Application of probiotic composition in preparation of medicine for preventing and treating ulcerative colitis

Abstract

The invention belongs to the field of microorganisms. The invention provides an application of a probiotic composition in preparation of medicines for preventing and treating ulcerative colitis, the probiotic composition comprises a combination of bacillus coagulans and clostridium butyricum, and the combination of bacillus coagulans and clostridium butyricum comprises a combination of bacillus coagulans and clostridium butyricum. The bacillus coagulans is bacillus coagulans JSSW-LA-07-1 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.10182, and the clostridium butyricum is clostridium butyricum JSIM-MCB20040312 with the preservation number of CGMCC No.1647. The bacillus coagulans and the clostridium butyricum are used for preparing the bacillus coagulans and the clostridium butyricum. The probiotic composition has the capacity of preventing and treating ulcerative colitis, the ulcerative colitis treating effect is better than that of positive control medicine salazosulfapyridine, the obvious ulcerative colitis treating effect is achieved, the inflammatory factor content and the oxidative stress level are remarkably reduced, inflammatory cell infiltration is reduced, intestinal flora imbalance is improved, and the effect of preventing and treating ulcerative colitis is achieved. Meanwhile, the effect of promoting planting of probiotics is achieved.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to the use of a probiotic composition in preparing a medicine for preventing and treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is an important type of inflammatory bowel disease. It starts with inflammation of the rectal mucosa and spreads to the proximal colon in a persistent manner. The scope of inflammation is usually limited to the mucosal layer, resulting in severe damage to the surface of the intestinal wall. The clinical manifestations of UC patients include diarrhea, blood in the stool, weight loss, and diffuse inflammation of the colon mucosa. Due to its high incidence, easy recurrence, and high risk of developing colorectal cancer, it has become an important health problem worldwide.

[0003] The pathogenesis of UC is still unclear, but it may be related to genetic susceptibility, dysregulated immune response to environmental factors, and changes in intestinal microbiota. Studies have shown that intestinal flora imbalance and intestinal barrier dysfunction can seriously affect gastrointestinal function, and UC patients have increased intestinal permeability, damaged intestinal mucosal barrier structure, disturbed intestinal flora homeostasis, and damaged intestinal symbiotic bacteria, which can trigger more severe immune responses and lead to recurrence of UC.

[0004] Key risk factors for UC include factors that affect the intestinal microbiota, such as antibiotic use, dietary changes, and the use of food additives. In the fecal microbiota of patients with irritable bowel syndrome (IBS), the relative abundance of pro-inflammatory bacteria of the Enterobacteriaceae family increases, the numbers of Lactobacillaceae and Bifidobacterium decrease, and the proliferation of specific bacterial species is associated with IBD symptoms. Clinical intervention treatments mainly focus on the use of immunosuppressive drugs, aminosalicylates, and corticosteroids to relieve the symptoms of IBS by inhibiting inflammation and regulating disordered immune responses. However, these conventional drugs can cause systemic toxic side effects and lead to serious complications, such as microbial infections, colorectal tumors, and autoimmune diseases. The current treatment goal for UC has shifted from treating symptoms to mucosal healing to change the natural course of the disease and improve long-term outcomes.

[0005] Probiotics can regulate intestinal metabolite products, increase intestinal mucosal proteins, and reduce intestinal permeability, so changing the intestinal microbial composition by supplementing probiotics is considered a potential therapeutic strategy. Clostridium butyricum (C. butyricum) is one of the most studied probiotics. It is a Gram-positive anaerobic bacterium that can produce abundant butyrate, which has also been shown to protect the epithelial function of pig small intestinal epithelial cells from the effects of LPS. Clinical trials have shown that Clostridium butyricum can effectively improve the symptoms, quality of life, and bowel movement frequency of patients with diarrhea-type irritable bowel syndrome (IBS-D), and the treatment response rate is high. In the mouse intestinal allergy model, administration of Clostridium butyricum can promote intestinal immune tolerance by increasing the abundance of Tregs, which can be used as a supplement or adjuvant therapy for food allergies. In addition to Clostridium butyricum, Bacillus coagulans (B. coagulans) as a food additive has also been shown to improve symptoms such as constipation and antibiotic-associated diarrhea. B. coagulans is a lactic acid-producing bacterium with spore-forming and facultative anaerobic properties, which enables it to better survive in the acidic environment of the stomach and adapt to the hypoxic intestinal environment, thereby inhibiting pathogens. It has been reported in the literature that Bacillus coagulans can improve intestinal damage in mice fed a high-fat diet.

[0006] In recent years, researchers have gradually paid attention to the function of probiotics in enhancing intestinal homeostasis, thereby reducing intestinal inflammation. Many single-strain probiotics have been shown to be effective in treating intestinal diseases, but due to the complexity of intestinal microorganisms, it is believed that the use of multiple strains may be more beneficial in restoring intestinal flora after dysbiosis occurs.

[0007] Although there have been reports of probiotics treating inflammatory bowel disease, different strains may also have different mechanisms of action. There is still uncertainty about the therapeutic effects and optimal timing of probiotics as dietary supplements or drugs. The use of specific combinations of probiotics to more effectively treat inflammatory bowel disease is worth further exploration. Summary of the invention

[0008] In order to solve the above-mentioned problems of the prior art, the present invention provides the use of a probiotic composition in the preparation of a medicament for the prevention and treatment of ulcerative colitis. The probiotic composition has a positive and significant effect on the prevention and treatment of ulcerative colitis. Its effect in treating ulcerative colitis is better than that of the positive control drug sulfasalazine, significantly reduces the content of inflammatory factors and the level of oxidative stress, reduces inflammatory cell infiltration, can improve intestinal flora imbalance, protect the intestinal mucosal barrier, and at the same time has the effect of promoting probiotic colonization.

[0009] The object of the present invention is to provide an application of a probiotic composition in the preparation of a medicament for preventing and treating ulcerative colitis. In the present invention, the probiotic composition comprises a combination of Bacillus coagulans and Clostridium butyricum, wherein the Bacillus coagulans is Bacillus coagulans JSSW-LA-07-1 with a preservation number of CGMCC No.10182, and the Clostridium butyricum is Clostridium butyricum JSIM-MCB20040312 with a preservation number of CGMCC No.1647.

[0010] The above-mentioned biological material sample with the deposit number CGMCC No.10182 has been disclosed in the patent entitled "A Bacillus coagulans with antagonistic properties against aquatic pathogens and its application" (publication number: CN105524855A), and the biological material sample with the deposit number CGMCC No.1647 has been disclosed in the patent entitled "A microecological preparation-quadruple live bacteria preparation and its preparation method" (publication number: CN1844364A).

[0011] Furthermore, the concentration of Bacillus coagulans and Clostridium butyricum in the probiotic composition is 1×10 6 Up to 1×10 10 CFU / g.

[0012] Further, Bacillus coagulans accounted for 60-95% of the total CFU composed of Bacillus coagulans and Clostridium butyricum.

[0013] Furthermore, the above-mentioned probiotic composition also comprises at least one of food raw materials, food-acceptable auxiliary materials or additives, and medically acceptable auxiliary materials or additives.

[0014] Furthermore, in the present invention, the product for preventing and treating ulcerative colitis is a health product or an oral pharmaceutical preparation.

[0015] Furthermore, the above-mentioned prevention and treatment of ulcerative colitis specifically includes: reducing the formation of ulcers in the mucosa and submucosal layer; reducing the inflammatory state; and reducing the level of oxidative stress.

[0016] Furthermore, the dosage forms of the above-mentioned oral preparations include oral liquid, drops, tablets, powders, capsules and granules.

[0017] Compared with the prior art, the use of the probiotic composition of the present invention in the preparation of a medicament for preventing and treating ulcerative colitis has the following beneficial effects:

[0018] (1) The probiotic composition has the ability to prevent and treat ulcerative colitis, and has a significant therapeutic effect on ulcerative colitis. Its effect in treating ulcerative colitis is better than that of the positive control drug sulfasalazine;

[0019] (2) The present invention significantly reduces the content of inflammatory factors and oxidative stress levels, reduces inflammatory cell infiltration, has the effect of actively regulating intestinal flora, can improve intestinal flora imbalance, protect the intestinal mucosal barrier, and has the effect of promoting the colonization of beneficial bacteria;

[0020] (3) The therapeutic method of regulating intestinal flora in the present invention has few serious adverse reactions in ulcerative colitis, and its efficacy is increasingly confirmed under the premise of ensuring the safety of treatment, which provides application value for the diagnosis and treatment of ulcerative colitis in clinical application and basic research, and supports the clinical transformation of the treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Statistical graphs of daily weight change rate, DAI score, macroscopic manifestations, and colon length of the DSS group, Life enhancer group, C. butyricum group, and B. coagulans group;

[0022] Figure 2 H&E staining images and histopathological scoring data of colon pathological histological changes in the DSS group, Life enhancer group, C. butyricum group, and B. coagulans group;

[0023] Figure 3 The graphs show the MDA content in colon tissue of mice in the DSS group, Life enhancer group, C. butyricum group, and B. coagulans group;

[0024] Figure 4 Statistical graphs of daily weight changes, DAI scores, macroscopic manifestations, and colon length of the Control group, DSS group, Life enhancer group, SASP group, and C. butyricum group;

[0025] Figure 5 H&E staining and histopathological scoring images of colon pathological histological changes in the Control group, DSS group, Life enhancer group, SASP group, and C. butyricum group;

[0026] Figure 6 It is the detection data graph of TNF-α content and IL-6 content in serum of Control group, DSS group, Life enhancer group, SASP group and C.butyricum group;

[0027] Figure 7 The data graph of MDA content in colon tissue of Control group, DSS group, Life enhancer group, SASP group and C.butyricum group;

[0028] Figure 8 Principal coordinate analysis (PCoA) and linear discriminant analysis plots for the Control and DSS groups;

[0029] Fig. 9 Analyze cladograms for LEfSe;

[0030] Fig.10 The principal coordinates analysis (PCoA) plots are for the DSS group and the Life enhancer group, and for the Life enhancer group and the Control group;

[0031] Fig.11 Linear discriminant analysis plots for the DSS and Life enhancer groups;

[0032] Fig.12 This is the principal coordinate analysis diagram of the Life enhancer group and the SASP group;

[0033] Fig.13 This is the linear discriminant analysis diagram of the Life enhancer group and the SASP group. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1 UC prevention experiment

[0036] 1. Experimental Animals

[0037] Healthy male Balb / c mice, 7 weeks old, weighing (22±2) g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0008, animal batch number: No.110322221103634448, animal ethics number: CMU2023166. Mice were raised under non-pathogenic conditions in the Experimental Animal Department of China Medical University (SPF grade), with an indoor temperature of (23±2)℃, 12h day and night cycle lighting, mice were housed in separate cages, and free access to food and water. Experiments were carried out after one week of adaptation. All experimental procedures were in accordance with the animal experiment welfare and ethical standards of China Medical University.

[0038] 2. Sources of experimental reagents and test kits (partial)

[0039] SPF grade rat maintenance feed (Beijing Huafukang Biotechnology Co., Ltd.)

[0040] Clostridium butyricum (Qingdao Donghai Pharmaceutical Co., Ltd.)

[0041] Bacillus coagulans (Qingdao Donghai Pharmaceutical Co., Ltd.)

[0042] Sulfasalazine (Shanghai MacLean Biochemical Technology Co., Ltd.)

[0043] IL-6 ELISA kit (Jiangsu ELISA Biotechnology Co., Ltd.)

[0044] TNF-α ELISA kit (Jiangsu ELISA Biotechnology Co., Ltd.)

[0045] Malondialdehyde (MDA) detection kit (Wanlei Biotechnology Co., Ltd.)

[0046] The probiotic composition Life enhancer of this embodiment includes the following components: live bacteria of Bacillus coagulans JSSW-LA-07-1 and live bacteria of Clostridium butyricum JSIM-MCB20040312; wherein the concentration of live bacteria of Bacillus coagulans JSSW-LA-07-1 is 5×10 8 CFU / g, the concentration of live bacteria of Clostridium butyricum JSIM-MCB20040312 was 5×10 7 CFU / g, Bacillus coagulans accounted for 90.9% of the total CFU composed of Bacillus coagulans and Clostridium butyricum.

[0047] 3. Solution preparation

[0048] (1) Preparation of 3% DSS drinking water: Weigh 3 g of DSS powder and dissolve it in 100 ml of sterile distilled water to prepare a DSS aqueous solution with a concentration of 3 g / 100 mL.

[0049] (2) Preparation of PBS: Add 4.0 g of sodium chloride, 0.1 g of potassium chloride, 1.98 g of disodium hydrogen phosphate, and 0.1 g of potassium dihydrogen phosphate to every 500 ml of distilled water and mix well.

[0050] (3) Preparation of the probiotic composition Life enhancer: Add 52 mg of Life enhancer to every 10 ml of normal saline and mix well.

[0051] (4) Preparation of Clostridium butyricum: Add 48.1 mg of Clostridium butyricum to every 10 ml of normal saline and mix well.

[0052] (5) Preparation of Bacillus coagulans: Add 573.3 mg of Bacillus coagulans to every 10 ml of normal saline and mix well.

[0053] 4. Grouping

[0054] Twenty-four male Balb / c mice adapted for 7 days were randomly divided into four groups, with 6 mice in each group, including 3% DSS-induced colitis model group (DSS), probiotic composition life enhancer group (DSS+Life enhancer, 52 mg / kg), Clostridium butyricum group (DSS+C.butyricum, 48.1 mg / kg), and Bacillus coagulans group (DSS+B.coagulans, 582.4 mg / kg).

[0055] The mouse ulcerative colitis model was established by drinking 3% DSS drinking water. The model was established on the first day of drinking 3% DSS drinking water, and the drinking water was changed every two days.

[0056] The drug-treated group received intragastric administration of the corresponding bacterial flora once a day starting from the first day of modeling for 7 consecutive days, while the model group received intragastric administration of an equal volume of normal saline.

[0057] The dosages of the three groups were:

[0058] Probiotic composition life enhancer group: 52 mg of probiotic composition per kg of mouse body weight was intragastrically administered;

[0059] C. butyricum group: 48.1 mg of C. butyricum per kg of mouse body weight was intragastrically administered;

[0060] Bacillus coagulans group: 582.4 mg of B. coagulans per kg of mouse body weight was intragastrically administered.

[0061] 5. Draw materials

[0062] First, sterilize the surgical instruments and Ep tubes required for the experiment in advance with high pressure, fix the mice with one hand, cut the mouse whiskers if necessary to prevent blood contamination, gently press the eye skin on the blood collection side to make the eyeball congested and protruding, use elbow forceps to clamp the eyeball and quickly remove it, and press the mouse heart with the middle finger of the left hand to speed up the heart pumping blood. When the blood runs out, kill the mouse by dislocation. After the mouse dies, open the abdominal cavity, take 0.5-1cm of the colon 1cm away from the anus, and use it for colon pathological tissue sections. The rest of the colon tissue is used for other experiments, but the same location must be taken for the same experiment. For fecal samples, take 3-5 pieces of feces from a single mouse and place them in a sterilized Ep tube, immediately freeze them in liquid nitrogen for 15 minutes, store them at -80℃ (or keep them in liquid nitrogen), and transport them on dry ice.

[0063] Example 2 UC prevention effect test

[0064] 1. Disease Activity Index (DAI) score and gross morphology

[0065] The disease activity index (DAI) score was calculated based on the weight, stool consistency and blood in stool of the mice as shown in Table 1.

[0066] Table 1 DAI score table

[0067] Scoring Weight loss % diarrhea Blood in the stool 0 0 0 = Normal 0 = No bleeding 1 1-5 2 6-10 2 = Loose stools 2 = Mild bleeding 3 11-20 4 >20 4 = Watery diarrhea 4 = severe bleeding

[0068] Daily weight change rate and DAI score of DSS group, Life enhancer group, C. butyricum group and B. coagulans group Figure 1 As shown in parts A and B in the figure. The results showed that as time went on, the mice in the DSS group ate less, had messy hair, and continued to lose weight. Most of them had watery diarrhea, purulent stools, and even bloody stools. The weight of the mice in the DSS group dropped significantly on the 6th and 7th days. However, the weight of the mice in the Life enhancer group did not change much, and most of them did not have symptoms such as diarrhea and bloody stools. The Lifeenhancer group and the Clostridium butyricum group significantly improved the symptom of weight loss in ulcerative colitis (P<0.01), while the Bacillus coagulans group showed no improvement.

[0069] The statistics of macroscopic manifestations and colon length in the DSS group, Life enhancer group, C. butyricum group and B. coagulans group were as follows: Figure 1 The results showed that the colon tissue of mice in the DSS group was edematous, with a large amount of inflammatory fluid in the intestinal cavity, accompanied by a certain degree of bleeding and congestion in the intestinal cavity. The colon length of DSS mice was significantly shorter than that of mice in other groups, while the colon length of the Life enhancer group and C.butyricum group was significantly longer than that of the DSS group, and the degree of bleeding and edema was reduced compared with the DSS group, but there was no significant improvement in the B.coagulans group compared with the DSS group.

[0070] 2. Colon pathological histological scoring

[0071] DSS-induced colonic damage includes the destruction of the intestinal mucosal barrier. Chronic inflammation is the basic characteristic of histological lesions of ulcerative colitis and is directly related to the progression of the disease. The colon was collected and histologically analyzed. The pathological changes of the colon tissues of the above groups of mice were observed under an optical microscope using HE staining.

[0072] The scoring criteria are shown in Table 2 , based on the percentage of tissue damage, degree of tissue damage, degree of crypt damage, and degree of inflammation.

[0073] Table 2 Histopathological scoring table

[0074]

[0075] The H&E staining images and histopathological scoring data of colonic histopathological changes in the DSS group, Life enhancer group, C. butyricum group, and B. coagulans group are shown in Figure 2 . Figure 2 As shown in A and B in the figure. The results showed that the mucosal layer of mice in the DSS group showed local atrophy, and extensive ulcers formed in the mucosa and submucosal layers. With the repeated onset of inflammation, the colon mucosal structure was repeatedly destroyed and could not be completely regenerated and repaired, resulting in deformation and continuous destruction of the crypt structure, a decrease in the number of goblet cells, resulting in reduced mucus secretion, a large number of lymphocytes infiltrating at the base, and abnormal inflammatory infiltration in the submucosal layer or muscular layer. In contrast, the colon tissues of mice in the Life enhancer group and the C. butyricum group were relatively intact, with very few inflammatory cell infiltrations and no large number of inflammatory cell infiltrations, and the above situation was significantly alleviated. The pathological histological score of the Life enhancer group was significantly reduced, and there was a trend of being lower than that of the C. butyricum group, while no obvious preventive effect was observed in the B. coagulans group compared with the DSS model group.

[0076] 3. Detection of MDA content in colon tissue

[0077] Long-term non-enzymatic free radical oxidation of lipids can lead to a dramatic destruction of the physicochemical structure of the membrane, especially related to the permeability and stability of lipid-protein complexes and the inactivation of lipid-dependent membrane-bound enzymes. Detecting changes in oxidative stress helps to clarify its impact on pathological processes and its regulation, and understand the mechanism of action of lipid peroxidation. Oxidative stress plays an important role in the occurrence of ulcerative colitis, and one of the most commonly used markers for evaluating oxidative stress is malondialdehyde (MDA). Colon tissues of mice in the above groups were collected to measure the degree of oxidative stress.

[0078] MDA content was determined using a malondialdehyde (MDA) detection kit (Wanlei Biotechnology Co., Ltd.). MDA content in colon tissue of mice in the DSS group, Life enhancer group, C. butyricum group, and B. coagulans group was as follows: Figure 3 As shown, the MDA activity in the intestinal tissue of mice in the DSS group was high, indicating a severe degree of oxidative stress, while the MDA level in the colon tissue of mice in the Life enhancer group was significantly reduced and was lower than that in the C. butyricum group and the B. coagulans group.

[0079] Example 3 UC treatment experiment

[0080] The probiotic composition Life enhancer of this embodiment includes the following components: live bacteria of Bacillus coagulans JSSW-LA-07-1 and live bacteria of Clostridium butyricum JSIM-MCB20040312; wherein the concentration of live bacteria of Bacillus coagulans JSSW-LA-07-1 is 5×10 8 CFU / g, the concentration of live bacteria of Clostridium butyricum JSIM-MCB20040312 was 5×10 7 CFU / g, Bacillus coagulans accounted for 90.9% of the total CFU composed of Bacillus coagulans and Clostridium butyricum.

[0081] Thirty male Balb / c mice adapted for 7 days were randomly divided into 5 groups, with 6 mice in each group, including healthy control group (Control), DSS-induced colitis model group (DSS), Life enhancer group (DSS+Life enhancer, 52 mg / kg), Clostridium butyricum group (DSS+Clostridium butyricum, 48.1 mg / kg) and sulfasalazine group (DSS+SASP, 150 mg / kg).

[0082] The mice were fed 3% DSS drinking water to establish the mouse ulcerative colitis model. The first day of drinking 3% DSS drinking water was the first day of modeling, and the drinking water was changed every two days. The healthy control group was given an equal volume of normal saline by gavage. After 7 days of modeling, the drug-treated group was given a daily gavage of the corresponding flora for 5 consecutive days. The model group and the healthy control group were given an equal volume of normal saline by gavage.

[0083] The dosages of the three groups were:

[0084] Life enhancer group: 52 mg of probiotic composition was intragastrically administered per kg of mouse body weight;

[0085] C. butyricum group: 48.1 mg of C. butyricum per kg of mouse body weight was intragastrically administered;

[0086] Sulfasalazine group: 150 mg of sulfasalazine per kg of mouse body weight was intragastrically administered.

[0087] The materials were collected according to the steps described in Example 1.

[0088] Example 4 UC treatment effect test

[0089] 1. Disease Activity Index (DAI) score and gross morphology

[0090] The scoring method is the same as in Example 2.

[0091] The statistics of daily weight change, DAI score, macroscopic appearance, and colon length of the control group, DSS group, Life enhancer group, SASP group, and C. butyricum group were as follows Figure 4 As shown in the A, B, C, and D parts of the figure. The results showed that from day 1 to day 7, the body weight of the DSS group, Life enhancer group, C.butyricum group, and SASP group all showed a similar degree of gradual decrease, and the DAI score showed a similar increase on day 7. All four groups met the DSS modeling criteria under the same conditions. From day 8 to day 12, the body weight of the DSS group continued to decrease, and the DAI score was still increasing. The body weight of the SASP group and C.butyricum group was moderated compared with the DSS group, while the Life enhancer group began to increase on day 10. The Life enhancer group significantly reduced the DAI score, and the improvement trend appeared earlier than the SASP group and C.butyricum group. The colon length of the Life enhancer group and the SASP group was significantly longer than that of the DSS group. The C.butyricum group showed an improvement trend, but no difference was observed.

[0092] 2. Colon pathological histological scoring

[0093] The scoring criteria are the same as those described in Example 2.

[0094] H&E staining images and histopathological scores of colon in the control group, DSS group, Life enhancer group, SASP group and C. butyricum group Figure 5 As shown. Figure 5 It can be seen that the colon mucosa of the mice in the Control group showed a clear structure, the epithelium was relatively intact, the glands were neatly arranged, and the goblet cells were clearly visible. In contrast, the colon epithelial cells of the mice in the enteritis model DSS group were damaged, the number of epithelial cells decreased, the glands were incomplete, and the crypts were damaged. Compared with the Control group, the colon inflammation pathology score in the DSS group was significantly increased, and the above pathological changes were significantly improved after treatment with Life enhancer, C. butyricum and SASP. Among them, the colon pathological damage in the Life enhancer group was the mildest. Therefore, Life enhancer can effectively improve the colon pathological damage caused by DSS. Combined with the above-mentioned DAI score and the improvement of colon shortening and the results of the prevention part experiment, Life enhancer has a good therapeutic effect.

[0095] 3. Detection of pro-inflammatory factors TNF-α and IL-6

[0096] To further evaluate colonic injury, the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum were quantified. The inflammatory factors IL-6 and TNF-α in mouse serum were detected by ELISA. IL-6 was detected using the IL-6 ELISA kit (Jiangsu ELISA Biotechnology Co., Ltd.), and TNF-α was detected using the TNF-α ELISA kit (Jiangsu ELISA Biotechnology Co., Ltd.).

[0097] The detection data of serum TNF-α content and IL-6 content in Control group, DSS group, Life enhancer group, SASP group and C. butyricum group are shown in Figure 6 As shown. Figure 6 It can be seen that compared with the Control group, the levels of IL-6 and TNF-α in the serum of the DSS group were significantly increased, while the levels of IL-6 and TNF-α in the serum of the Life enhancer group were significantly lower than those of the DSS group. Therefore, Life enhancer can reduce the systemic inflammatory state of mice with ulcerative colitis. The C. butyricum group can also reduce the concentration of TNF-α, while the SASP group is not as obvious as the first two treatment groups.

[0098] 4. Detection of MDA content in colon tissue

[0099] The data of MDA content in colon tissue of Control group, DSS group, Life enhancer group, SASP group and C. butyricum group are shown in Figure 7 As shown. Figure 7 It can be seen that the MDA content in the intestinal tissue of mice in the DSS group was the highest, indicating that the degree of oxidative stress was the most severe, while the MDA level in the colon tissue of mice in the Life enhancer group was lower than that in the DSS group, but still higher than that in the Control group, indicating that after DSS induction, the tissue was still damaged and could not return to normal levels, and tended to be lower than that in the SASP group and C.butyricum group.

[0100] Example 5 Analysis of changes in β diversity of intestinal flora

[0101] Intestinal flora sequencing method: This part was technically supported by Shanghai Institute of Biomedical Technology. Mouse feces were extracted using QIAamp DNA Stool Mini Kit (QIAGEN, Germany), and then the 16s rRNA gene V3-V4 region was amplified using primers 338F and 806R; TransStart Fastpfu DNA Polymerase (Quanshijin, China) was used for amplification, and the PCR reaction was 20 cycles. The PCR amplification product was purified using Agencourt AMPure beads (Beckman Coulter, USA), quantified by Qubit, and then all PCR products were mixed in equal amounts. After adding Illumina sequencing adapters on both sides of the mixed product, 2x300 bp bidirectional sequencing was performed using Illumina MiSeq sequencer (Illumina, USA).

[0102] β diversity refers to the difference in biodiversity between different ecosystems, and the comparison of the microbial structure between samples or groups. The calculation of the β diversity index is related to the presence or absence of species and species abundance. The present invention uses the Bray_curtis method to calculate the species distance, and further uses PCoA analysis to intuitively display the degree of difference in the microbial structure between samples. Principal coordinate analysis (Principle Coordinate Analysis, PCoA) is based on the dimensionality reduction analysis method, which aims to use low-dimensional space (two-dimensional plane) to display the distance between samples. The grouping characteristics are presented in the form of a scatter plot, and the percentage (number in brackets of the axis title) is used to evaluate the degree of explanation of each coordinate axis for the overall microbial structure difference. Similarity analysis (ANOSIM, Analysis of Similarities) was used to determine whether there were significant differences between the groups. The species classification information between different groups was compared, and the online Lefse software was used to analyze http: / / huttenhower.sph.harvard.edu / lefse / , using default parameters.

[0103] This example analyzes the changes in β diversity of the intestinal flora of mice among the Control group, DSS group, Life enhancer group, SASP group and C. butyricum group, displays the changes in β diversity of each group of mice through PCoA analysis, and determines whether there are significant differences among the groups through ANOSIM analysis.

[0104] (1) β-diversity analysis of control and DSS groups

[0105] The principal coordinates analysis (PCoA) and linear discriminant analysis of the Control and DSS groups were Figure 8 As shown, the LEfSe analysis phylogenetic tree is shown in Fig. 9 As shown. In the figure, red represents the Control group and green represents the DSS group. The red and green nodes represent the microbial groups that play an important role in their respective groups, while the yellow nodes represent the microbial groups that do not play an important role in both groups. The species names represented by the English letters in the figure are displayed on the right side of the figure. From the inner circle to the outer circle, they are species at the phylum, class, order, family, and genus levels. Linear discriminant analysis (LDA) and effect size (LEfSe) are used to reveal the dominant bacterial groups in each group to determine the bacterial groups with significant differences between the treatment groups.

[0106] Depend on Figure 8 , Fig. 9 It can be seen that the distance between the Control and DSS groups was the farthest, indicating that the intestinal flora of the DSS-induced UC group changed significantly compared with the Control group (P<0.01). LEfSe comparative analysis found that a total of 40 taxa were significantly enriched in the Control group at different taxonomic levels, while 16 taxa were significantly enriched in the DSS group.

[0107] (2) β-diversity analysis of DSS group and life enhancer group

[0108] The principal coordinates analysis (PCoA) of the DSS group and the Life enhancer group, and the principal coordinates analysis (PCoA) of the Life enhancer group and the Control group were as follows Fig.10 As shown in the A and B parts of the figure, D represents the DSS group, L represents the Life enhancer group, and C represents the Control group. The linear discriminant analysis of the DSS and Life enhancer groups is shown in Fig.11 shown.

[0109] Depend on Fig.10 It can be seen that the Life enhancer and DSS groups are far apart, indicating that the difference in the microbiota between the Life enhancer and DSS groups is also large (P<0.01). At the same time, it is observed that there is a certain distance between the Life enhancer group and the Control group (P<0.01), but it is smaller than the distance between the Life enhancer and DSS groups. This shows that DSS has an impact on the intestinal flora of mice, but after the intervention of Life enhancer, this change is reduced. Fig.11It can be seen that there are 17 taxa significantly enriched in the Life enhancer group at different taxonomic levels. The top five bacterial groups in relative abundance at the genus level are: Ligilactobacillus, Streptophyta, Paramuribaculum, Duncanilla, and Odoribacter.

[0110] (3) β-diversity analysis of the life enhancer group and the SASP group

[0111] The principal coordinates analysis of the Life enhancer group and the SASP group was as follows Fig.12 As shown in Figure 2, the linear discriminant analysis of the Life enhancer group and the SASP group is shown in Figure 2. Fig.13 As shown. Fig.12 , Fig.13 It can be seen that the Life enhancer group and the SASP group were far apart, and the intestinal flora between the two groups was significantly different (P<0.01). At the genus level, the top five bacterial communities in the SASP group were: Lactobacillus, Lactobacillus taiwanensis, Paraprevotella, Limosilactobacillus, and Limosilactobacillus reuteri.

[0112] β-diversity analysis results

[0113] The microbial characteristics of the Control group and the DSS group were relatively independent and the two-dimensional space distance was the farthest. DSS had a certain impact on the intestinal flora. After the Life enhancer intervention, the distance between the Life enhancer group and the DSS group was farther, and the microbial flora of the Life enhancer group and the DSS group was quite different. Although there was a certain gap between the distance between the Life enhancer group and the Control group, it was smaller than the distance between the Life enhancer group and the DSS group. This shows that the effect of DSS on the intestinal flora of mice is significant, but this effect was alleviated after the Life enhancer intervention.

[0114] In the DSS group, the abundance of Prevotellamassilia was higher, and studies have shown that Prevotellamassilia colonization can lead to metabolic changes in the microbiota and reduce the production of IL-18, thereby exacerbating intestinal inflammation and potential systemic autoimmunity. Ruminococcus gnavus is associated with Crohn's disease, and Alistipes is considered to be associated with diseases such as colorectal cancer and autism. This shows that DSS-induced UC mice upregulated the abundance of pathogenic bacteria. Treatment with Life enhancer can upregulate the abundance of Ligilactobacillus, Streptophyta, Paramuribaculum, Duncaniella, and Odoribacter. It shows that the probiotic composition can increase the abundance of beneficial bacteria and improve the characteristics of the fecal microbiota. The probiotic composition Life enhancer has the effect of promoting the colonization of beneficial bacteria.

[0115] Judging from the macroscopic indicators and inflammatory factor levels, the improvement effect of the Life enhancer group was better than that of the positive control drug SASP group, and the difference in intestinal flora between the Life enhancer group and the SASP group was also relatively obvious. In the LEfSe comparative analysis of SASP and Lifeenhancer, it was found that Ruminococcus was upregulated in the SASP group, and this flora was also relatively enriched in the DSS group.

[0116] MDA is an important indicator of oxidative stress. The degree of oxidative stress in SASP increased. Studies have shown that oxidative stress may be the main factor leading to microbial dysbiosis in IBD, and that Ruminococcus may be an important member of the altered intestinal community in IBD. In mice treated with Life enhancer and SASP after DSS treatment, the level of IL-6 in the serum was significantly reduced compared with the ulcerative colitis group, among which the effect of Life enhancer was more significant, and C. butyricum did not show significant improvement; TNF-α increased in the DSS group, and Life enhancer and C. butyricum reduced this increase, while the TNF-α level of mice in the SASP group did not decrease significantly, indicating that SASP mice had a high level of inflammation throughout the body, which was consistent with the gross morphological indicators of the SASP group. The range and severity of the lesions in the group treated with SASP were weakened, but the improvement of UC in the group treated with Life enhancer was more obvious, which could inhibit the expansion of lesions. Therefore, the Life enhancer treatment regimen can more effectively treat UC.

[0117] In summary, the probiotic mixture composed of Clostridium butyricum and Bacillus coagulans has the ability to prevent and treat UC. Life enhancer has a significant therapeutic effect on UC, and its therapeutic effect on UC is better than that of the positive control drug sulfasalazine. It has the effect of reducing inflammation. The levels of IL-6 and TNF-α in the serum of the Life enhancer group were significantly reduced, and the systemic inflammatory state of mice with ulcerative colitis was reduced. It also has an effect on the intestinal flora of mice with DSS-induced colitis. Microbial diversity analysis confirmed that Life enhancer can improve intestinal flora imbalance, protect the intestinal mucosal barrier, increase the abundance of beneficial bacteria, and promote the colonization of beneficial bacteria. These findings support the clinical transformation of Life enhancer for the treatment of UC.

[0118] The present invention provides evidence that the probiotic composition Life enhancer is a potential intestinal flora regulator for the treatment of UC. The impact of UC on the patient's quality of life and family social burden is very long-lasting. The treatment method of regulating intestinal flora with probiotics has few serious adverse reactions in UC, and its efficacy is increasingly confirmed under the premise of ensuring the safety of treatment. The present invention is intended to provide a certain reference value for the diagnosis and treatment of UC in clinical applications and basic research.

[0119] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those skilled in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also belong to the scope of the present invention and should be defined by the claims.

Claims

1. Use of a probiotic composition in the preparation of a medicament for preventing and treating ulcerative colitis, characterized in that: The probiotic composition comprises a combination of Bacillus coagulans and Clostridium butyricum, wherein the Bacillus coagulans is Bacillus coagulans JSSW-LA-07-1 with a preservation number of CGMCC No.10182, and the Clostridium butyricum is Clostridium butyricum JSIM-MCB20040312 with a preservation number of CGMCC No.1647.

2. The use according to claim 1, characterized in that: The concentration of Bacillus coagulans and Clostridium butyricum in the probiotic composition is 1×10 6 Up to 1×10 10 CFU / g.

3. The use according to claim 2, characterized in that: The Bacillus coagulans accounted for 60-95% of the total CFU consisting of Bacillus coagulans and Clostridium butyricum.

4. The use according to claim 1, characterized in that: The probiotic composition further comprises at least one of a food raw material, a food-acceptable auxiliary material or additive, and a medically acceptable auxiliary material or additive.

5. The use according to any one of claims 1 to 4, characterized in that: The medicine is a health product or an oral pharmaceutical preparation.

6. The use according to any one of claims 1 to 4, characterized in that: The prevention and treatment of ulcerative colitis includes: reducing the ulcer formation in the mucosa and submucosal layer; reducing the inflammatory state; and reducing the oxidative stress level.

7. The use according to claim 5, characterized in that: The dosage forms of the oral pharmaceutical preparation include oral liquid, drops, tablets, powders, capsules and granules.

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