CN115838662A - Bifidobacterium bifidum with potential of relieving Crohn's disease - Google Patents

Abstract

The invention discloses a bifidobacterium bifidum with potential of relieving Crohn's disease, belonging to the technical field of microorganisms and medicines. The invention provides a bifidobacterium bifidum CCFM1284, which is preserved in Guangdong province microorganism strain preservation center at 10-14 months in 2022, wherein the preservation number is GDMCC No:62886. the bifidobacterium bifidum CCFM1284 provided by the invention has anti-inflammatory property; the bifidobacterium bifidum CCFM1284 can relieve macroscopic disease characterization symptoms of a TNBS-induced Crohn's Disease (CD) mouse; reducing pathological damage to colon, inhibiting fibrotic deposition in colon, inhibiting intestinal inflammation, and enhancing intestinal barrier in Crohn's Disease (CD) mice; the aim of effectively preventing and treating the mouse Crohn disease induced by TNBS is achieved.

Description

Bifidobacterium bifidum with potential of relieving Crohn's disease

Technical Field

The invention relates to bifidobacterium bifidum with potential of relieving Crohn's disease, belonging to the technical field of microorganisms and medicines.

Background

Crohn's Disease (CD) is characterized by chronic recurrent intestinal inflammation with a markedly increasing incidence worldwide, particularly in emerging economies. The pathogenic factors are very complex, including gene mutation, poor diet, intestinal flora imbalance, hyperfimmunity and the like. The disease can also lead to severe complications such as strictures, fistulas and even tumors. Although aminosalicylic acids, steroids or immunosuppressive agents and biologicals (such as infliximab and vedolizumab) are widely used to treat patients, these measures do not completely prevent CD or its postoperative recurrence. Currently, these therapies are often expensive and also cause adverse reactions such as infections, liver and bone toxicity (disclosed in Cushing et al 2021,325 (1): 69-80.). Therefore, early effective inflammation management and prevention is of paramount importance.

In view of the limitations of conventional treatment methods and the various problems involved, it is necessary to find a new intervention or treatment for crohn's disease. At present, probiotics gradually enter the field of the public along with the development of intestinal microecology science, the price is relatively low, the side effect is less, the probiotics are easily accepted by the general public, and the research of CD resistant strains and the development of related preparations become the current research hotspots. For example, the published Bacteroides cellulolyticus DSM 14838T, bacillus amyloliquefaciens TITI, bacillus subtilis, bifidobacterium longum DM21062 and the like can prevent CD or relieve enteritis symptoms; probiotic combination formulation VSL #3 has anti-inflammatory effects and reduces endoscopic recurrence after crohn's disease surgery (disclosed in Fedorak et al clinical surgery and Hepatology 2015,13 (5): 928-935.e 2.); probiotic and prebiotic combination therapy is safe and effective for use in alleviating the clinical symptoms of active CD patients (disclosed in Fujimmori et al journal of gastroenterology and hepatology,2007,22 (8): 1199-1204.). Probiotics are expected to benefit more and more CD patients.

Bifidobacterium bifidum is also a probiotic which is widely concerned, and has stronger adaptability to gastric acid and bile salt of a human body and good utilization capacity for host source mucin, so that the Bifidobacterium bifidum can better play a beneficial role in the using process of the Bifidobacterium bifidum as a probiotic. A great deal of evidence shows that bifidobacterium bifidum or inactivated thalli thereof relieve a plurality of gastrointestinal tract diseases and achieve the effect of improving the gastrointestinal tract health of a host. Bifidobacterium bifidum CGMCC NO.13632 can obviously improve the pathological indexes of constipation of mice and has the effects of preventing and treating constipation (disclosed in CN 106834187B); bifidobacterium bifidum GDMCC No.60255 significantly reduced the inflammatory response at the colorectal cancer site and decreased the number of tumors in colorectal cancer model mice (published in CN 107629988A). Bifidobacterium bifidum BGN4 inhibits oxidative stress, alleviates epithelial barrier damage and exerts disease symptom relief in ulcerative colitis mice (disclosed in Lee et al. Journal of medical food,2022,25 (2): 146-157.). IN addition, bifidobacterium bifidum MIMBb75 relieves pain, bloating, urgency, digestive disturbances IN humans IN a variety of gastrointestinal disease states (disclosed IN AU2012213576B2; IN2013DN06748A; CN103476273A; andrescen V, gschossmann J, layer P. The Lancet Gastroenterology & hepatology,2020,5 (7): 658-666.); non-viable Bifidobacterium bifidum DSM 24514 significantly improved gastrointestinal symptoms in IBS patients (disclosed in CN 112312920A).

Probiotics have strain specificity in disease alleviation, and strains with disease alleviation effects must be effectively screened. TNBS-induced chronic colitis is characterized by Th1 and Th17 type inflammatory responses, and the pathological manifestations of intestinal tissue are thickening of the intestinal wall and fibrosis (disclosed in Nature protocols,2017,12 (7): 1295-1309.). Compared to the acute model, it resembles the clinical response of human crohn's disease. Chronic colitis of the TNBS type has become a major animal model for screening drugs for the treatment of crohn's disease. In addition, the development of probiotics capable of treating Crohn's disease is very limited, so that the patent has wide market application prospect.

Disclosure of Invention

The invention provides a Bifidobacterium bifidum (Bifidobacterium bifidum) CCFM1284, which is preserved in Guangdong province microorganism strain preservation center 10 and 14 months 2022 with the preservation number of GDMCC No:62886.

the bifidobacterium bifidum CCFM1284 is used for collecting a fecal sample of a healthy male in Wuxi city of Jiangsu province, extracting the genome of the screened bifidobacterium, amplifying and sequencing the 16S rDNA (finished by Shanghai biological engineering Co., ltd.), and comparing the sequence in GenBank to show that the strain is bifidobacterium bifidum CCFM1284 as the 16S rDNA sequence of the strain is shown in SEQ ID No. 1.

The thallus characteristics of the bifidobacterium bifidum CCFM1284 are as follows: gram-positive rod-shaped bacteria rarely appear in a bifurcated or bent rod-shaped form and do not form spores. Colony characteristics: the bacterial colony on the MRSc solid culture medium is porcelain white, round and convex, moist and neat and smooth in edge. Growth characteristics: the strain is strict anaerobe, is sensitive to oxygen and grows optimally at 37 ℃. The bifidobacterium bifidum CCFM1284 has an immunoregulation effect in an MLN cell model and can induce the mass production of IL-10.

The invention provides a microbial agent which contains the bifidobacterium bifidum CCFM1284 or fermentation liquor thereof, or freeze-dried powder or lysate containing the bifidobacterium bifidum CCFM1284.

In an embodiment of the invention, the freeze-dried powder is powder prepared by preparing a bacterial solution containing the bifidobacterium bifidum CCFM1284 by a conventional freeze-drying process or other processes.

In one embodiment of the present invention, in the microbial agent, the viable count of bifidobacterium bifidum CCFM1284 is not less than 10 ⁹ CFU/g or 10 ⁹ CFU/mL。

The invention also provides a product which contains the bifidobacterium bifidum CCFM1284 or contains the microbial agent.

In one embodiment of the invention, the product is a food, pharmaceutical or nutraceutical product.

In one embodiment of the present invention, the food may be a beverage, yogurt, jelly, probiotic powder, or the like.

In one embodiment of the present invention, the pharmaceutical product contains bifidobacterium bifidum CCFM1284, and pharmaceutically acceptable carrier and/or pharmaceutical excipients.

In one embodiment of the invention, the viable count of bifidobacterium bifidum CCFM1284 in the product is at least: 10 ⁹ CFU/g。

In one embodiment of the present invention, the pharmaceutically acceptable carrier is one or more carriers selected from the group consisting of fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents, which are generally used pharmaceutically. The selected carrier can meet the survival requirement of the bifidobacterium, and is particularly according to the actual requirement.

In one embodiment of the present invention, the pharmaceutical preparation is in the form of granules, capsules, tablets, pills, oral liquids, or the like.

In one embodiment of the invention, the bulking agent is understood to be an excipient diluent to increase the weight and volume of the tablet to facilitate tableting, or an excipient absorbent to absorb excess liquid components of the raw material.

In one embodiment of the invention, the filler is selected from starch, sucrose, lactose, calcium sulfate or microcrystalline cellulose.

In one embodiment of the invention, the binder is understood to mean that the starting drug is not sticky or not sufficiently sticky in nature, and a sticky substance is added to facilitate granulation. The binder is selected from cellulose derivatives, alginates, gelatin or polyvinylpyrrolidone.

In one embodiment of the invention, the wetting agent is understood to be a liquid which is not sticky to the drug substance itself, but which wets its pharmaceutical excipients and induces its stickiness to form granules. The wetting agent is selected from water, ethanol, starch or syrup.

In one embodiment of the invention, the disintegrant is understood to be an excipient capable of being incorporated into a tablet to facilitate rapid disintegration of the tablet into fine particles in gastrointestinal fluids. The disintegrating agent is selected from sodium carboxymethyl starch, carboxypropylcellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate or sodium bicarbonate.

In one embodiment of the invention, the lubricant is understood to be a chemical substance that facilitates the flow of the tablet during granulation and facilitates the de-molding of the tablet. The lubricant is selected from talcum powder, calcium stearate, magnesium stearate, aerosil or polyethylene glycol.

In one embodiment of the invention, the flavoring agent is understood to be a pharmaceutical excipient used in pharmaceuticals to improve or mask the unpleasant taste and odor of the drug. The flavoring agent is selected from sweetener such as simple syrup, sucrose, lecithin, orange peel syrup or cherry syrup; a fragrance of lemon, anise or peppermint oil; mucilages of sodium alginate, acacia, gelatin, methylcellulose, or sodium carboxymethylcellulose; effervescent agents of citric acid, tartaric acid or sodium bicarbonate mixtures.

The invention provides application of the bifidobacterium bifidum CCFM1284 or the microbial agent in preparing products for preventing and/or relieving chronic colon injury or chronic Crohn's disease.

In one embodiment of the invention, the product is a food, pharmaceutical or nutraceutical product.

In one embodiment of the present invention, the food may be a beverage, yogurt, jelly, probiotic powder, or the like.

In one embodiment of the invention, the medicament contains the bifidobacterium bifidum CCFM1284, a pharmaceutically acceptable carrier and/or a pharmaceutic adjuvant.

In one embodiment of the present invention, the pharmaceutically acceptable carrier is one or more carriers selected from the group consisting of fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents, which are generally used pharmaceutically. The selected carrier can meet the survival requirement of the bifidobacterium, and is particularly according to the actual requirement.

In one embodiment of the present invention, the pharmaceutical preparation is in the form of granules, capsules, tablets, pills, oral liquids, or the like.

In one embodiment of the invention, the viable count of bifidobacterium bifidum CCFM1284 in the product is at least: 10 ⁹ CFU/g。

The invention also provides application of the bifidobacterium bifidum CCFM1284 or the microbial agent in preparing products for preventing and/or relieving TNBS induced inflammation and reducing pathological damage of colon.

In one embodiment of the invention, the product is a food, pharmaceutical or nutraceutical product.

In one embodiment of the invention, the medicament contains the bifidobacterium bifidum CCFM1284, a pharmaceutically acceptable carrier and/or a pharmaceutic adjuvant.

In one embodiment of the invention, the viable count of bifidobacterium bifidum CCFM1284 in the product is at least: 10 ⁹ CFU/g。

In one embodiment of the present invention, the food may be a beverage, yogurt, jelly, probiotic powder, or the like.

In one embodiment of the present invention, the pharmaceutically acceptable carrier is one or more carriers selected from the group consisting of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents, which are generally used pharmaceutically. The selected carrier can meet the survival requirement of the bifidobacterium, and is particularly according to the actual requirement.

In one embodiment of the present invention, the pharmaceutical preparation is in the form of granules, capsules, tablets, pills, oral liquids, or the like.

The invention also provides a preparation method of the microbial agent, which comprises the following steps:

(1) Inoculating bifidobacterium bifidum CCFM1284 into MRS liquid culture medium containing 0.5% cysteine, carrying out anaerobic culture at 37 ℃, and harvesting the thallus reaching the stationary phase;

(2) Centrifuging to collect thalli, and washing 3 times by using sterile PBS;

(3) Adjusting the cell concentration to 5X 10 with sterile PBS ⁹ cfu/mL；

Advantageous effects

The bifidobacterium bifidum CCFM1284 provided by the invention has the following properties:

1. in an in vitro Mesenteric Lymph Node (MLN) co-culture model, the bifidobacterium bifidum CCFM1284 has unique anti-inflammatory properties;

2. since Crohn's Disease (CD) is a chronic disease, in order to get closer to the disease, the present invention obtains a Crohn's Disease (CD) model by first sensitizing with TNBS induction and then clystering with TNBS solution, and the symptoms of Crohn's Disease (CD) can be alleviated with bifidobacterium bifidum CCFM1284 of the present invention as follows:

(1) The bifidobacterium bifidum CCFM1284 is capable of alleviating the macro disease characterization symptoms of TNBS-induced Crohn's Disease (CD) mice;

(3) The bifidobacterium bifidum CCFM1284 reduces pathological damage to the colon of a Crohn's Disease (CD) mouse and inhibits fibrotic deposition on the colon;

(4) The bifidobacterium bifidum CCFM1284 reduces the expression of inflammatory factors of intestinal tracts of mice with Crohn's Disease (CD) and inhibits the intestinal tract inflammation;

(5) Promoting the expression of colon tight junction protein and mucin in Crohn's Disease (CD) mice, thereby enhancing intestinal barrier;

(6) The bifidobacterium bifidum CCFM1284 promotes the proliferation of Treg cells in the MLN of a mouse with Crohn's Disease (CD), thereby achieving the aim of effectively preventing and treating the mouse with the Crohn's disease induced by TNBS.

In conclusion, the bifidobacterium bifidum CCFM1284 provided by the invention can effectively relieve TNBS-induced Crohn's Disease (CD).

Biological material preservation

A Bifidobacterium bifidum (CCFM 1284) is classified and named as Bifidobacterium bifidum, is preserved in Guangdong province microorganism strain preservation center 10.14.2022, and has the preservation number of GDMCC No. 62886, and the preservation address of Michelia furiosa No. 100, dazhou No. 59, lou 5, guangdong province scientific microbiological research institute.

Drawings

FIG. 1: schematic diagram of animal model modeling.

FIG. 2: effect of different bifidobacterium bifidum on weight change in CD mice.

FIG. 3: effect of different bifidobacterium bifidum on survival of CD mice.

FIG. 4: effect of different bifidobacterium bifidum on colon length in CD mice.

FIG. 5: effect of different bifidobacterium bifidum on colon histopathology in CD mice.

FIG. 6: effect of different bifidobacterium bifidum on colonic tissue fibrosis in CD mice.

FIG. 7: effect of different bifidobacterium bifidum on colonic claudin tight in CD mice.

FIG. 8: effect of different bifidobacterium bifidum on colonic tissue inflammatory factors in CD mice.

FIG. 9: effect of different bifidobacterium bifidum on CD mouse mesenteric lymph node regulatory T cells.

Detailed Description

The bifidobacterium bifidum FXJWS17M4, FHeNJZ3M6 and enterococcus faecalis FJSWX25M1 referred to in the following examples were deposited in the industrial microbial resources and information center of colleges and universities in china, university, south of the river.

The media involved in the following examples are as follows:

bifidobacterium specific screening medium (g/L): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 1mL tween 80, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, distilled water: 1000mL; pH:6.2-6.4; sterilizing at 115 deg.C for 20 min. When preparing a solid culture medium, 15g of agar is added, and sterile mupirocin and sterile nystatin are added according to the volume of 1 per thousand and 0.5 per thousand of the culture medium respectively before pouring the plate, namely the final concentrations of the mupirocin and the nystatin are respectively 100 mu g/mL and 25U/mL.

Preparing a cysteine-containing MRS culture medium: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 5g/L of anhydrous sodium acetate, 2g/L of citric acid hydrogen diamine, 2g/L of dipotassium phosphate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 1mL/L of tween-80, 1% of cysteine, pH 6.4, 115 ℃ and 20-minute sterilization.

Example 1: screening and identification of bifidobacterium bifidum CCFM1284

1. Sample collection

Collecting feces samples of healthy men in Wuxi city, jiangsu province, placing in a sample collecting tube filled with 30% glycerol, storing in a heat preservation box filled with an ice bag, taking back to a laboratory, and rapidly placing in a refrigerator at-80 ℃ for separation and screening.

2. Separation and purification of bacterial strains

(1) Dilution coating: taking about 0.5g of the fecal sample stored in the 30% glycerin tube, and mixing with 4.5mL of normal saline in a 10mL centrifuge tube under the aseptic condition to obtain 10 ^-1 Diluting the solution, repeating the above operation to obtain 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Diluting the solution;

(2) Coating culture: sucking 10 μ L of the solution obtained in step (1) ^-4 、10 ^-5 、10 ^-6 Uniformly coating three gradient diluents on a bifidobacterium specificity screening culture medium by using a coating rod, and culturing for 48 hours at 37 ℃ under an anaerobic condition to obtain a diluted coating plate;

(3) And (3) purification and culture: and (3) taking a diluted coating plate with the colony number within a range of 30-300, randomly selecting 10 ceramic white colonies from each sample, marking the colonies with smooth and wet surfaces and regular edges on an improved MRS solid culture medium, and then culturing for 48 hours under an anaerobic condition at 37 ℃ to obtain single colonies. And then inoculating the single colony in an improved MRS liquid culture medium, and culturing for 24h under the anaerobic condition of 37 ℃ to obtain the purified culture solution.

3. Strain preservation and identification

Extracting the genome of the screened bifidobacterium, amplifying and sequencing the 16S rDNA (finished by Shanghai biological engineering Co., ltd.), comparing the sequence in GenBank after sequencing analysis, wherein the sequence of the 16S rDNA of the strain is shown as SEQ ID NO.1, and the result shows that the strain is bifidobacterium bifidum which is named as bifidobacterium bifidum CCFM1284.

Example 2: preparation of Bifidobacterium bifidum microbial inoculum for animal experiments.

The method comprises the following specific steps:

(1) Activation of bifidobacterium bifidum CCFM 1284: sucking 10 μ L of Bifidobacterium bifidum suspension preserved in glycerol, coating on MRS solid culture medium containing 0.1% cysteine, and performing anaerobic culture at 37 deg.C for 48 hr;

(2) And (3) selecting an activated CCFM1284 single colony, inoculating the single colony into 10mL of liquid MRS medium containing cysteine, and carrying out anaerobic culture at 37 ℃ for 48h to obtain a seed solution.

Centrifuging the seed solution of Bifidobacterium bifidum CCFM1284 at 7000g for 10min, collecting fresh thallus, washing with sterile PBS for 3 times, and adjusting thallus concentration to 5 × 10 with sterile PBS ⁹ cfu/mL, and preparing the bifidobacterium bifidum CCFM1284 microbial inoculum.

The bifidobacterium bifidum FXJWS17M4 microbial agent and the bifidobacterium bifidum FHENJZ3M6 microbial agent are prepared according to the method.

Example 3: in vitro immunoregulatory capacity of bifidobacterium bifidum CCFM1284

The method comprises the following specific steps:

(1) MLN (mesenteric lymph node) was taken out from SPF-grade 6-8 week-old male C57BL/6 mice, ground and filtered, and then RPIM1640 complete medium containing fetal bovine serum (10%), gentamicin (1%) and diabody (1%) was added, and the cells were counted using a hemocytometer, and the concentration of MLN cells (mesenteric lymphocytes) was adjusted to about 1X 10 ⁶ Per mL, preparation of an intestinal lineMembrane lymphocyte bacterial suspension;

(2) Preparation of bacterial suspension: respectively washing bifidobacterium and enterococcus faecalis thallus by sterile PBS for 3 times, and then adjusting the thallus concentration to OD 600nm=0.5 by sterile PBS, so as to respectively prepare bifidobacterium suspension and enterococcus faecalis suspension;

(3) Supplementing HEPES (10 mmol/L), beta-mercaptoethanol (0.05 mmol/L), L-glutamine (3 mmol/L), anti-CD3 antibody (1 mmol/L) and anti-CD28 antibody (1 mmol/L) to the mesenteric lymphocyte bacterial suspension obtained in the step (1), fully mixing uniformly, and inoculating MLN cells to a 96-well plate according to 200 mu L per well;

and respectively adding the prepared bifidobacterium suspension and the prepared enterococcus faecalis suspension into each hole according to the volume of 20 mu L (MLN cell number: thallus cell number =1 10), uniformly mixing, putting into a carbon dioxide constant-temperature incubator at 37 ℃, and culturing for 72h.

Co-culture of PBS and MLN single cell suspensions was used as a blank.

After the culture is finished, centrifuging for 5min at 4 ℃ and 12000R/min, collecting supernatant, and using the supernatant for measuring the contents of the cytokines IL-10, IL-17 and IL-12, wherein the specific measuring method refers to the specification of an R & D mouse cytokine ELISA detection kit, and the results are shown in Table 1.

Table 1: comparison of immunoregulatory capacities of different Bifidobacterium bifidum in MLN coculture model

As can be seen from Table 1, all Bifidobacterium bifidum can induce a certain amount of IL-10 compared with enterococcus faecalis FJSWX25M1, wherein the inducing ability of Bifidobacterium bifidum CCFM1284 is strongest; there was no significant difference in the IL-12 inducing ability of the three strains.

Example 4: establishment of chronic Crohn's disease mouse model by chronic TNBS and intervention experiment design

1. The method comprises the following specific steps:

(1) 50 male BALB/c mice at 6-8 weeks were randomly divided into 5 groups (blank group, building group and three probiotic intervention groups: bifidobacterium bifidum FXJWS17M4 group, bifidobacterium bifidum FHENJZ3M6 group, bifidobacterium bifidum CCFM1284 group), 10 mice per group.

After all mice were fasted for 24h, they were placed one by one in an isoflurane respiratory anesthesia machine, after they were fully anesthetized, the backs (1.5 × 1.5 cm) of the mice were shaved with a shaver, and 150 μ L of a pre-sensitization solution was applied for pre-sensitization. Preparing a pre-sensitive solution: acetone and olive oil were mixed at a volume ratio of 4:1, and a 5% TNBS aqueous solution was diluted to 1% by mass. The blank control group was smeared with the corresponding solution without TNBS.

(2) One week later, a plastic hose was connected using a 1mL sterile syringe for rectal infusion of TNBS solution (50 μ L/mouse), and after completion of the infusion, the mice were quickly inverted for 1-2 min to prevent outflow of the drug solution, and then the mice were returned to the cage.

As shown in figure 1, TNBS infusion was performed once a week for 4 weeks with sequentially increasing TNBS concentration (i.e., intragastric concentration 1% for the first week, intragastric concentration 1.5% for the second week, intragastric concentration 2% for the third week, and intragastric concentration 2.5% for the fourth week); 3-4 cm of rectum, and the perfusion solution of the experimental group and the perfusion solution of the blank control group are respectively 35 percent ethanol water solution and 35 percent ethanol water solution containing TNBS.

The bifidobacterium bifidum bacterial agent (200 mu L/day) prepared in the gastric lavage example 2 is started one week ahead, the whole experiment period is continued, and the concentration of the bacterial agent is 5 multiplied by 10 ⁹ CFU/mL。

(3) After the last molding, observing the condition of the mouse every day, weighing, measuring occult blood, and recording disease activity index; euthanasia was performed after 3 days;

taking the colon of each group of mice, and measuring the length of the colon; selecting a distal colon of 1cm, and storing the colon until 4% paraformaldehyde fixing solution (used for pathological sections) is obtained; selecting 0.5cm colon, and preserving in Trizol protective solution (for extracting RNA); the remaining colon tissue was preserved in PBS (for cytokine determination); lymph nodes were taken and stored in RPMI 1640 cell culture medium (for Treg cell assay).

All experimental animal mice, purchased from Peking Wintonlifa laboratory animals Co., ltd, were raised in an SPF-grade barrier environment at an ambient temperature of 23. + -. 2 ℃ and a relative humidity of 50. + -. 10%.

2. The mice in each group after the experiment was finished were detected: body weight change, colon length, survival rate, inflammatory factors, treg cells, pathological section making and Masson staining, and the experimental results are as follows:

(1) As can be seen from fig. 2, the weights of the blank mice were: 23.60g, model group mice weight: 19.90g, the weight is reduced by 16 percent, and the weight of mice in the Bifidobacterium bifidum FXJWS17M4 group, the Bifidobacterium bifidum FHENJZ3M6 group and the Bifidobacterium bifidum CCFM1284 group is reduced by 6.8 percent, 1.8 percent and 0.8 percent respectively.

It can be seen that after chronic TNBS modeling, the body weight of the mice is significantly reduced, and the weight reduction is significantly recovered due to bifidobacterium CCFM1284 intervention. Whereas bifidobacterium FXJWS17M4 intervention was not significantly restored.

(2) As can be seen from fig. 3, chronic TNBS modeling resulted in 50% mortality in mice, 40%, and 30% mortality in FXJWS17M4, FHeNJZ3M6, and CCFM1284 groups, respectively. It can be seen that CCFM1284 intervention significantly reduced mortality compared to other bifidobacteria.

(3) As can be seen from fig. 4, in the blank group of mice, the colon length was 11.11cm, in the model group 8cm, chronic TNBS modeling resulted in a significant reduction in the colon length in the mice, and significant recovery of the colon length was obtained after intervention with bifidobacterium CCFM1284 and FHeNJZ3M6 (10.39 and 9.77cm, respectively). Whereas bifidobacterium FXJWS17M4 intervention was not significantly restored (8.98 cm).

(4) As can be seen from FIG. 5, after chronic TNBS modeling, most intestinal glands in the colon of the mice disappear and are replaced by hyperplastic connective tissue, the number of goblet cells is obviously reduced, and a large amount of lymphocyte infiltration (blue arrows) is carried out around the goblet cells; the connective tissue of the local lamina propria was slightly hyperplastic, proliferating to the muscular layer (black arrow), the muscular layer was locally ruptured (orange arrow), and a small amount of infiltration of granulocytes was seen in the mucosal layer, submucosa, muscular layer and serosal layer (green arrow).

Compared with the model group mice, the CCFM1284 group mice have intact intestinal mucosal epithelium; only a small amount of granulocyte infiltration can be seen in the local lamina propria, and the pathological condition is obviously improved.

Local intestinal glands in the lamina propria of mice in the FXJWS17M4 group disappeared and were replaced by hyperplastic connective tissue, the number of goblet cells was slightly reduced, more lymphocytes were focally infiltrated (blue arrows), and the arrangement of peripheral intestinal glands was irregular. The local connective tissue of the indigenous layer of the mice in the FHENJZ3M6 group is slightly proliferated with a small amount of granulocyte infiltration (blue arrow), the arrangement of the surrounding intestinal glands is irregular, and individual granulocytes infiltrate into the submucosa (green arrow); the lamina propria is occasionally slightly dilated in the intestinal gland and the epithelium is flattened.

(5) As can be seen from the results of Masson staining in FIG. 6, no collagen infiltration is observed in the colon tissue of the mice in the blank group, after the model is made by the chronic TNBS, the collagen infiltration degree of the intestinal tissue is deeper, the intestinal tissue morphology is abnormal, villous edema can be observed, the intestinal wall is thickened, and the symptoms are similar to the pathological features of the intestinal tract of the CD patients.

Collagen infiltration was reduced in the intervention groups: the tissue collagen infiltration degree is reduced most remarkably after CCFM1284 intervention, which shows that the microbial inoculum has the potential of relieving Crohn disease most.

(6) As can be seen from FIG. 7, after chronic TNBS modeling, the relative expression levels of the colon Mucin Mucin-2, claudin ZO-1 and Claudin-1 genes are significantly reduced (0.19, 0.55 and 0.29 respectively), and the expression levels of the CCFM1284 group are significantly increased (1.27, 0.98 and 0.64 respectively) after probiotic dry-prognosis. It can be seen that bifidobacterium bifidum CCFM1284 reduces the damage of TNBS to the physical barrier of the mouse intestinal tract.

(7) As can be seen from FIG. 8, the IL-17 content in the colon homogenate of the blank group was 1825pg/mL, the modeling group was 3942pg/mL, and the respective intervention groups of FXJWS17M4, FHENJZ3M6 and CCFM1284 were 2207pg/mL, 1177pg/mL and 1920pg/mL; the content of IL-17 in ileum homogenate of the blank group is 1294pg/mL, the model group is 2039pg/mL, and the dry pre-groups of FXJWS17M4, FHENJZ3M6 and CCFM1284 are 1650pg/mL, 1859pg/mL and 1044pg/mL respectively.

Therefore, after the chronic TNBS modeling, the colon and ileum inflammatory factors are obviously increased, the content of the inflammatory factors is reduced after probiotic intervention, and the CCFM1284 has the best intervention effect.

(8) As can be seen from fig. 9, the content of mesenteric lymph node regulatory T cells (tregs) in the mice in the blank group was 15%, tregs were significantly reduced (7.67%) after chronic TNBS modeling, and after intervention, the Treg ratios in mesenteric lymph nodes in the FXJWS17M4 group, FHeNJZ3M6 group, and CCFM1284 group were all improved to some extent, and were 12.7%, 14.9%, and 17.9%, respectively, and only the intervention effect of CCFM1284 was significant. Therefore, compared with other two strains of bacteria, the microbial inoculum has the potential of controlling inflammation and relieving Crohn's disease.

Example 5: preparation of tablets containing the Microecological preparation of the invention

Respectively weighing 25.7 parts by weight of bifidobacterium CCFM1284 powder preparation prepared by a freeze drying method, 55.0 parts by weight of yeast beta-glucan, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 part by weight of talcum powder, 1.0 part by weight of cane sugar and 1.0 part by weight of water, mixing, preparing wet granules by a conventional method, tabletting by a tabletting machine produced by the pharmaceutical machinery factory in the south-middle province, drying by a small-sized medicine drying machine produced by Yikang traditional Chinese medicine machinery limited company in the city of Qingzhou, and packaging to obtain the tablet.

Example 6: preparation of Capsule products containing the Microecological preparation of the invention

Culturing the bifidobacterium CCFM1284 in an improved MRS culture medium at 37 ℃ for 24 hours in an anaerobic way, centrifuging for 15 minutes at 4 ℃ under the condition of 5000rpm, washing for 1-2 times by using a sterile phosphate buffer solution (pH 7.4), and resuspending the thalli by using a protective agent to ensure that the final concentration of the thalli reaches 10 ¹⁰ CFU/mL. Freeze drying for 48 hr to obtain powder containing Bacillus bifidus CCFM1284, and making into commercially available medicinal capsule.

The components of the protective agent comprise: 100g/L skimmed milk powder, 30mL/L glycerin, 100g/L maltodextrin, 150g/L trehalose and 10g/L sodium glutamate.

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.

Claims (10)

1. Bifidobacterium bifidum (CCFM 1284) is preserved in Guangdong province microorganism strain collection center at 10 months and 14 days of 2022, and the preservation number is GDMCC No:62886.

2. a microbial agent, comprising Bifidobacterium bifidum CCFM1284 or fermentation broth thereof according to claim 1, or lyophilized powder of Bifidobacterium bifidum CCFM1284.

3. The microbial agent according to claim 2, wherein the viable count of bifidobacterium bifidum CCFM1284 in the microbial agent is at least: 10 ⁹ CFU/g。

4. A product comprising bifidobacterium bifidum CCFM1284 according to claim 1 or comprising a microbial inoculant according to claim 2 or 3.

5. The product of claim 4, wherein the product is a food, pharmaceutical or nutraceutical product.

6. The product according to claim 4 or 5, wherein the viable count of Bifidobacterium bifidum CCFM1284 is at least: 10 ⁹ CFU/g。

7. Use of a bifidobacterium bifidum CCFM1284 as defined in claim 1 or a microbial agent as defined in claim 2 or 3 for the preparation of a product for preventing and/or alleviating chronic colonic damage or chronic crohn's disease.

8. Use according to claim 7, wherein the product is a pharmaceutical or nutraceutical product.

9. The use according to claim 8, wherein the product has a viable count of Bifidobacterium bifidum CCFM1284 of at least: 10 ⁹ CFU/g。

10. Use of bifidobacterium bifidum CCFM1284 as defined in claim 1 or a microbial agent as defined in claim 2 or 3 for the preparation of a product for preventing and/or alleviating TNBS-induced inflammation and for reducing pathological damage to the colon.

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