CN110448577B - Probiotic microcapsule preparation for repairing ulcerative colitis - Google Patents
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- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
- A61K9/5042—Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
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Abstract
The invention relates to a microecological microcapsule composition for repairing colonitis, wherein the composition for treating colonitis is a combination of bifidobacterium adolescentis microcapsule and clostridium butyricum microcapsule. The microecological microcapsule composition disclosed by the invention intervenes on rats, and the DSS is utilized to induce ulcerative colitis, so that the changes of the weight, colon length, inflammatory factors, intestinal microorganisms and the like of each group of rats are evaluated, and the effect of the composition on preventing the ulcerative colitis is researched. The test result shows that the composition can improve the physiological index of colonitis rats, reduce the content of intestinal inflammatory factors such as TNF-alpha, IL-6 and the like, reduce the activity of myeloperoxidase and regulate the structure of intestinal flora. The invention is characterized in that the microecological microcapsule is safe, has no toxic and side effects, and has certain advantages compared with the drug treatment such as antibiotics. Therefore, the invention provides important technical support for developing a novel preparation for preventing colonitis.
Description
Technical Field
The invention belongs to the technical field of enteritis treatment preparations, and in particular relates to a probiotic microcapsule preparation for repairing ulcerative colitis.
Background
Ulcerative Colitis (UC) is a chronic, non-specific inflammatory disease of the colon and rectum. At present, the incidence rate of the disease is higher and higher, and the incidence rate of the disease is increased in China along with the change of the diet structure in the past. The main clinical manifestations are abdominal pain, diarrhea, bloody pus, etc. Studies have shown that the onset of UC may be related to inheritance, immunomodulation, environmental and intestinal microorganisms. However, the exact pathogenesis of UC is still unclear, and existing treatments, such as antibiotics, corticosteroids and immunomodulators, have met with some success; because of the side effects of medication, it is not suitable for long-term treatment.
The number and activity of probiotics are reduced under the severe strong acid and alkali environment which is processed, stored and transported to the gastrointestinal tract, and the microcapsule technology is an effective means for protecting the probiotics from the external adverse environment, and is paid more attention to and widely applied.
In recent years, the relationship between the onset of ulcerative colitis and the intestinal flora has become a research hotspot, and research has demonstrated that the intestinal flora of people and animals suffering from ulcerative colitis is significantly different from that of healthy people, and ulcerative colitis patients often show intestinal flora imbalance to different extents and often involve changes in the number of various flora. Experiments show that the probiotic drug therapy, intestinal flora transplantation and other methods are used for improving intestinal microecology of organisms, and the probiotic drug therapy can have better auxiliary treatment effect on people and animals suffering from chronic ulcerative colitis.
Disclosure of Invention
The invention provides a colonitis ecological restoration microecological preparation, namely a bifidobacterium adolescentis microcapsule and clostridium butyricum microcapsule preparation for colonitis restoration, which uses a bifidobacterium adolescentis microcapsule and clostridium butyricum microcapsule composition for adjusting intestinal immunity and a microbial system of chronic ulcerative enteritis; thereby making up for the deficiencies of the prior art.
The invention firstly provides a microecological microcapsule, wherein the core material is bifidobacterium adolescentis and clostridium butyricum;
the number of the bifidobacterium adolescentis microcapsules and clostridium butyricum microcapsules is 1x10 8 ～5×10 8 CFU/g；
The microcapsule has wall material comprising 1% chitosan, 1% sodium carboxymethylcellulose and 1% fructo-oligosaccharide;
wherein the adding proportion of 1% chitosan, 1% sodium carboxymethyl cellulose and 1% fructo-oligosaccharide is 2:1:1, a step of;
preferably, 4% of glycerol is also added to the wall material as a cryoprotectant.
The application of the microecological microcapsule in preparing a product for treating ulcerative colitis is provided.
The microecological microcapsule composition has the advantages of improving physiological indexes of rats with enteritis, reducing intestinal inflammatory factors such as TNF-alpha, IL-6 and the like, reducing the activity of myeloperoxidase and regulating intestinal flora. Is expected to develop a novel high-efficiency microecological preparation for auxiliary treatment of ulcerative colitis.
Drawings
Fig. 1: myeloperoxidase (MPO) activity profile;
fig. 2: each group of colon histological evaluation charts, wherein a: baCb (1:1) group; b: baCb (5:1) group; c: baCb (1:5) group; d: baCb (5:5) group; e: a wall material group; f: DSS group; g: a control group;
fig. 3: a tissue injury score;
fig. 4: microbial profile of the cecal content of each group at portal level, wherein a: baCb (1:1) group; b: baCb (5:1) group; c: baCb (1:5) group; d: baCb (5:5) group; e: a wall material group; f: DSS group; g: a control group;
fig. 5: microbial profile at the genus level for each group of cecal contents, wherein a: baCb (1:1) group; b: baCb (5:1) group; c: baCb (1:5) group; d: baCb (5:5) group; e: a wall material group; f: DSS group; g: a control group;
fig. 6: each set of LDA value distribution histograms, where A: baCb (1:1) group; b: baCb (5:1) group; c: baCb (1:5) group; e: a wall material group; f: DSS group; g: control group.
Detailed Description
The microencapsulation provided by the invention can effectively lead bifidobacterium adolescentis and clostridium butyricum to resist the strong acid and strong alkali environment of the gastrointestinal tract, so that the bifidobacterium adolescentis and clostridium butyricum have higher bacterial activity after reaching the intestinal tract. The invention combines the bifidobacterium adolescentis microcapsule and the clostridium butyricum microcapsule, and can effectively inhibit the release of inflammatory factors and regulate the intestinal flora structure.
The present invention will be described in detail with reference to examples.
Example 1: preparation of bifidobacterium adolescentis microcapsule and clostridium butyricum microcapsule
1) Selecting wall materials:
the wall material used for the strain microcapsule coating comprises: chitosan, sodium carboxymethyl cellulose and fructooligosaccharides. Wherein chitosan is a biological natural polymer and has mucoadhesive property. Sodium carboxymethyl cellulose (NaCMC) is an anionic derivative of cellulose with mucoadhesive properties. Fructo-oligosaccharide is a prebiotic and can promote the growth and propagation of probiotics such as bifidobacteria and the like. The agent with chitosan matrix exists in gel form at low pH value to prevent diffusion of bioactive agent, and can make the embedded probiotic bacteria pass through the severe environment of gastrointestinal tract smoothly, and maintain sufficient strain activity.
2) Selection and culture of strains:
the strain adopts Bifidobacterium adolescentis (BNCC 134301) and Clostridium butyricum (BNCC 337239). The butyric acid and other substances produced by clostridium butyricum can promote the growth and reproduction of bifidobacteria, so as to promote the probiotic effect of bifidobacteria and clostridium butyricum on intestinal tracts. Culturing bifidobacteria and clostridium butyricum in MRS culture medium and enhanced clostridium sporogenes culture medium at 37 ℃ for 24-48 h, centrifuging the bacteria-containing culture solution at 6000rmp/min for 10min at 4 ℃, collecting cells, washing 2-3 times with physiological saline, and then re-suspending in the physiological saline. The activity of the strain is regulated to be 1 multiplied by 10 9 CFU。
3) Preparation of microcapsules: respectively preparing 1% chitosan (deacetylation degree is more than or equal to 85%) solution, 1% sodium carboxymethyl cellulose solution and 1% fructo-oligosaccharide solution, wherein the adding ratio is 2:1:1, a step of; bifidobacterium adolescentis (BNCC 134301) and clostridium butyricum (BNCC 337239) are respectively core materials, and the ratio of the core materials to the wall materials is 1:4, a step of; 4% glycerol is added as a cryoprotectant, and the microcapsule is prepared by a vacuum freeze drying method. The embedded probiotics activity is 1 multiplied by 10 measured by a plate counting method 8 CFU。
Example 2: effect detection of compositions
1) Compounding of the compositions
The microbial activities of the bifidobacterium adolescentis (BNCC 134301) microcapsule and the clostridium butyricum (BNCC 337239) microcapsule adopted in the invention are respectively 1 multiplied by 10 8 CFU/mouse/day-5×10 8 CFU/mouse/day, the addition ratio was 1:1. 5: 1. 1:5 and 5:5.
2) Experimental animals and experimental design
Test selection of 7 week old adult male SD rats 126Only, the random was divided into 7 groups of 3 replicates each, 6 replicates each. The 7 groups were BaCb (1:1), baCb (5:1), baCb (1:5), baCb (5:5), wall material, DSS and normal (see Table 1), respectively. Rats were subjected to a gastric lavage composition on days 1-14, baCb (1:1), baCb (5:1), baCb (1:5) and BaCb (5:5), wall material groups were subjected to gastric lavage without probiotic microcapsules, and DSS and normal groups were subjected to gastric lavage with PBS. The addition level of the bifidobacterium adolescentis microcapsule is 1 multiplied by 10 8 CFU/mouse/day and 5×10 8 CFU/mouse/day, clostridium butyricum microcapsules were added at a level of 1X10 8 CFU/mouse/day and 5×10 8 The treatment method of CFU/mouse/day and lavage is shown in Table 1. On days 8-14, 5% DSS was added to the drinking water of each group except the normal group, which resulted in ulcerative colitis, and the enteritis formation was observed. The test design is shown in Table 1.
Table 1: experimental design of synergistic intervention of bifidobacterium adolescentis microcapsule and clostridium butyricum microcapsule in rats
3) Sampling and processing
Rats were weighed after 24 hours of fasting. The eyeball is killed after blood is taken out. Blood was centrifuged to obtain serum, which was stored at-80℃for subsequent index determination. After dissection, tissues such as colon, cecum and the like are collected for subsequent index measurement.
4) Detection index
(1) The body weight, colon length and weight of each rat were weighed.
(2) The content of inflammatory factors such as TNF-alpha, IL-6, IL-10, IL-1 beta and the like is measured according to the kit.
(3) Myeloperoxidase (MPO) activity was measured.
(4) H & E staining.
(5) The method is characterized in that a 16SrRNA sequencing technology is adopted, and based on an Illumina Hiseq sequencing platform, the structure and the flora number of the cecum microorganisms are determined by a double-End sequencing (Paired-End) method.
5) Test results
(1) Effects of groups on physiological growth of ulcerative enteritis rats
The change in physiological index of each group of rats after DSS application is shown in table 2. Compared with the normal group, the weight gain and the diet of rats in the DSS group and the wall material group are obviously reduced, the colon length and the colon weight/length are obviously increased, which indicates that ulcerative colitis caused by DSS leads to slow weight gain and reduced food intake of the rats; at the same time, causes hyperemia of the colon to thicken, or forms ulcer surfaces, etc., resulting in shortening of the colon and an increase in the ratio of weight/length of the colon. Compared with the DSS group and the wall material group, the weight gain and the diet of the BaCb (1:1), the BaCb (5:1), the BaCb (1:5) and the BaCb (5:5) are obviously increased, and the colon length and the colon weight/length are obviously increased, so that the microecological microcapsule composition has obvious repairing effect on ulcerative colitis.
Table 2: influence of synergy of bifidobacterium adolescentis microcapsules and clostridium butyricum microcapsules on physiological indexes of enteritis rats
(2) Effects of Myeloperoxidase (MPO) Activity
The Myeloperoxidase (MPO) activity of the colon reflects the extent of neutrophil infiltration. The test result shows that the MPO activity of the DSS group and the wall material group is obviously higher than that of the control group, which indicates that the DSS causes intestinal inflammation; whereas the application of microcapsules without probiotics had no restorative effect on DSS-induced intestinal inflammation. The BaCb (1:1), baCb (5:1), baCb (1:5) and BaCb (5:5) have significantly increased MPO activity compared with the DSS and wall material groups, and have no significant difference from the normal group, so that the microecological microcapsule composition provided by the invention can inhibit MPO activity generated by DSS induced ulcerative colitis of rats.
(3) Histological evaluation
To investigate the prophylactic effect of the composition on ulcerative enteritis, histopathological lesions were assessed using paraffin embedding and H & E staining, histological analysis and histological lesion scores are shown in figures 2, 3. Inflammatory reactions such as crypt disappearance, epithelial structural damage, severe infiltration of inflammatory cells of the colonic mucosa, etc., occurred in DSS and wall material (fig. 2, E, F) treated samples. Whereas no significant inflammatory response was observed in the samples of the microecological microcapsule composition after drying (fig. 2A, B, C and D), the epithelial cell morphology was similar to the control. The tissue damage scores (fig. 3) were significantly higher for the DSS and wall material groups than for the control group, indicating that DSS induced ulcerative enteritis in rats caused intestinal tissue damage. Meanwhile, the tissue injury score of the composition group is obviously lower than that of the DSS group and the wall material group, and the composition group has no obvious difference from the control group, which shows that the micro-ecological microcapsule composition can relieve intestinal tissue injury caused by DSS after dry prognosis.
(4) Effects on DSS-induced ulcerative enteritis rat inflammatory factors
The changes in inflammatory factors in rats of each group after DSS administration are shown in Table 3, and the amounts of IL-1β, IL-17, IL-6 and TNF- α in serum of the DSS group and the wall material group are significantly increased as compared with the control group, indicating that ulcerative colitis caused by DSS leads to an increase in inflammatory factors in serum. Compared with the DSS group and the wall material group, the BaCb (1:1) group, the BaCb (5:1) group, the BaCb (1:5) group and the BaCb (5:5) group promote the secretion of the proinflammatory factor IL-10, inhibit the excessive secretion of the inflammatory factors IL-1 beta, IL-17, IL-6 and TNF-alpha, and show that the microecological microcapsule composition has the effect of preventing ulcerative colitis.
Table 3: effects of Bifidobacterium adolescentis microcapsule and Clostridium butyricum microcapsule on inflammatory factors of ulcerative colitis SD rat
(5) Alpha diversity analysis
Samples were assessed for species abundance and species diversity by Alpha diversity analysis, with the measures being Chao1, ace, shannon and Simpson, where the Chao1 and Ace indices measure species abundance and the Shannon and Simpson indices are used to measure species diversity. At a similarity level of 97%, the number of OTUs per group was obtained. Compared with the normal group, the number of OUT, chao1, ace and Shannon indexes of the wall material group and the DSS group are reduced, and the Simpson indexes are obviously increased due to obvious difference, which indicates that the DSS induces the wall material group and the DSS group rats to form ulcerative colitis, so that the species abundance and diversity of intestinal microorganisms are reduced. Compared with the wall material group and the DSS group, the number of OUT, chao1, ace and Shannon indexes of the BaCb (1:1), baCb (5:1), baCb (1:5) and BaCb (5:5) groups are increased and have obvious differences, and the Simpson index is obviously reduced; indicating that the abundance and diversity of intestinal microorganisms can be regulated under the intervention of the microecological microcapsule composition. In table 4, the Coverage of OUT is counted, and the values of the Coverage are all above 0.998, which indicates that the result represents the actual situation of the microorganism in the sample. The results show that the composition has a preventive effect on ulcerative enteritis.
Table 4: analysis of the Alpha diversity of the colonic flora of rats at different levels
(6) Relative abundance of major microbiota components at the portal level
FIG. 4 is a graph showing the relative abundance of major microbiota components at the portal level. The dominant flora on the phylum level was mainly Firmicutes, bacteroides (bacterioides) and proteus (Proteobacteria) by colony bar graph analysis. The relative abundance of Firmicutes (DSS) and wall groups was significantly reduced compared to the control group, and increased significantly after treatment with the composition. In contrast, the relative abundance of bacteroides (bacterioides) increased significantly after DSS and wall material treatment, but decreased significantly after treatment with the probiotic microcapsule composition. The relative abundance of Proteus (Proteus) was significantly higher in the DSS and wall groups than in the control group, while significantly reduced after treatment with the probiotic microcapsule composition. The results show that the microecological microcapsule composition can regulate intestinal flora.
(7) Relative abundance of major microbiota components at the genus level
FIG. 5 is a graph showing the relative abundance of major microbiota components at the genus level. After DSS treatment, the relative abundance of Bacteroides (bacteria) was significantly increased and the relative abundance of Lactobacillus (Lactobacillus) was significantly decreased in the DSS group and the wall material group compared to the control group. Under the intervention of the composition, the relative abundance of Bacteroides (bacterioides) is significantly reduced, and the relative abundance of Lactobacillus (Lactobacillus) is significantly increased. The results show that the intervention of the microecological microcapsule composition can regulate the abundance of beneficial bacteria and harmful bacteria in the intestinal tract.
(8) Group sample LEfSe analysis
To further investigate the differences in probiotic populations, at each phylogenetic level (from phylum to genus), the probiotic groups were compared with the control, wall material, DSS groups. LEfSe was used to identify specific bacterial lines altered by probiotic treatment. Fig. 5 is a histogram of LDA value distribution, showing species with LDA values greater than a set value (set to 4.0), where the length of the histogram represents the magnitude of the effect of the different species (i.e., LDA values), and different colors represent different groupings of species. The relative abundance of Bacteroides (bacterioides), bacteroidaceae (bacterioidaceae), erysipelas (erysiphe) is higher in the rat intestinal samples of the wall material group compared to the other treatment groups, the relative abundance of Bacteroides (bacteriodia) in the DSS group is higher, and clostridium (clostridium), firmicutes (clostridium) and clostridium (clostridium) in the samples show higher relative abundance after the probiotic composition treatment. The results show that the microecological microcapsule composition can regulate intestinal microbial flora.
The invention combines the bifidobacterium adolescentis microcapsule and the clostridium butyricum microcapsule to prepare the microecological preparation, can relieve intestinal mucosa injury caused by inflammation, reduce the activities of inflammatory factors and myeloperoxidase, regulate intestinal flora, reduce intestinal pathogenic bacteria, improve diarrhea, hematochezia and other symptoms, has better auxiliary treatment effect on ulcerative colitis, and is hopeful to develop the ulcerative colitis repair microecological preparation.
Claims (3)
1. The microecological microcapsule is characterized by comprising a wall material and a core material, wherein the core material is bifidobacterium adolescentis and clostridium butyricum; the number of the bifidobacterium adolescentis and clostridium butyricum is 1 multiplied by 10 8 ～5×10 8 CFU/g; the wall material is 1% of chitosan, 1% of sodium carboxymethyl cellulose and 1% of fructo-oligosaccharide; and the ratio of 1% chitosan, 1% sodium carboxymethyl cellulose and 1% fructo-oligosaccharide is 2:1:1.
2. The micro-ecological microcapsule according to claim 1, wherein 4% of glycerol is added to the wall material as a cryoprotectant.
3. Use of a micro-ecological microcapsule according to any of claims 1-2 for the preparation of a product for the treatment of ulcerative colitis.
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