CN112111422B - Bifidobacterium pseudocatenulatum capable of relieving colitis and application thereof - Google Patents
Bifidobacterium pseudocatenulatum capable of relieving colitis and application thereof Download PDFInfo
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- CN112111422B CN112111422B CN202010902877.5A CN202010902877A CN112111422B CN 112111422 B CN112111422 B CN 112111422B CN 202010902877 A CN202010902877 A CN 202010902877A CN 112111422 B CN112111422 B CN 112111422B
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Abstract
The invention discloses bifidobacterium pseudocatenulatum capable of relieving colitis and application thereof, belonging to the technical field of biology. The invention provides bifidobacterium pseudocatenulatum MY40C which has immunoregulatory capacity and can improve intestinal barrier and relieve colitis. Compared with the model group mice, the levels of MPO and COX-2 in colon tissues of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C dry-treated mice are remarkably reduced, and the tissue damage is remarkably reduced.
Description
Technical Field
The invention relates to bifidobacterium pseudocatenulatum capable of relieving colitis and application thereof, belonging to the technical field of biology.
Background
Ulcerative colitis is a diffuse inflammatory response characterized by the presence of abscesses, neutrophil, eosinophil and plasma cell infiltrates within the crypts, attacking the intima of the colon. The most common symptoms of patients with UC are diarrhea, rectal bleeding, tenesmus and external heaviness, mucus outflow and abdominal pain.
The cause of ulcerative colitis is still unknown, and is currently thought to be the result of interactions between foreign substances causing host responses, genes and immune influences.
Bloody diarrhea is the most common early symptom of ulcerative colitis, and other symptoms are abdominal pain, hematochezia, weight loss, tenesmus, vomiting and the like in sequence. Occasionally, arthritis, iridocyclitis, liver dysfunction and skin lesions are mainly manifested. The disease manifests itself in chronic, low malignancy in most patients and in acute, catastrophic outbreaks in a few patients (about 15%).
Ulcerative colitis directly affects quality of life, resulting in changes in social, psychological and professional areas. The selection of an appropriate treatment is critical to improving the quality of life of patients with ulcerative colitis. There is an increasing search for conventional treatments using drugs with the aim of reducing symptoms and inflammation. The currently used therapeutic drugs are: sulfasalazine salicylic acid preparations such as adisha, mesalamine, etc.; the corticosteroid is prednisone or dexamethasone, but long-term use of the corticosteroid may cause hypertension, diabetes, osteoporosis and other diseases, thereby affecting the success of treatment.
In view of the various problems of the existing treatment schemes, a scheme for relieving colitis, which replaces the traditional method, is particularly important, and the new treatment schemes comprise monoclonal antibodies, prebiotics, probiotics, and microbial metabolites such as unsaturated fatty acids, short-chain fatty acids and the like. The feasibility of the above-mentioned therapies has also prompted us to continue to search for dietary supplements with broader application and greater potential, and at the same time, with a colitis-alleviating effect.
Disclosure of Invention
The invention provides an application of bifidobacterium pseudocatenulatum in preparing a product for preventing and/or treating colitis, and the bifidobacterium pseudocatenulatum can be used for preparing the product for preventing and/or treating colitis.
The Bifidobacterium pseudocatenulatum is Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) CCFM1115 which is preserved in Guangdong province microbial strain collection center with the preservation number of GDMCC No:60954 and the preservation date of 2020, 07, 16 days.
The Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) CCFM1115 is derived from healthy infant feces, the sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence obtained through sequencing is compared in an NCBI database, the result shows that the strain is the Bifidobacterium pseudocatenulatum and is named as Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) CCFM1115, and meanwhile, the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) CCFM1115 is named as Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C and is retained in the food biotechnology strain collection center of south China university.
The colony of the Bifidobacterium pseudocatenulatum MY40C on the MRS solid culture medium is protruded, smooth, round, milky white and translucent, and the diameter of the colony is 1-2 mm.
In one embodiment of the invention, the colitis is ulcerative colitis.
In one embodiment of the invention, the viable count of Bifidobacterium pseudocatenulatum in the product is not less than 1X 1010CFU/g。
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the pharmaceutical product is a lyophilized powder.
In one embodiment of the invention, the freeze-dried powder is prepared by inoculating the bifidobacterium pseudocatenulatum into a culture medium for culture to obtain a seed solution; inoculating the seed solution into a culture medium for culturing to obtain a culture solution; centrifuging the culture solution, and collecting bacterial sludge; washing the bacterial sludge with normal saline, and then resuspending to obtain a resuspension solution; adding a freeze-drying protective agent into the heavy suspension to obtain a mixed solution; and (4) carrying out vacuum freeze drying on the mixed solution to obtain freeze-dried powder.
In one embodiment of the present invention, the seed solution is inoculated into a culture medium in an inoculum size of 2-4% (v/v) for culture.
In one embodiment of the invention, the components of the lyoprotectant include skim milk powder, trehalose, sucrose and water.
In one embodiment of the invention, the components of the lyoprotectant are 80-120g/L skimmed milk powder, 80-140g/L trehalose, 140-180g/L sucrose and water.
In one embodiment of the invention, the components of the lyoprotectant include 100g/L skim milk powder, 100g/L trehalose, 160g/L sucrose and water.
In one embodiment of the invention, the addition amount of the lyoprotectant in the resuspension solution is 2-4 times of the total weight of the bacterial sludge.
In one embodiment of the present invention, the seed culture medium is MRS solid culture medium and the fermentation culture medium is MRS liquid culture medium.
In one embodiment of the present invention, the MRS liquid medium is a cysteine hydrochloride-added MRS liquid medium.
In one embodiment of the present invention, the cysteine hydrochloride is added in an amount of 0.04 to 0.1% by mass.
In one embodiment of the invention, the seed solution is inoculated into the MRS liquid culture medium by an inoculum size of 2-4% for culture under the culture conditions that: anaerobic culture is carried out for 24-36 h at 34-38 ℃, 7000-00 rmp centrifugation is carried out for 20-30 min, bacterial sludge is collected, and the bacterial sludge is washed for 3-4 times by normal saline and then resuspended.
The invention also provides a product for preventing and/or treating colitis, which contains the bifidobacterium pseudocatenulatum.
In one embodiment of the invention, the viable count of Bifidobacterium pseudocatenulatum in the product is not less than 1X 1010CFU/g。
Advantageous effects
(1) The Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C provided by the invention is separated from intestinal flora of healthy people, and the strain has no toxic or side effect on human bodies, so that the medicine prepared from the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C provided by the invention has certain advantages compared with the traditional medicine for treating colitis, and the strain can be used for preparing probiotic preparations and the like, thereby having wide market prospect.
(2) By adopting the Bifidobacterium pseudocatenulatum MY40C provided by the invention, the disease activity index of a mouse during DSS-induced colitis can be obviously reduced, and the reduction of the weight and the shortening of the colon can be reduced.
(3) Compared with the model group mice, the activity of MPO, COX-2 in colon tissues of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C dry-treated group mice is obviously reduced, and the tissue damage is obviously reduced.
(4) The intervention of Bifidobacterium pseudocatenulatum MY40C can reduce the apoptosis of intestinal epithelial cells and reduce the activity of Caspase-3 which is a key enzyme in the apoptosis of the epithelial cells. In a Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C dry pre-group, proteins such as tight junction proteins ZO-1, Claudin-3, Occludin and the like related to intestinal barriers are obviously higher than those in a model group, and colon tissue ultrastructural analysis shows that the tight junction structure of a model group mouse is damaged, while Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C intervenes that the tight junction structure of the model group mouse is complete and villus is neat.
(5) Under the intervention of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C, the concentration of mucin MUC2 is increased, the number of goblet cells is obviously increased, and the colon mucus layer is also obviously repaired.
(6) Bifidobacterium pseudocatenulatum MY40C intervention significantly reduced the concentration of the pro-inflammatory cytokines TNF- α and IL-6 and up-regulated the concentration of the anti-inflammatory cytokines IL-10 and PPAR- γ, as well as up-regulated the concentration of immunoglobulin IgE in the serum. The observation result of the colon pathological section also shows that the colon tissue structure of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C dry-pre-group mice is relatively complete, the goblet cells are rich, and no serious damage is caused.
(7) The intervention of Bifidobacterium pseudocatenulatum MY40C obviously reduces the concentrations of TLR 4/NF-kB signal channel key proteins TLR4, p-p65 and p-IkB, inhibits the activity of a TLR 4/NF-kB signal channel, and relieves the immune response.
Biological material preservation
A Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) is classified and named as Bifidobacterium pseudocatenulatum, is deposited in Guangdong province microorganism strain collection center at 16.07.2020, and has the deposit number of GDMCC No. 60954, and the deposit address of Guangzhou city Michelia Tokyo No. 100, No. 59, building 5.
Drawings
FIG. 1: body weight change during DSS modeling of different groups of mice.
FIG. 2: disease activity index DAI index changes in different groups of mice.
FIG. 3: colon length for different groups of mice.
FIG. 4: colon morphology in different groups of mice.
FIG. 5: colon tissue HE staining of different groups of mice.
FIG. 6: colon histopathology scores were performed on different groups of mice.
FIG. 7: activity of MPO in Colon tissue of different groups of mice.
FIG. 8: activity of cyclooxygenase COX-2 in colon tissue of different groups of mice.
FIG. 9: different groups of mice colon tissue goblet cell PAS staining pattern.
FIG. 10: colon tissue mucus layer aliskiren staining pattern of different groups of mice.
FIG. 11: the expression level of MUC2 protein in colon tissues of different groups of mice.
FIG. 12: number of goblet cells in colon tissue of different groups of mice.
FIG. 13: expression level of alpha-catenin 1 protein in colon tissues of different groups of mice.
FIG. 14: expression level of beta-catenin protein in colon tissues of mice of different groups.
FIG. 15: expression level of E-Cadherin1 protein in colon tissue of mice of different groups.
FIG. 16: the expression level of ZO-1 protein in colon tissues of different groups of mice.
FIG. 17: expression level of Occludin protein in colon tissue of mice of different groups.
FIG. 18: expression level of claudin-3 protein in colon tissue of mice of different groups.
FIG. 19: immunohistochemical staining pattern of tight junction protein in colon tissue of different groups of mice.
FIG. 20: ultrastructural image of colon tissue of different groups of mice.
FIG. 21: different groups of mice colon tissue epithelial cell apoptosis staining pattern.
FIG. 22: expression level of Caspase-3 protein in colon tissue of mice of different groups.
FIG. 23: expression of TNF-alpha protein in colon tissue of different groups of mice.
FIG. 24: expression level of IL-6 protein in colon tissue of different groups of mice.
FIG. 25: expression level of IL-10 protein in colon tissue of different groups of mice.
FIG. 26: the expression level of PPAR-gamma protein in colon tissue of different groups of mice.
FIG. 27 is a schematic view showing: the expression level of IgE in serum of mice of different groups.
FIG. 28: different groups of mice colon tissues TRL 4/NF-kB signal pathway key protein immunohistochemical staining.
FIG. 29: mean optical density values of TRL 4/NF-kappa B signal pathway key proteins in colon tissues of different groups of mice.
In fig. 1 to 29, "" and "all" indicate significant differences from the DSS Model group, and the more the "more" the significant differences are.
Detailed Description
The invention is further elucidated with reference to a specific embodiment and a drawing.
The mice referred to in the examples below were male SPF (Specific pathogen free) grade C57BL/6J mice 8 weeks old, purchased from the university of tokyo institute of model animals; the ELISA kits referred to in the following examples were purchased from Shanghai enzyme-linked Biotechnology Ltd; the skim milk powder, trehalose, sucrose, and paraformaldehyde referred to in the following examples were purchased from national pharmaceutical group chemical agents, ltd; dextran Sulfate Sodium (DSS) referred to in the examples below was purchased from tokyo pockets, south kyo; the Allilyn blue, the nuclear fast red staining solution, and the fluorescent dye Hoechst 33258 referred to in the examples below were purchased from Nanjing Senega technologies, Inc.
The media involved in the following examples are as follows:
MRS liquid medium: 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 0.5g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate monohydrate, 2g/L of diammonium hydrogen citrate, 2.6g/L, Tween 801 mL/L of dipotassium hydrogen phosphate trihydrate and 0.5g/L of cysteine hydrochloride.
MRS solid medium: 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 0.5g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate monohydrate, 2g/L of diammonium hydrogen citrate, 2.6g/L, Tween 801/801 mL g/L of dipotassium hydrogen phosphate trihydrate, 0.5g/L of cysteine hydrochloride and 20g/L of agar.
The detection methods referred to in the following examples are as follows:
method for detecting Disease Activity Index (DAI):
the DAI score was based on Murthy's scoring system and included three aspects of weight change, hematochezia status and stool characteristics (specific scoring criteria are shown in Table 1). During modeling, the weight of the mouse is measured every day, the hematochezia condition and the stool character of the mouse are detected, and the score is calculated according to the table 1, wherein DAI is the sum of the weight change score, the hematochezia score and the stool character score. The fecal occult blood condition is measured by a Pirami hole fecal occult blood reagent, the specific operation is carried out according to a reagent instruction, and if reddish brown or bright red blood can be seen by naked eyes in the feces, the feces are bloody by naked eyes. Stool traits are divided into three grades: normal, loose and loose feces, and normal feces of mice are formed into granules; feces are loose if they are viscous and loose, but do not adhere to the anus; if the feces are unformed or watery and adhere to the anus, it is loose stool.
TABLE 1 disease Activity index Scoring criteria
Weight loss (%) | Occult/macroscopic bloody stool | Stool character | Score of | |
0 | Negative in occult blood | Is normal | 0 | |
1~5 | Negative in | Loosening | 1 | |
6~10 | Positive | Loosening | 2 | |
11~15 | Positive occult blood | |
3 | |
>15 | Bloody stool with naked eyes | |
4 |
The detection method of the colon length comprises the following steps:
after the mice were sacrificed, the entire colon (end of cecum to anus) was removed and the length was measured.
The detection method of the colon histopathology characteristics comprises the following steps:
soaking a 1cm far-end colon (1 cm away from the anus) in 4% paraformaldehyde solution at 4 ℃ for 24h to obtain a fixed far-end colon tissue; sequentially dehydrating, transparentizing and waxing the fixed distal colon tissue, and embedding the tissue in a wax block by using a leica paraffin embedding machine to obtain the wax block embedded with the colon tissue; the method comprises the following steps of dehydration, transparency and wax impregnation: (1) and (3) dehydrating: dehydrating the fixed tissue by 70%, 80% and 90% (v/v) gradient ethanol solutions for 30min, respectively, and adding 95% and 100% (v/v) ethanol solutions for 2 times, 20min each time; (2) and (3) transparency: putting the tissue into mixed solution of alcohol and xylene at equal volume ratio for 15min, and then putting xylene I and xylene II for 3min respectively; (3) wax dipping: the tissue samples were placed in paraffin I and paraffin II liquids at 62 ℃ for 30min each.
Slicing the wax block embedded with the colon tissue by a Leica manual rotary slicer, wherein the slicing thickness is 5 mu m, and obtaining a colon tissue slice; spreading and fishing out the colon tissue slices, baking the slices, staining with hematoxylin, differentiating, rinsing, redyeing, dehydrating, transparentizing and sealing the slices to obtain H & E colon slices; the method comprises the following specific operations of spreading and fishing slices, baking slices, hematoxylin staining, differentiation, rinsing, eosin counterstaining, dehydration, transparency and sealing slices: (1) and (3) displaying and fishing pieces: placing the slices in a water bath with constant temperature of 42 ℃ for spreading, and carefully fishing out the slices by using a glass slide; (2) baking slices: putting the slices into a 65 ℃ oven for baking for 1 h; (3) and (3) hematoxylin staining: hydrating the slices firstly (namely placing the slices in dimethylbenzene I and dimethylbenzene II for 5min respectively, then sequentially placing the slices in 100%, 95%, 90%, 80% and 70% (v/v) gradient alcohol solutions for 5min respectively, and finally placing the slices in distilled water for 3min), then dyeing (namely placing the slices in hematoxylin dyeing solution for about 20s), and finally washing with water (namely washing the slices with tap water for about 30 min); (4) differentiation: placing the slices into 1% (v/v) hydrochloric acid ethanol solution for 7s for fading; (5) rinsing: washing the slices with tap water for about 20 min; (6) counterdyeing: immersing the slices in eosin staining solution, and immediately taking out; (7) and (3) dehydrating: putting the slices into a 95% (v/v) ethanol solution I, a 95% (v/v) ethanol solution II and a 70% (v/v) ethanol solution in sequence, taking out immediately after putting, then soaking in an 80% (v/v) ethanol solution for 50s, and finally soaking in 100% (v/v) ethanol for 2 min; (8) and (3) transparency: immersing slices into a mixed solution of ethanol and xylene in an equal volume ratio for 1min, and then immersing slices into xylene I and xylene II for 2min respectively; (9) sealing: : the slices were mounted with neutral gum.
The prepared H & E colon sections are scanned by a Pannoramic MIDI digital section scanner for photographing, and a scoring system of Dieleman is adopted to score tissue damage of each group of colon tissue sections, wherein the scoring of the tissue damage comprises four aspects of inflammation degree, lesion depth, crypt destruction and lesion range (the specific standard is shown in a table 2).
TABLE 2 tissue damage score criteria
The detection method of the pathological features of the mucus layer of the colon tissue comprises the following steps:
the colon epithelium is covered with a protective mucus layer, the colon mucus layer mainly consists of mucin, and the mucin and cell nucleus can be dyed blue and red respectively by alisin blue staining (the alisin blue staining is used for detecting the change of the colon mucus layer (the concrete method refers to Yan comfortable. Bifidobacterium longum genetic and phenotypic diversity and correlation research of the Bifidobacterium longum genetic and phenotypic diversity and immunoregulation function. 2019. Jiangnan university, doctor academic paper).
And (3) determination of colon tissue biochemical indexes:
colon tissue is prepared according to the following steps of 1: 9 adding tissue lysate (containing 1% (v/v) protease inhibitor and 1% (v/v) phosphatase inhibitor), crushing the colon tissue by a high-throughput crusher to obtain homogenate, centrifuging at 12000g and 4 ℃ for 15min, and collecting the supernatant to obtain the supernatant of the colon tissue; the activities of Myeloperoxidase (MPO), cyclooxygenase (COX-2) and Caspase-3 in the supernatant of colon tissue were measured by ELISA kit (Shanghai enzyme-linked biosciences, Ltd.), wherein the activities of MPO and COX-2 were in U/g of colon protein, and Caspase-3 was in U/mg of colon protein. The concentration of total protein in the colon tissue supernatant was measured in mg/ml using a BCA kit (pecan biotechnology limited).
The concentration of MUC2, cytokines TNF- α, IL-6, IL-10, PPAR- γ, and IgE in the supernatant of colon tissue were determined by ELISA kits (Shanghai enzyme-linked biosciences, Inc.), wherein the concentration of MUC2, the cytokines TNF- α, IL-6, IL-10, PPAR- γ were measured in pg/mg of colonic protein, and IgE was measured in pg/ml of serum.
The expression level of claudin-3, ZO-1, occludin, alpha-catenin 1, beta-catenin and E-Cadherin1 in the supernatant of colon tissue was determined by ELISA kit (Shanghai enzyme-linked biosciences Co., Ltd.), wherein the expression level of claudin-3, ZO-1, occludin, alpha-catenin 1, beta-catenin and E-Cadherin1 was in pg/mg of colon protein.
Example 1: screening, identification, culture, observation and preservation of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C
1. Screening
Taking 1g of healthy infant feces samples from tin-free areas, coating the samples after gradient dilution in an MRS solid culture medium (containing 10 mug/mL mupirocin antibiotic), placing the samples in an anaerobic environment at 37 ℃ for culturing for 72 hours, and observing and recording colony morphology; selecting a colony with a wet surface, a bulge and white and yellow color, streaking on an MRS solid culture medium, carrying out purification culture under the anaerobic condition at 37 ℃, and repeating the operation for 3 times to obtain a purified single colony; selecting single colonies, streaking on an MRS solid culture medium, carrying out anaerobic culture at 37 ℃ for 36h, carrying out gram staining on the obtained colonies (the gram staining method refers to the author of Industrial microbiology and Breeding in textbook of textbooks: Zhuge healthcare), recording the morphology of the colonies, examining the physiological and biochemical characteristics of the strains (the examination result is shown in table 3) according to the evaluation manual of common bacteria systems (the author: Dongxu pearl) in the textbook of textbooks, keeping the strains which are gram negative, have raised and white yellowing shapes on the colonies, are catalase negative and are fructose-6-phosphokinase positive, and obtaining only one strain in the screening.
2. Identification
Extracting the genome of the screened strain, amplifying and sequencing the 16S rDNA of the strain (the nucleotide sequence of the 16S rDNA obtained by amplification is shown as SEQ ID NO. 1), and comparing the obtained sequence with the nucleic acid sequence in NCBI-Blast to show that the strain is Bifidobacterium pseudocatenulatum and is named as Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY 40C;
the primers used for 16S rDNA amplification are as follows:
27F:5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID NO.2);
1492R:5’-TACGGCTACCTTGTTACGACTT-3’(SEQ ID NO.3);
the 16S rDNA amplification procedure was as follows:
5min at 95 ℃; 35 cycles (95 ℃ 30 s; 55 ℃ 30 s; 72 ℃ 2 min); 10min at 72 ℃.
TABLE 3 physiological and biochemical Properties of the strains
Experimental project | Results | Experimental project | Results |
Catalase assay | - | Melezitose | - |
Catalase test | - | Fucose sugar | - |
Fructose-6-phosphate phosphoketolase | + | Inulin powder | - |
Glucose | + | Cellobiose | - |
Fructose | + | Sodium gluconate | + |
Sucrose | + | Glucuronic acid sodium salt | - |
Note: "-" indicates negative, and "+" indicates positive.
3. Cultivation and Observation
A single colony of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C is picked and inoculated on an MRS solid culture medium, and after culturing for 48h at 37 ℃, the colony characteristics of the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C on the mMRS solid culture medium are observed. The bacterial colony of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C on the MRS solid culture medium is protruded, is smooth, circular, milky white and semitransparent, and has the diameter of 1-2 mm.
4. Preservation of
Selecting a single colony of Bifidobacterium pseudocatenulatum (MY 40C) to be inoculated into an MRS liquid culture medium, and culturing for 24h under the anaerobic condition at 37 ℃ to obtain a bacterial liquid; placing the bacterial liquid in a centrifuge tube, centrifuging at 3000rpm for 10min, and collecting thalli; adding the sterilized PBS buffer solution into the thallus, placing the thallus in a centrifuge tube, centrifuging at 3000rpm for 10min, washing to obtain the washed thallus, repeating the operation for 3 times, adding the sterilized 30% (v/v) glycerol into the obtained thallus, and storing the thallus in a glycerol tube at the temperature of minus 80 ℃.
Example 2: preparation of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C bacterial suspension
(1) Dipping a bacterial liquid of Bifidobacterium pseudocatenulatum (MY 40C) from a glycerol tube, streaking on an MRS solid culture medium, and culturing at 37 ℃ for 48h in an anaerobic environment to obtain a single bacterial colony; and selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 48h at 37 ℃ in an anaerobic environment for activation culture, and repeating the operation for 3 times to obtain activated bacteria liquid.
(2) Inoculating the activated bacterial liquid obtained in the step (1) into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), culturing at 37 ℃ for 24h to obtain a fermentation liquid, centrifugally collecting the bacteria from the fermentation liquid, re-suspending the bacteria by using normal saline, and adjusting the viable count to be 5 multiplied by 109CFU/mL, and preparing a bacterial suspension.
Example 3: bifidobacterium pseudocatenulatum MY40C for relieving symptoms of DSS-induced colitis mice
The method comprises the following steps:
(1) preparing 2.5% DSS solution: dextran Sulfate Sodium (DSS) was made up in sterile tap water to a concentration of 2.5% (w/v) DSS solution.
(2) 24 healthy male C57BL/6J mice at 8 weeks of age were randomly divided into 3 groups, 3 groups were designated as: a blank Control group (Control), a modeling group (DSS), a Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C dry Control group (MY40C + DSS); the experimental protocol and treatment regimen for each group of mice using 8 mice per group are shown in table 4.
(3) Treating a blank Control group (Control), a building block (DSS), and a Bifidobacterium pseudocatenulatum (MY40 pseudomonas 40C dry Control group (MY40C + DSS);
wherein the processing method of Bifidobacterium pseudocatenulatum (MY40C + DSS) MY40C stem group comprises the following steps: the experiment is carried out on days 1-7, and Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C dry group (MY40C + DSS) is administered with gastric lavage 5 × 109CFU/mL of pseudosmall chain bifidobacterium suspension of 200 mu L and freely drinking distilled water; the experiment is carried out on 8-14 days, and Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C dry group (MY40C + DSS) is administered with gastric lavage 5 × 109200 mu L of pseudosmall chain bifidobacterium bacterial suspension with the bacterial quantity of CFU/mL, and freely drinking 2.5 percent DSS solution;
the processing method of the building module (DSS) comprises the following steps: on the 1 st to 7 th days of the experiment, the DSS group drenches 200 mu L of normal saline every day and freely drinks distilled water; on 8-14 days of the experiment, 200 mu L of the normal saline solution for gastric lavage is administered to the DSS group every day, and 2.5 percent DSS solution is freely drunk;
the blank Control group (Control) processing method comprises the following steps: in the experimental process, the control group was infused with 200 μ L of normal saline daily and freely drunk with distilled water.
During the molding period, the body weight of each group of mice (the detection result is shown in figure 1) and the disease activity index DAI index change of each group of mice (the detection result is shown in figure 2) are detected.
After the molding is finished, the blank Control group (Control), the molding group (DSS), and the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C pretreatment group (MY40C + DSS) were killed, the entire colon (cecum end to anus) was taken, the length of the colon was measured (see fig. 3 for the test results), and the appearance of the colon was observed (see fig. 4 for the test results).
Table 4 experimental mouse treatment protocol
The disease activity index of each group of mice in the DSS modeling process is shown in fig. 2; on day 7 of modeling, the weight of mice in the DSS group was reduced by 20.72%, while the weight of mice in the MY40C intervention group (MY40C + DSS) was reduced by only 6.58%, so the weight loss of mice in the MY40C intervention group (MY40C + DSS) was significantly lower than that of the modeling group (see fig. 1 for the test results).
At the end of the molding, the DAI index of the mice in the DSS group increased to 11.63, while the intervention in the MY40C intervention group (MY40C + DSS) reduced the DAI index of the mice to 5.125, so that the DAI index of the MY40C intervention group (MY40C + DSS) was significantly lower than that of the model group (see fig. 2 for the test results).
The colon length of the normal group of mice is 7.275cm, the colon length of the DSS model group of mice is only 4.463cm, and the intervention of the MY40C intervention group (MY40C + DSS) significantly increases the colon length to 5.70cm (the detection result is shown in figure 3).
The colon of the control mouse is normally red, and the feces are granular; the dark red intestinal wall, swelling and bleeding can be seen in the colons of the model group; while the MY40C intervention group (MY40C + DSS) colon was closer to the blank group (see FIG. 4 for the test results).
Therefore, the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C has a better improvement effect on mice with DSS-induced colitis.
Example 4: bifidobacterium pseudocatenulatum MY40C protective action against colon tissue in colitis mice
The detailed implementation manner is the same as the steps (1) - (3) in the example 3;
after the molding obtained in step (3) in example 3 is finished, the mouse is sacrificed, 1cm of distal colon tissue is taken for fixing, dehydrating, embedding and HE staining, HE staining of colon tissue of different groups of mice is observed (the detection result is shown in figure 5), and the activity of Myeloperoxidase (MPO) and the activity of cyclooxygenase (COX-2) of the colon tissue (the detection result is shown in figure 7) are detected (the detection result is shown in figure 8).
From FIG. 5, it can be seen that the colon tissue structure of MY40C intervention group (MY40C + DSS) is similar to that of the normal group, with the glands and crypts more intact, the goblet cells more abundant, and no inflammatory cell infiltration and submucosal edema. After induction of DSS, the colon tissues of mice were significantly damaged, including inflammatory cell infiltration, destruction of crypt structures, disappearance of goblet cells, submucosal edema, etc. The colonic mucosal layer structure of the building block is thus almost completely destroyed, inflammatory cell infiltration is severe, crypt and gland structures are almost completely eliminated, and goblet cells are eliminated.
The histopathological scoring criteria were referenced to relevant literature (see in particular J Agr Food Chem,2019,67(48): 13282-.
Histopathological scores showed that the pathology score of the building block reached 12.5 points, whereas the score of the MY40C intervention group (MY40C + DSS) was significantly lower than that of the building block, only 6.75 points (see figure 6 for test results).
The contents of enzymes MPO (test result is shown in figure 7) and COX-2 (test result is shown in figure 8) related to the colon tissue inflammation in the modeling group are 14.15U/g protein and 9.672U/g protein respectively; in the MY40C intervention group (MY40C + DSS), the contents of enzymes MPO (detection result is shown in figure 7) and COX-2 (detection result is shown in figure 8) related to colon tissue inflammation are 9.829U/g protein and 6.967U/g protein respectively, so that the strain Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C has a good protection effect on colon tissues of mice.
Example 5: bifidobacterium pseudocatenulatum MY40C for protecting colon mucous layer of colitis mouse
The detailed implementation manner is the same as the steps (1) - (3) in the example 3;
after the molding obtained in step (3) in example 3 is completed, the mouse is sacrificed, and 1cm of distal colon tissue is taken for fixing, dehydrating, embedding and alizarin blue staining, and the distribution of mucin MUC2 of different groups of colon tissues and the expression amount of MUC2 protein of different groups of colon tissues are observed (the detection result is shown in FIG. 11).
Determining the distribution and number of goblet cells by PAS staining (see J Agr Food Chem,2019,67(48):13282-
Results of the alisin blue staining and PAS staining showed that both goblet cells (see fig. 9) and colon tissue mucus layer (see fig. 10) were destroyed in DSS-treated mice, whereas MY40C intervention had a clear protective effect on goblet cells and colon tissue mucus layer.
As can be seen from fig. 11, the concentration of MUC2 in DSS group mice was significantly reduced to 23.4pg/mg protein, while treatment with MY40C intervention group (MY40C + DSS) resulted in a significant increase in MUC2 content to 37.41pg/mg protein (see fig. 11 for the results of the test); moreover, the number of goblet cells in colon tissues of mice in the DSS group was reduced to 0.875 per crypt, and treatment with MY40C intervention group (MY40C + DSS) significantly reduced the loss of goblet cells, increasing the number of goblet cells to 8.25 per crypt (see fig. 12 for the test results). Therefore, the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C has a remarkable protective effect on colonic mucus layers of colitis mice.
Example 6: bifidobacterium pseudocatenulatum MY40C protective action against colonic mucosal barrier in colitis mice
The detailed implementation manner is the same as the steps (1) - (3) in the example 3;
after the modeling obtained in the step (3) in the example 3 is finished, the mouse is sacrificed, and the colon tissue is taken to detect biochemical indexes in the colon tissue supernatant according to a colon tissue biochemical index measuring method, wherein the biochemical indexes comprise the expression quantity of the tight junction protein alpha-catenin 1 (the detection result is shown in figure 13), the expression quantity of the tight junction protein beta-catenin (the detection result is shown in figure 14), the expression quantity of the tight junction protein E-Cadherin1 (the detection result is shown in figure 15), the expression quantity of the tight junction protein ZO-1 (the detection result is shown in figure 16), the expression quantity of the tight junction protein Occludin (the detection result is shown in figure 17), and the expression quantity of the tight junction protein claudin-3 (the detection result is shown in figure 18).
The distribution was measured by immunohistochemical method, and the microstructure of the tight junctions was measured by microtome and transmission electron microscopy (see FIGS. 19 and 20) (see J Agr Food Chem,2019,67(48): 13282-.
Immunohistochemical experiments also demonstrated that treatment with MY40C significantly increased the concentration of claudin-3, ZO-1, a tight junction protein.
In addition, as can be seen from fig. 20, the tight connection structure of the model-building mouse was broken, the tight connection, the adhesive connection, and the desmosomes were all broken, the pitch was increased, and the naps were broken. The colon ultrastructure of the MY40C trunk group (MY40C + DSS) mice showed similar to the blank group, tight junctions, adhesive junctions, dense desmosomes, and complete structure with neat and intact villi.
The Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C of the invention is proved to remarkably improve the tight junction structure of colon epithelial cells after intervention, so that the mucosal epithelial barrier is kept intact.
Example 7: effect of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C on apoptosis of colonic epithelial cells in colitis mice
The detailed implementation manner is the same as the steps (1) - (3) in the example 3;
after the modeling obtained in the step (3) in the example 3 is finished, the mouse is sacrificed, the colon tissue is taken to detect the biochemical index in the supernatant of the colon tissue according to the colon tissue biochemical index measuring method, the expression quantity of Caspase-3 protein of the colon tissue is detected (the detection result is shown in figure 22), and the epithelial cell apoptosis of different groups of colon tissues is observed (the detection result is shown in figure 21).
The colon tissue is subjected to apoptosis staining by using a fluorescent dye Hoechst 33258 (the specific method is referred to J Agr Food Chem,2019,67(48): 13282-.
The results show that: DSS treatment caused cell canonical morphological changes, nuclear fragmentation, chromosome condensation, and cell contraction (see figure 21 for test results); bifidobacterium pseudocatenulatum MY40C can significantly improve colonic cell apoptosis after dried.
As can be seen from FIG. 22, Caspase-3 is a key enzyme of apoptosis, the activity of Caspase-3 in colon tissue of mice in DSS group reaches 2.183U/mg protein, and the treatment of MY40C stem group (MY40C + DSS) can significantly reduce the activity of Caspase-3, which is only 1.495U/mg protein (the detection result is shown in FIG. 22). Thus, Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C intervention significantly reduced apoptosis of colonic epithelial cells leaving the mucosal epithelial barrier intact.
Example 8: bifidobacterium pseudocatenulatum MY40C regulating effect on colitis mouse immunity
The detailed implementation manner is the same as the steps (1) - (3) in the example 3;
after the model is made, the mouse obtained in the step (3) in the example 3 is sacrificed, and the colon tissue is taken to detect the biochemical indexes in the colon tissue supernatant according to the colon tissue biochemical index measuring method, wherein the biochemical indexes comprise the expression level of TNF-alpha in the colon tissue (the detection result is shown in figure 23), the expression level of IL-6 (the detection result is shown in figure 24), the expression level of IL-10 (the detection result is shown in figure 25), the expression level of PPAR-gamma (the detection result is shown in figure 26) and the expression level of serum IgE protein (the detection result is shown in figure 27).
As can be seen from FIGS. 23 to 25, DSS treatment increased the concentration of proinflammatory cytokines TNF- α and IL-6 in colon tissue to 9.144pg/mg protein and 1.741pg/mg protein, respectively, and reduced the concentration of the anti-inflammatory cytokine IL-10 to 4.187pg/mg protein. While Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C treatment can significantly reduce the concentrations of TNF-alpha and IL-6 to 7.891.33pg/mg protein and 1.33pg/mg protein and increase the concentration of IL-10 to 6.374pg/mg protein.
Furthermore, as is clear from fig. 26 to 27, the intervention of MY40C intervention group (MY40C + DSS) significantly increased PPAR- γ concentration compared to DSS treatment. In addition, Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C intervention increased serum immunoglobulin IgE concentration from 245.3pg/mL to 320.5 pg/mL. Therefore, the Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C significantly improved the intestinal immune response after intervention.
Example 9: bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C regulating TLR 4/NF-kB signaling pathway in colitis mice
The detailed implementation manner is the same as the steps (1) - (3) in the example 3;
killing the mouse after the modeling obtained in the step (3) in the embodiment 3 is finished, taking 1cm of distal colon tissue for fixing, dehydrating and embedding, and carrying out immunohistochemical staining on NF-kB signal channel key proteins TLR4, p65, p-p65, I kB and p-I kB; and (4) analyzing the average optical density of the protein (the detection result is shown in figure 28).
The results of immunohistochemistry show that the mean optical density of TRL4, p-p65 and p-IkB proteins of colon tissues of mice in the DSS-based model group is remarkably increased compared with that of mice in the blank group, and the mean optical density of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) MY40C of the intervening mice in the group is remarkably lower than that of the TRL4, p-p65 and p-IkB proteins in the DSS group and is close to that of the blank group (the detection results are shown in a graph from 28 to 29). Therefore, after intervention of Bifidobacterium pseudocatenulatum MY40C, the TLR 4/NF-kB signal pathway of the intestinal tract is obviously inhibited, and the intestinal tract immune response is reduced.
Example 10: preparation of Bifidobacterium pseudocatenulatum MY40C lyophilized preparation
The method comprises the following specific steps:
(1) activation of the strain: dipping a bacterial liquid of Bifidobacterium pseudocatenulatum (MY 40C) from a glycerol tube, streaking on an MRS solid culture medium, and culturing at 37 ℃ for 48h in an anaerobic environment to obtain a single bacterial colony; and selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 48h at 37 ℃ in an anaerobic environment for activation culture, and repeating the operation for 3 times to obtain activated bacteria liquid.
(2) Inoculating the bacterial liquid obtained in the step (1) into an MRS liquid culture medium according to the inoculation amount of 3%, carrying out anaerobic culture at 37 ℃ for 28h to obtain a fermentation liquid, centrifuging the obtained fermentation liquid at 10000rpm for 20min, collecting bacterial sludge, washing the bacterial sludge for later use by using normal saline for 3 times, and adjusting the viable count to be 1 multiplied by 1011CFU/mL。
(3) Preparing a freeze-drying protective agent: 100g/L of skim milk powder, 100g/L of trehalose, 160g/L of sucrose and the balance of water are mixed to obtain the freeze-drying protective agent.
(4) Adding the prepared freeze-drying protective agent into the bacterial sludge obtained in the step (2), wherein the weight of the freeze-drying protective agent is 3 times of that of the bacterial sludge, uniformly mixing, performing vacuum freeze-drying, and finally performing vacuum packaging on the preparation obtained by freeze-drying.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> bifidobacterium pseudocatenulatum capable of relieving colitis and application thereof
<130> BAA200701A
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1037
<212> DNA
<213> Bifidobacterium pseudocatenulatum
<400> 1
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aaacgggtgg taatgccgga tgctccagtt gatcgcatgg tcttctggga aagctttcgc 180
ggtatgggat ggggtcgcgt cctatcagct tgacggcggg gtaacggccc accgtggctt 240
cgacgggtag ccggcctgag agggcgaccg gccacattgg gactgagata cggcccagac 300
tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga tgcagcgacg 360
ccgcgtgagg gatggaggcc ttcgggttgt aaacctcttt tatcggggag caagcgagag 420
tgagtttacc cgttgaataa gcaccggcta actacgtgcc agcagccgcg gtaatacgta 480
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cggtgtgaaa gtccatcgct taacggtgga tccgcgccgg gtacgggcgg gcttgagtgc 600
ggtaggggag actggaattc ccggtgtaac ggtggaatgt gtagatatcg ggaagaacac 660
caatggcgaa ggcaggtctc tgggccgtta ctgacgctga ggagcgaaag cgtggggagc 720
gaacaggatt agataccctg gtagtccacg ccgtaaacgg tggatgctgg atgtggggcc 780
cgttccacgg gttccgtgtc ggagctaacg cgttaagcat cccgcctggg gagtacggcc 840
gcaaggctaa aactcaaaga aattgacggg ggcccgcaca agcggcggag catggggatt 900
aattcgatgc aacgcgaaga accttactgg gctgacatgt cccgacggtc gtaaagatac 960
ggcttccctc ggggcgggtt cacaggtggt gcatgtcgtc gtcagcctcg tgtcggagaa 1020
tgttgggtta gtcgcac 1037
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
tacggctacc ttgttacgac tt 22
Claims (9)
1. The application of Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) CCFM1115 in preparing products for preventing and/or treating colitis is characterized in that the Bifidobacterium pseudocatenulatum is preserved in Guangdong province microorganism strain preservation center with the preservation number of GDMCC No:60954, and the preservation date is 2020, 07, 16 days.
2. Use of Bifidobacterium pseudocatenulatum in the manufacture of a product for the prevention and/or treatment of colitis according to claim 1, characterized in that the viable count of Bifidobacterium pseudocatenulatum is not less than 1X 1010CFU/g。
3. Use of bifidobacterium pseudocatenulatum for the preparation of a product for the prevention and/or treatment of colitis according to claim 2, characterized in that said product is a food product, a pharmaceutical product or a nutraceutical product.
4. Use of Bifidobacterium pseudocatenulatum for the preparation of a product for the prevention and/or treatment of colitis according to claim 3, characterized in that said medicament is a lyophilized powder.
5. The use of Bifidobacterium pseudocatenulatum for the preparation of a product for the prevention and/or treatment of colitis according to claim 4, wherein the lyophilized powder is prepared by a method comprising: inoculating the bifidobacterium pseudocatenulatum of claim 1 into a culture medium for culture to obtain a seed solution; inoculating the seed solution into a culture medium for culture to obtain a culture solution; centrifuging the culture solution, and collecting bacterial sludge; washing the bacterial sludge with normal saline, and then resuspending to obtain a resuspension solution; adding a freeze-drying protective agent into the heavy suspension to obtain a mixed solution; and (4) carrying out vacuum freeze drying on the mixed solution to obtain freeze-dried powder.
6. The use of Bifidobacterium pseudocatenulatum for the preparation of a product for the prevention and/or treatment of colitis as claimed in claim 5, wherein the constituents of said lyoprotectant include skim milk powder 80-120g/L, trehalose 80-140g/L, sucrose 140-180g/L and water.
7. Use of bifidobacterium pseudocatenulatum for preparing a product for preventing and/or treating colitis according to claim 5 or 6, wherein the addition amount of the lyoprotectant in the resuspension is 2 to 4 times of the total weight of the bacterial sludge.
8. A product for use in the prevention and/or treatment of colitis, characterized in that it contains bifidobacterium pseudocatenulatum according to claim 1.
9. The product according to claim 8, wherein the viable count of Bifidobacterium pseudocatenulatum in the product is not less than 1 x 1010CFU/g。
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CN112592865B (en) * | 2020-12-31 | 2022-04-29 | 江南大学 | Rumen lactobacillus capable of protecting intestinal barrier and application thereof |
CN113897302B (en) * | 2021-08-06 | 2022-08-09 | 东北农业大学 | Bifidobacterium capable of relieving colitis and application thereof |
CN114854639B (en) * | 2022-05-24 | 2023-08-08 | 江南大学 | Bifidobacterium pseudolongum capable of high-yield inosine and relieving ulcerative colitis and application thereof |
CN117363543B (en) * | 2023-10-30 | 2024-04-23 | 中国农业大学 | Exopolysaccharide pseudocatenin bifidobacterium pseudocatenin with enteritis relieving function and application thereof |
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