promega, USA) performs PCR quantification. Finally use->
The Rapid DNA-Seq Kit (bio Scientific, USA) and Miseq PE300 platform from Illumina corporation were pooled and sequenced. All of the above procedures, including DNA extraction and sequencing, were delegated to be done by shanghai meg corporation. />
As shown in fig. 21, the abundance of intestinal pathogenic bacteria Alistipes, odoribacter and Helicobacter in the DSS group was significantly increased (p < 0.05) compared to the CD group, while the abundance of pathogenic bacteria parisutterella and mucispirum associated with IBD was significantly increased (p < 0.05) and the abundance of pathogenic bacteria romiboutsia was slightly increased (p > 0.05) in the DSS group; the decreased abundance of intestinal probiotics Akkermansia, prevotellaceae _ucg_001, bifidobacterium, desulfovibrio and lactobacillus indicates that the intestinal flora of mice in the DSS group has been disturbed. Compared with the DSS group, the other groups have certain regulation effect on the small intestinal flora. Compared with the DSS group, the A (F.prausnitzii/sC-CPP-2) group can remarkably reduce the abundance of intestinal pathogenic bacteria Alistipes, odoribacter, helicobacter, parasutterella and Mucispirillum and increase the abundance of intestinal probiotics Akkermansia, lachnospiraceae, prevotellaceae _UCG_001, bifidobacterium, faecalinacterium, desulfovibrio and Lactobacillus. In addition, the abundance of Faecarinbacteria was significantly increased in the A (F.prausnitzii/sC-CPP-2), A (F.prausnitzii) and Free F.prausnitzii groups compared to the other groups, and the abundance of A (F.prausnitzii/sC-CPP-2) was significantly higher than in the A (F.prausnitzii) and Free F.prausnitzii groups (p < 0.05), indicating that the probiotic F.prausnitzii had colonized the gut.
(2) Detection of short chain fatty acids in the colon contents of mice
Taking 100-200mg of colon contents of each group of mice, adding 500-1000 mu l of diethyl ether, sufficiently shaking and uniformly mixing, then carrying out ultrasonic treatment for 30min, and detecting the content of short chain fatty acid by using GC-MS through a 0.22 mu m organic filter membrane.
The results are shown in figure 22, where the Total short chain fatty acid content (Total acid) is significantly reduced (p < 0.05) in DSS and a (Empty) groups compared to CD groups. The other groups all showed different increases in short chain fatty acid content compared to the DSS group, with the total short chain fatty acid content of the a (f.prausnitzii/sC-CPP-2) group being highest, the a (f.prausnitzii) group and the Free f.prausnitzii group. By comparing the butyric acid content of each group, the butyric acid content of group A (F.prausnitzii/sC-CPP-2), group A (F.prausnitzii) and Free F.prausnitzii was higher than that of the other groups, indicating that supplementation with F.prausnitzii increased the content of short chain fatty acids, especially butyric acid, in the intestinal tract of the colon inflammatory mice. It can be seen that the F.prausnitzii/sC-CPP-2 microcapsule provided by the invention can alleviate the symptoms of mouse colitis induced by DSS by increasing the content of short chain fatty acid.
In conclusion, the probiotic microcapsule provided by the invention can be used for targeted delivery of the sulfhydryl radix codonopsis polysaccharide and the probiotics to the colon, wherein the sulfhydryl radix codonopsis polysaccharide and the probiotics are combined for use, the adhesion efficiency of the probiotics in the colon is obviously improved, and the effective colonization of the probiotics in the colon and the effect of the probiotics are realized. The probiotic may be selected from any one or a combination of two or more of the genera bifidobacterium, lactobacillus, streptococcus, lactococcus, leuconostoc, propionibacterium, saccharomyces, pediococcus, staphylococcus, preferably any one or a combination of two or more of bifidobacterium longum, bifidobacterium adolescentis, bifidobacterium breve, bifidobacterium infantis, bifidobacterium animalis, bifidobacterium bifidum, lactobacillus acidophilus, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus reuteri, lactobacillus fermentum, lactobacillus bulgaricus, streptococcus thermophilus, kluyveromyces marxianus, pediococcus acidilactici, pediococcus pentosaceus, staphylococcus calf, staphylococcus xylosus, staphylococcus intestinal leuconostoc mesenteroides, lactobacillus lactis subspecies lactis, lactobacillus diacetyl subspecies lactis.
Claims (8)
1. The application of the sulfhydryl radix codonopsis polysaccharide in the production of foods, health-care products or medicines is characterized in that the sulfhydryl radix codonopsis polysaccharide is prepared by the following method:
(1) Preparation of carboxymethyl codonopsis pilosula polysaccharide
Dissolving 1-2 g of codonopsis pilosula polysaccharide in 50-100mL of deionized water, adding 150-300 mL of 3mol/L NaOH solution after full dissolution, and stirring for 10min; adding 20-40 g of monochloroacetic acid, and reacting for 4 hours at 65 ℃; cooling to room temperature, adjusting pH to 7.0, and dialyzing; vacuum drying the dialyzed product to obtain carboxymethyl pilose asiabell root polysaccharide;
(2) Preparation of sulfhydryl pilose asiabell root polysaccharide
Dissolving 100-200 mg of carboxymethyl codonopsis pilosula polysaccharide in 20-40 mL of distilled water, and fully dissolving; then adding 100-500 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, activating carboxyl in the reaction system, and stirring for 1h at room temperature; adding N-acetyl-L-cysteine under the protection of nitrogen, and stirring for reaction for 24 hours; dialyzing with distilled water; and (3) freeze-drying the product obtained by dialysis to obtain the sulfhydryl pilosula polysaccharide.
2. A probiotic preparation comprising a probiotic and the mercaptodangshen polysaccharide of claim 1.
3. The probiotic preparation of claim 2, wherein the probiotic is any one or a combination of two or more of the genera bifidobacterium, lactobacillus, streptococcus, lactococcus, leuconostoc, propionibacterium, saccharomyces, pediococcus, staphylococcus.
4. A probiotic preparation according to claim 3, wherein the probiotic is any one or a combination of two or more of bifidobacterium longum, bifidobacterium adolescentis, bifidobacterium breve, bifidobacterium infantis, bifidobacterium animalis, bifidobacterium bifidum, lactobacillus acidophilus, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus reuteri, lactobacillus fermentum, lactobacillus bulgaricus, streptococcus thermophilus, kluyveromyces marxianus, pediococcus acidilactici, pediococcus pentosaceus, staphylococcus calf, staphylococcus xylosus, staphylococcus meat, leuconostoc mesenteroides, lactococcus lactis subspecies lactis, lactococcus lactis diacetyl subspecies lactis.
5. The probiotic preparation according to any one of claims 2 to 4, characterized in that the probiotic preparation is a probiotic microcapsule.
6. The probiotic microcapsule is characterized by being prepared by the following steps:
(1) Preparing sodium alginate microcapsules comprising probiotics and the sulfhydryl radix codonopsis polysaccharide according to claim 1;
(2) Coating for the first time by chitosan;
(3) A second coating was performed using Eudragit S100.
7. The probiotic microcapsule of claim 6, wherein the probiotic microcapsule is prepared by the following method:
(1) Preparation of sodium alginate microcapsule containing probiotics and sulfhydryl radix codonopsis polysaccharide
Adding the sulfhydryl radix Codonopsis polysaccharide of claim 1 into probiotics according to the proportion of 0.5-1.0% g/mLPreparing probiotic bacteria/sulfhydryl radix Codonopsis polysaccharide suspension in the suspension; mixing sodium alginate solution with the concentration of 3-5% g/mL with probiotic bacteria/sulfhydryl radix codonopsis polysaccharide suspension according to the weight ratio of 1: 1-5, and preparing into a bacterial gel mixture, wherein the viable count of the probiotics is 10 8 ～10 9 CFU/mL; spraying 20-50 mL of fungus gel mixture into 1-2L of calcium chloride solution with the concentration of 2-4%g/mL by using an atomizer, and mechanically stirring at 300rpm for 30min for calcification; centrifuging the obtained solution at 4deg.C and 5000r/min for 10min, and washing with distilled water to obtain microcapsule A;
(2) First coating with chitosan
Adding the microcapsule A into chitosan solution with the concentration of 4% -10% according to the proportion of 0.5% mg/mL, stirring for 40min at 100rpm, centrifuging, collecting precipitate, and washing with distilled water to obtain microcapsule B;
(3) Secondary coating with Eudragit S100
And adding the microcapsule B into Eudragit S100 coating liquid with the concentration of 2-5% g/mL according to the proportion of 0.5% mg/mL, fully shaking for 4 hours for coating, centrifuging, collecting precipitate, washing with distilled water, and removing residual solution, thus obtaining the probiotic microcapsule.
8. Use of the probiotic microcapsules of claim 6 or 7 in the preparation of a food, a health product or a pharmaceutical product.
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