Lactobacillus fermentum GLF-217 and its applications in ulcerative colitis, hyperlipidemia and reducing organ damage

Abstract

The present invention discloses a strain of Lactobacillus fermentum GLF-217. The Lactobacillus fermentum GLF-217 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 4, 2023, with the deposit number CGMCC No. 28336. The Lactobacillus fermentum GLF-217 provided by the present invention can effectively treat ulcerative colitis, hyperlipidemia and reduce organ damage at the same time, providing a technical solution for the combined treatment of ulcerative colitis, hyperlipidemia and organ damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation. More specifically, it relates to Lactobacillus fermentum GLF-217 and its application in ulcerative colitis, hyperlipidemia, and reduction of organ damage. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic non-specific inflammatory disease that occurs in the gastrointestinal tract, mainly including ulcerative colitis (UC) and Crohn's disease (CD). Its pathogenesis is affected by multiple factors such as genetic susceptibility, impaired intestinal barrier function, intestinal flora imbalance, and immune response disorders. There is currently no consensus on the specific pathogenesis. The clinical manifestations of patients with ulcerative colitis are diarrhea, bloody stools, weight loss, and diffuse inflammation of the colonic mucosa. Due to its chronic, recurrent, high incidence, and high risk of developing colorectal cancer (CRC), ulcerative colitis has become a major health problem worldwide.

[0003] Traditionally, the conventional drugs used for ulcerative colitis are sulfasalazine salicylate preparations, corticosteroids, etc. However, long-term use of these drugs will produce side effects, including hypertension, diabetes, osteoporosis, and organ damage, etc., greatly reducing the quality of life of patients. Therefore, it is particularly important to find a healthy and effective method for treating ulcerative colitis. The difference in intestinal flora (type and quantity) between patients with colitis and healthy people is considered to be one of the key factors of the disease. Among them, probiotics, as an alternative to antibiotics, have attracted much attention because they can improve the damaged mucosal barrier function, regulate the imbalance of intestinal flora, inhibit pathogenic bacteria, and enhance the immunity of the intestinal system, thus alleviating ulcerative colitis to a certain extent. Compared with ordinary drugs, probiotics have the advantages of high safety, no side effects, no drug resistance, and low cost.

[0004] Hyperlipidemia (HLP) is a disease caused by abnormal lipid metabolism disorders in the body, mainly manifested by abnormal levels of various blood lipid components such as cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) in plasma being too high, and high-density lipoprotein cholesterol (HDL-C) level being too low. HLP is an important cause of chronic metabolic diseases such as atherosclerosis, cardiovascular diseases, and fatty liver diseases. Preventing and controlling hyperlipidemia is of great significance for preventing and treating metabolic diseases and ensuring public health.

[0005] Common drugs for treating hyperlipidemia in clinical practice include statins, which mainly reduce cholesterol in serum, liver, and aorta, and lower the levels of very low-density lipoprotein cholesterol and low-density lipoprotein cholesterol. Commonly used drugs include lovastatin, simvastatin, atorvastatin, etc. However, statins have obvious side effects, mainly manifested in liver function damage: elevated levels of serum glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST), and myopathy, etc.

[0006] Organ tissue injury refers to pathological damage to human skin, subcutaneous tissue, synovial bursa, intervertebral disc, peripheral nerves, and blood vessels caused by various activities. Organ injury is a special critical state between deterioration and recovery. If diagnosed and treated in a timely manner, there is hope for cure; otherwise, it may deteriorate and even cause organ failure, bringing serious damage to the body. Therefore, the diagnosis and treatment of organ tissue injury are extremely important. In recent years, research on the mechanism of liver injury, as well as its prevention and treatment, has become one of the hotspots in biological and medical research at home and abroad.

[0007] Currently, whether it is the treatment of ulcerative colitis, hyperlipidemia, or organ injury, complications will occur and worsen during the actual treatment process. When taking drugs for treating ulcerative colitis, it may cause elevated blood pressure and exacerbate organ damage; when taking drugs for treating hyperlipidemia, it will obviously lead to complications such as liver function damage. In addition, for patients with multiple complications such as ulcerative colitis, hyperlipidemia, and organ injury, how to carry out targeted treatment simultaneously is also an important problem that needs to be solved urgently.

[0008] Patent CN116004442A discloses a Lactobacillus fermentum and its application in improving hyperlipidemia preparations, which can significantly reduce the levels of triglycerides, cholesterol, and low-density lipoproteins in serum, and can significantly promote the secretion of short-chain fatty acids in the intestine, and significantly improve the pathological conditions of the mouse liver, thereby effectively improving hyperlipidemia. However, this strain only has the effect of improving hyperlipidemia and does not have the effect of treating other complications, such as ulcerative colitis. Its functional effects and application scenarios are limited. Summary of the Invention

[0009] Aiming at the above existing technical problems, the primary object of the present invention is to provide a strain of Lactobacillus fermentum GLF-217, which simultaneously has the effects of preventing and / or treating ulcerative colitis, helping to reduce the contents of triglycerides, total cholesterol, and low-density lipoproteins in serum, and / or reducing organ injury, and has broad market prospects.

[0010] The second object of the present invention is to provide the use of the above-mentioned Lactobacillus fermentum GLF-217 or its bacterial suspension in the preparation of a pharmaceutical composition or health product that helps to reduce the contents of triglyceride, total cholesterol and low-density lipoprotein in serum or has an adjuvant protective effect on reducing liver injury. And the use of the above-mentioned Lactobacillus fermentum GLF-217 or its bacterial suspension in the preparation of a pharmaceutical composition for preventing and / or treating ulcerative colitis.

[0011] The third object of the present invention is to provide a bacterial agent comprising the above-mentioned Lactobacillus fermentum GLF-217 and / or its bacterial suspension.

[0012] The fourth object of the present invention is to provide the use of the above-mentioned bacterial agent in the preparation of a pharmaceutical composition or health product that helps to reduce the contents of triglyceride, total cholesterol and low-density lipoprotein in serum or has an adjuvant protective effect on reducing liver injury. And the use of the above-mentioned bacterial agent in the preparation of a pharmaceutical composition for preventing and / or treating ulcerative colitis.

[0013] The above objects of the present invention are achieved by the following technical solutions:

[0014] The present invention screened and obtained a strain of Lactobacillus fermentum GLF-217, and the Lactobacillus fermentum GLF-217 was deposited in the General Microbiological Center of the China National Center for Biotechnology Development on September 4, 2023, with the deposit number of CGMCC No. 28336.

[0015] The present invention used shotgun metagenomics and transcriptome analysis to explore the preventive and therapeutic effects and mechanisms of action of Lactobacillus fermentum GLF-217 on ulcerative colitis. The results showed that treatment with Lactobacillus fermentum GLF-217 could significantly improve dextran sulfate sodium-induced ulcerative colitis in mice, specifically manifested as increased body weight, increased water intake, increased food intake, increased colon length, and decreased disease activity index, immune organ index, inflammatory factors and histopathological scores in mice. Further research found that Lactobacillus fermentum GLF-217 could prevent and treat ulcerative colitis in mice by optimizing intestinal microbial diversity, improving the intestinal mucosal barrier and alleviating inflammation. Specifically, Lactobacillus fermentum GLF-217 prevented and treated ulcerative colitis in mice by regulating the intestinal microbiota and increasing the production of short-chain fatty acids. Lactobacillus fermentum GLF-217 improved the intestinal barrier by increasing mucin-2 and zonula occludens-1. Lactobacillus fermentum GLF-217 optimized the immune barrier response by reducing the contents of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), and increasing interleukin-10 (IL-10).

[0016] The inventors also found that Lactobacillus fermentum GLF-217 can effectively prevent and treat hyperlipidemia caused by a high-fat diet. In an experimental atherosclerosis model, the plasma adiponectin level is negatively correlated with triglycerides and low-density lipoproteins and positively correlated with high-density lipoproteins. A high-fat diet reduces the ADP concentration in the serum of golden hamsters, while GLF-217 can maintain and stimulate the secretion of ADP, thereby reducing the incidence of atherosclerosis. The relative constancy of blood lipids in the human body is mainly regulated by hepatocytes. When hepatocytes are damaged, abnormal blood lipid metabolism occurs in the body. The experimental results show that a long-term high-fat diet can cause relatively serious damage to the liver and increase the risk of developing fatty liver, while GLF-217 can relieve the liver tissue damage caused by a high-fat diet. In the later treatment of hyperlipidemia, GLF-217 can reduce blood lipids and relieve the elevation of AST and ALT, but has no obvious effect on the morphology of hepatocytes with severe fatty degeneration. Therefore, preventing the formation of hyperlipidemia and avoiding liver damage caused by high blood lipids are of great significance to human health.

[0017] In the preventive experimental results, GLF-217 regulates the abundances of Adlercreutzia equolifaciens, Asaccharobacter celatus, Lactobacillus fermentum, and Bifidobacterium pseudolongum. These bacteria downregulate the lipid synthesis pathway, thereby effectively inhibiting the occurrence of hyperlipidemia. In addition, GLF-217 promotes the fermentation of pyruvate to produce propionic acid, and propionic acid inhibits the elevation of blood lipids, inflammatory factors, AST, and ALT and promotes the production of adiponectin, thereby reducing the incidence of hyperlipidemia and its complications (inflammation, liver damage, atherosclerosis, etc.). In the treatment experimental results, GLF-217 regulates the abundances of Corynebacterium glucurono lyticum, Enterococcus faecalis, and Enterobacter cloacae complex. GLF-217 and its related strains upregulate the lipid metabolism pathway, thereby reducing the hyperlipidemia caused by hyperlipidemia in golden hamsters. In addition, although the intake of lovastatin can effectively reduce blood lipids, it also aggravates liver damage. Lactobacillus reuteri upregulates the pathway of pyruvate fermentation to butyric acid, promoting the production of short-chain fatty acids, thereby relieving liver damage. The content of Lactobacillus reuteri in the intestinal flora of golden hamsters taking lovastatin is significantly reduced, which has a negative impact on liver damage.

[0018] The present invention provides a strain of Lactobacillus fermentum GLF-217, which can effectively prevent and / or treat ulcerative colitis simultaneously, contribute to reducing the contents of triglyceride, total cholesterol and low-density lipoprotein in serum and / or reducing organ damage, and provides a technical solution for the combined treatment of ulcerative colitis, hyperlipidemia and / or reducing organ damage.

[0019] Furthermore, the present invention also claims the use of the above-mentioned Lactobacillus fermentum GLF-217 or its bacterial suspension in the preparation of a pharmaceutical composition or health product that helps to reduce the contents of triglyceride, total cholesterol and low-density lipoprotein in serum or has an auxiliary protective effect on reducing liver damage. And the use of Lactobacillus fermentum GLF-217 or its bacterial suspension in the preparation of a pharmaceutical composition for preventing and / or treating ulcerative colitis.

[0020] In some embodiments, the organ damage is liver damage.

[0021] In some embodiments, the ulcerative colitis is dextran sulfate sodium-induced ulcerative colitis.

[0022] In some embodiments, the prevention and / or treatment of ulcerative colitis includes at least one of the following: improving the intestinal mucosal barrier, alleviating the inflammatory response or regulating the structure of the intestinal microbiota.

[0023] In some embodiments, the alleviation of the inflammatory response is one or more of increasing the expression of IL-10, decreasing the expression of IL-1β, decreasing the expression of IL-6, decreasing the expression of IFN-γ or decreasing the expression of TNF-a.

[0024] In some embodiments, the regulation of the structure of the intestinal microbiota is one or more of promoting the growth of probiotics in the intestinal flora, inhibiting the growth of pathogenic bacteria in the intestinal flora, increasing the abundance of short-chain fatty acid-producing strains or increasing the diversity of the intestinal flora.

[0025] In some embodiments, the improvement of the intestinal mucosal barrier refers to promoting the expression of mucin 2 and tight junction protein-1.

[0026] In some embodiments, more specifically, the efficacy of the Lactobacillus fermentum GLF-217 in preventing and / or treating ulcerative colitis specifically includes at least one of the following:

[0027] (1) Reducing weight loss caused by ulcerative colitis;

[0028] (2) Reducing the reduction in food intake and water intake caused by ulcerative colitis;

[0029] (3) Significantly increasing the colon length;

[0030] (4) Significantly reduce the disease activity index;

[0031] (5) Significantly increase the levels of anti-inflammatory cytokines in the serum and significantly reduce the levels of pro-inflammatory cytokines in the serum;

[0032] (6) Significantly reduce the pathological score of intestinal tissue;

[0033] (7) Significantly increase the expression levels of tight junction protein ZO-1 and mucin MUC-2 in the intestine;

[0034] (8) Significantly increase the content of medium- and short-chain fatty acids;

[0035] (9) Significantly increase the intestinal flora diversity;

[0036] (10) Reduce the relative abundances of Escherichia coli, Aeromonas caviae, and Clostridium perfringens in the intestinal flora;

[0037] (11) Significantly increase the enrichment of immune response-related genes.

[0038] Since Lactobacillus fermentum GLF-217 has any one of the above-mentioned effects, the application of Lactobacillus fermentum GLF-217 in the above should also be within the protection scope of this application.

[0039] Furthermore, the present invention also claims protection for a bacterial agent, which comprises the above-mentioned Lactobacillus fermentum GLF-217 and / or its bacterial suspension.

[0040] Furthermore, the present invention also claims protection for the application of the above-mentioned bacterial agent in the preparation of a pharmaceutical composition or health product that helps to reduce the contents of triglyceride, total cholesterol, and low-density lipoprotein in the serum or has an adjuvant protective effect on reducing liver damage. And the application of the bacterial agent in the preparation of a pharmaceutical composition for preventing and / or treating ulcerative colitis.

[0041] The present invention has the following beneficial effects: The present invention provides a strain of Lactobacillus fermentum GLF-217. The Lactobacillus fermentum GLF-217 can play a preventive and therapeutic role in a dextran sulfate sodium-induced colitis mouse model by protecting the intestinal mucosal barrier, attenuating the inflammatory response, regulating the structure of the intestinal microbiota, etc.; furthermore, Lactobacillus fermentum GLF-217 can reduce the contents of triglyceride, total cholesterol and low-density lipoprotein in the serum, thereby playing a role in preventing and / or treating hyperlipidemia. At the same time, the Lactobacillus fermentum GLF-217 also has excellent therapeutic effects in reducing organ damage. The present invention provides a strain of Lactobacillus fermentum GLF-217, which can effectively treat ulcerative colitis, hyperlipidemia and reduce organ damage at the same time, and provides a technical solution for the combined treatment of ulcerative colitis, hyperlipidemia and organ damage. Description of the Drawings

[0042] Figure 1 It is the growth morphology of Lactobacillus fermentum GLF-217 on the medium.

[0043] Figure 2 It is the identification result of 16s rRNA of Lactobacillus fermentum GLF-217.

[0044] Figures 3 - 5 They are the graphs of the daily body weight, water intake and food intake changes of the mice, respectively.

[0045] Figure 6 It is the schematic diagram of the disease activity index of the mice.

[0046] Figure 7 It is the immune organ index of the mice.

[0047] Figure 8 It is the colon length of the mice.

[0048] Figure 9 They are the serum immune cytokine levels of each group of mice.

[0049] Figure 10 It is the H&E staining section of the mouse colon.

[0050] Figure 11 It is the histopathological score of the colon tissue of each group of mice.

[0051] Figure 12 It is the ZO-1 immunofluorescence section of the colon of each group of mice.

[0052] Figure 13 It is the MUC-2 immunofluorescence section of the colon of each group of mice.

[0053] Figure 14Colonic ZO-1 and MUC-2 surface density of each group of mice.

[0054] Figure 15 Shannon index of intestinal flora of each group of mice.

[0055] Figure 16 PCoA analysis of mouse intestinal flora.

[0056] Figure 17 Results of species-level analysis of mouse intestinal flora.

[0057] Figure 18 Schematic diagram of differential metabolic pathways in mice in the Lactobacillus fermentum GLF-217 prevention group.

[0058] Figure 19 Short-chain fatty acid levels of each group of mice.

[0059] Figure 20 Analysis of short-chain fatty acid correlations.

[0060] Figure 21 Correlation network analysis of Lactobacillus fermentum GLF-217.

[0061] Figures 22 - 24 Graphs of daily body weight, water intake, and food intake changes in mice, respectively.

[0062] Figure 25 Disease activity index of each group of mice.

[0063] Figure 26 Immune organ index of each group of mice.

[0064] Figure 27 Colonic length of each group of mice.

[0065] Figure 28 Serum immune cytokine levels of each group of mice.

[0066] Figure 29 Colonic H&E staining sections of each group of mice.

[0067] Figure 30 Colonic histopathological scores of each group of mice.

[0068] Figure 31 Colonic ZO-1 immunofluorescence sections of each group of mice.

[0069] Figure 32 Colonic MUC-2 immunofluorescence sections of each group of mice.

[0070] Figure 33 Colonic ZO-1 and MUC-2 surface density of each group of mice.

[0071] Figure 34 It is the Shannon index of the intestinal flora of each group of mice.

[0072] Figure 35 It is the PCoA analysis of the intestinal flora of mice.

[0073] Figure 36 It is the analysis result of the species level of the intestinal flora of mice.

[0074] Figure 37 It is the schematic diagram of the differential metabolic pathways of mice in the Lactobacillus fermentum GLF-217 prevention group.

[0075] Figure 38 It is the short-chain fatty acid level of each group of mice.

[0076] Figure 39 It is the correlation analysis of short-chain fatty acids.

[0077] Figure 40 It is the correlation network analysis of Lactobacillus fermentum GLF-217.

[0078] Figure 41 It is the schematic diagram of gene distribution analyzed by colon transcriptome data.

[0079] Figure 42 It is the result of the gene ontology (GO) enrichment analysis.

[0080] Figure 43 It is the weight change graph of golden hamsters in the Lactobacillus fermentum GLF-217 prevention group.

[0081] Figure 44 It is the weight change graph of golden hamsters in the Lactobacillus fermentum GLF-217 treatment group.

[0082] Figure 45 and 46 They are respectively the schematic diagrams of the contents of TC, TG, and LDL-C in the sera of golden hamsters in the Lactobacillus fermentum GLF-217 prevention group and treatment group.

[0083] Figure 47 and 48 They are respectively the schematic diagrams of the contents of AI, ADP, ALT, AST, TNF-α, and IL-6 in mice in the Lactobacillus fermentum GLF-217 prevention group and treatment group.

[0084] Figure 49 It is the schematic diagram of the short-chain fatty acid content in golden hamsters in the Lactobacillus fermentum GLF-217 prevention group.

[0085] Figure 50 It is the schematic diagram of the short-chain fatty acid content in golden hamsters in the Lactobacillus fermentum GLF-217 treatment group. Specific implementation method

[0086] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The following embodiments are preferred embodiments of the present invention, but do not limit the protection scope of the present invention in any form. The present invention mainly elaborates the strain and the application idea based on the strain. The replacement of simple parameters in the embodiments cannot be elaborated one by one in the embodiments, but this does not limit the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should be regarded as equivalent replacement methods and should be included in the scope of the present invention.

[0087] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0088] In this study, GraphPad and R software were used to perform statistical analysis on the data. All data were expressed as mean ± standard deviation (SD). Wilcoxon was used for significance analysis. When the value was *, P < 0.05; **, P < 0.01; ***, P < 0.001, it was considered to have significance, strong significance, and extremely strong significance. The ggplot2 package was used for principal coordinate analysis (PCoA) and to draw box plots and bubble plots respectively. The DESeq2 software package was used for P-value calculation and correction in the metabolic pathway bubble plots.

[0089] Example 1 Obtaining Lactobacillus fermentum GLF-217

[0090] 1. Screening and isolation of Lactobacillus fermentum:

[0091] Sampling personnel obtained samples from the feces of infants in West China Hospital of Sichuan University. The samples were diluted in a ten-fold gradient. 100 μL of samples were taken from the diluents of -2, -3, and -4 dilution gradients respectively and spread on MRS agar medium, and then placed in an incubator at 37 °C for inverted culture for 48 h. Pure single colonies were isolated by streaking from the colonies with obvious morphological differences on the -2 dilution gradient plate. One strain with good growth state was isolated and named GLF-217.

[0092] 2. Identification of Lactobacillus fermentum:

[0093] The GLF-217 isolation and purification plate was observed, the colony morphological characteristics were recorded, its physiological and biochemical characteristics were studied, and the 16srRNA technology was used to perform taxonomic identification on the strain.

[0094] GLF-217 has the following morphological, physiological and biochemical characteristics and molecular biology identification results:

[0095] (1) Morphological characteristics

[0096] The colonies of GLF-217 on MRS medium are milky white, with a smooth and moist surface, plump, and round (as Figure 1 shown). After Gram staining, it is Gram-positive and rod-shaped under the microscope, arranged individually.

[0097] (2) Physiological and biochemical characteristics

[0098] GLF-217 is a Gram-positive bacterium, negative for catalase. It has no flagella, is immotile, cannot form spores, and is facultatively anaerobic. It produces gas when cultured in 10% skim reconstituted milk. It can metabolize pentoses such as D-ribose, monosaccharides such as D-glucose and D-fructose, and disaccharides such as D-maltose and D-sucrose, but cannot metabolize lactose.

[0099] (3) Results of molecular biology identification

[0100] The 16S rRNA sequence of GLF-217 is shown in SEQ ID: No. 1. The molecular biology identification result of the 16S rRNA of GLF-217 is Lactobacillus fermentum, and the identification result is as Figure 2 shown. This strain was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 4, 2023, with the preservation number of CGMCC No. 28336.

[0101] Example 2 Construction of an experimental animal model of ulcerative colitis

[0102] Fifty 8-week-old C57BL / 6J mice (black male mice) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. After 1 week of adaptive feeding, they were randomly divided into 5 groups, with 10 mice in each group. The grouping of experimental animals is shown in Table 1. Among them, the model group, the drug treatment group, the Lactobacillus fermentum GLF-217 treatment group (Treat group), and the Lactobacillus fermentum GLF-217 prevention group (Pro group) drank drinking water containing 3% dextran sulfate sodium for 1 consecutive week until the body weight decreased by more than 10% or the mice showed characteristics such as loose stools and bloody stools, indicating successful modeling. The drinking water, feed, and bedding were changed every day. The environmental temperature of the animal laboratory was controlled at 23 ± 1 °C, the relative humidity was controlled at 52% - 60%, and a 12-hour light-dark cycle was maintained. The mice were all fed with standard normal commercial mouse feed (mainly composed of crude protein, crude fiber, crude fat, and trace elements).

[0103] After the experiment, the mice were euthanized by intraperitoneal injection of 1% sodium pentobarbital solution, and various tissue samples were collected, including immune organs, serum, proximal colon, feces, cecal contents, distal colon, and other tissues.

[0104] Table 1

[0105]

[0106] Note: In the Lactobacillus fermentum prevention group in Table 1, the daily gavage cycle of Lactobacillus fermentum for 1 - 2 weeks was determined according to indicators such as the body weight, food intake, water intake, and DAI score of the mice.

[0107] Improvement effect of Lactobacillus fermentum GLF - 217 prevention group on physiological indexes and DAI of mice in Example 3

[0108] (1) Record the body weight, fecal characteristics, food intake, and water intake of the mice in Example 2 every day. Evaluate the level of blood in the stool according to the instructions of the mouse fecal occult blood kit, and calculate the DAI score according to Table 2; (Comprehensively score the three situations of the percentage of body weight loss, stool viscosity, and stool bleeding, and the total score of the 3 results divided by 3 is the DAI value).

[0109] Table 2

[0110]

[0111] (2) Experimental results of the Lactobacillus fermentum GLF - 217 prevention group

[0112] Monitor the water intake, food intake, body weight, and disease activity index of the mice every day to evaluate the severity of the ulcerative colitis model establishment.

[0113] Figures 3 - 5 They are the graphs of the daily body weight, water intake, and food intake changes of the mice respectively. The results show that from the 1st to the 7th day, the water intake, food intake, and body weight of the model group and the Pro group decreased gradually to a similar extent. From the 8th to the 10th day, the water intake, food intake, and body weight of the mice in the model group still showed a downward trend, with extremely significant differences from the Control group (P < 0.01).

[0114] Figure 6 It is the schematic diagram of the disease activity index of the mice. As shown in the figure, compared with the Control group, the disease activity index of the mice in other groups gradually increased. Compared with the model group, the disease activity index score of the mice in the Pro group decreased significantly and there were significant differences from the model group.

[0115] Figure 7 It is the immune organ index of the mice. As shown in the figure, the kidney and liver coefficients of the mice in the model group decreased significantly, but were alleviated after ingesting Lactobacillus fermentum GLF - 217.

[0116] Figure 8 It is the colon length of the mice. As shown in the figure, compared with the Control group, the colon length of the mice in the model group was significantly shortened, while the colon length of the mice in the Pro group was significantly increased compared with the model group.

[0117] Effect of Lactobacillus fermentum GLF-217 preventive group on inflammatory cytokines in dextran sulfate sodium-induced ulcerative colitis mice

[0118] (1) Determination of serum cytokines

[0119] In Example 2, before euthanasia, blood was collected from the orbital venous plexus of mice using a capillary tube. First, the blood sample was allowed to clot naturally for 30 minutes. Then, the blood sample was centrifuged at 3000 rpm for 20 min at 4°C, and the serum was separated and collected. Finally, the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17A (IL-17A), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the serum were detected using the corresponding ELISA kits.

[0120] (2) Experimental results of Lactobacillus fermentum GLF-217 preventive group

[0121] Figure 9 The serum immune cytokine levels of mice in each group are shown in the figure. As shown, compared with the Control group, the levels of pro-inflammatory cytokines in the serum of model group mice increased significantly, while the levels of anti-inflammatory cytokines decreased significantly, and the opposite was true for the Pro group.

[0122] Effect of Lactobacillus fermentum GLF-217 preventive group on pathological lesions in dextran sulfate sodium-induced colitis mice

[0123] (1) Determination of immune organ index

[0124] The weight and length of the colon of mice in each group in Example 2 were measured and photographed. At the same time, the weights of the kidneys, livers, and spleens were measured, and the immune organ index was calculated according to the following formula.

[0125] Immune organ index = weight of target organ (mg) / body weight (g).

[0126] (2) Intestinal tissue pathological analysis and immunofluorescent protein

[0127] After euthanasia of mice in Example 2, the colon was weighed and photographed, and then the distal colon of the mice was taken and divided into three parts for H&E staining section, histopathological scoring, transcriptome, and immunofluorescent protein determination.

[0128] The first part was used for H&E staining section and histopathological scoring. The specific steps of staining were as follows: After washing the colon samples with PBS, they were first fixed in 4% (w / v) paraformaldehyde for 24 h, then dehydrated, paraffin-embedded, and sectioned (3 μm). Next, the sections were stained with hematoxylin-eosin, and the histopathological score was observed and evaluated under an optical microscope according to the colon tissue pathological scoring criteria in Tables 3 and 4.

[0129] In the second part, transcriptome sequencing was performed. The total RNA of the samples was extracted according to the Trizol extraction instructions. The extraction effect was detected by 0.8% agarose gel electrophoresis. The purified mRNA was fragmented and a sequencing library was prepared. Denaturation treatment produced single-stranded DNA fragments, which were subjected to PCR amplification to generate DNA clusters. The DNA amplicons were linearized into single strands. Modified DNA polymerase and dNTPs with four fluorescent labels were added to read the types of nucleotides polymerized in the first-round reaction of each template sequence, and the fluorescence signal results collected in each round were counted to obtain the sequence of the template DNA fragment and detect the change in expression level. Then, library construction and quality control were carried out, and the raw RNA-seq data were filtered. After constructing the RNA library, sequencing was performed using Illumina Novaseq 6000, and featucounts was used to estimate gene expression levels.

[0130] The third part was used for immunofluorescent protein determination. The colon tissues were labeled with antibodies against MUC-2 (mucin-2) and ZO-1 (zonula occludens-1) respectively, and further immunofluorescent staining was carried out. Fluorescein combined with antibodies ZO-1 and MUC-2 to form fluorescent antibodies. Multicomponent complexes were formed by specific binding with antigens, and a fluorescence microscope was used to study the characterization and localization of ZO-1 and MUC-2 in intestinal tissues. The surface densities of immunofluorescent ZO-1 and MUC-2 were measured and calculated, and according to the positive expression levels of the antibodies, the following formula was used to analyze and evaluate indicators such as areal density, positive cell density, and positive intensity. The improvement effect of the tested strains on the colonic intestinal membrane barrier was evaluated.

[0131] ① Areal density = integrated optical density value (IOD) / tissue area of the area to be measured: The integrated optical density value is the integral of the optical density of all positive signals. Dividing by the tissue area of the area to be measured can reflect the amount and depth of positivity, which is proportional.

[0132] ② Positive intensity = average value of the color depth of each positive point: In bright field, it is the average optical density, and in dark field, it is the average intensity. The larger the value, the stronger the positive degree.

[0133] ③ Positive cell density = number of positive cells / tissue area of the area to be measured: Analyze the ratio of the number of positive cells to the tissue area of the area to be measured. It reflects the number of positive cells per unit area, which is proportional.

[0134] Table 3

[0135]

[0136] Table 4

[0137]

[0138]

[0139] (3) Experimental results of the Lactobacillus fermentum GLF-217 prevention group

[0140] Figure 10 These are H&E stained sections of the colon of mice in each group. The section results show that the structures of each layer of the intestinal tissue of healthy mice in the Control group are clear and the boundaries are obvious; the mucosa is intact, the cell morphology is normal, and there is no obvious inflammatory cell infiltration. In contrast, the intestinal tissue of the Model group is almost completely ulcerated, the obvious intestinal gland structure has disappeared, the mucosal epithelium has fallen off, the intestinal glands have completely necrosed and disappeared, the damage has invaded the serosa, the connective tissue is arranged loosely, and there are many neutrophil and lymphocyte infiltrations; a small amount of neutrophil, lymphocyte infiltrations and proliferated connective tissue can be seen in the muscular layer and serosa. In contrast, in the intestinal tissue of the Pro group, small areas of ulceration can be seen, the mucosal epithelium at the damaged area is intact, a small number of intestinal glands have necrosed and disappeared, replaced by proliferated connective tissue, accompanied by a small amount of neutrophil and lymphocyte infiltrations; a small amount of neutrophil and lymphocyte infiltrations can be seen in the submucosa.

[0141] Figure 11 These are the histopathological scores of the colon tissues of mice in each group. As shown in the figure, there are significant differences between the Model group and the Control group, and there are significant differences in the scores between the Pro group and the Model group (P<0.01).

[0142] Figure 12 These are the immunofluorescence sections of ZO-1 in the colon of mice in each group. As shown in the figure, the immunofluorescence analysis is divided into ZO-1 and MUC-2. The fluorophore is linked to the antibody, and the antibody-antigen specific binding is used to localize its distribution. The results show that the contents of MUC-2 protein and ZO-1 protein are rich in the Control group. In the Model group, due to the damage of the intestinal mucosa, there are very few tight junction proteins left. In the sections of the Pro group mice, the distribution of ZO-1 can be clearly seen, and the expression level is significantly higher than that of other groups.

[0143] Figure 13 These are the immunofluorescence sections of MUC-2 in the colon of mice in each group. As shown in the figure, there are huge differences between the Model group and the Control group for the tight junction protein MUC-2. At the same time, the extensive distribution of MUC-2 in the Pro group is significantly more than that in the Model group.

[0144] Figure 14Colonic ZO-1 and MUC-2 surface density in each group of mice. As shown in the figure, the expression of tight junction protein ZO-1 and MUC-2 was further quantitatively analyzed, and a scatter plot of their surface density was drawn. It can be seen that the trends of the surface density of ZO-1 and MUC-2 are similar. Compared with other groups, the Pro group is closer to the Control group, and there is a significant difference from the Model group (P<0.05).

[0145] Determination of intestinal flora-related indicators in the Lactobacillus fermentum GLF-217 prevention group of Example 6

[0146] (1) Collect the feces of the mice in Example 2 every week. Extract the total DNA of the fecal samples according to the instructions of the fecal DNA extraction kit. After detecting the DNA purity and integrity by 0.8% agarose gel electrophoresis, perform shotgun metagenomic sequencing and prepare a sequencing library. Use the humann2 database to obtain the abundance data and metabolic pathway data of the intestinal microorganisms of each group of mice, and use R software to qualitatively and quantitatively analyze the α-diversity and β-diversity of the intestinal microorganisms of each group of mice. At the same time, calculate the differential flora and differential metabolic pathways between each group of mice.

[0147] (2) After freeze-drying the feces of the mice, extract short-chain fatty acids (SCFAs) and quantitatively analyze the levels of short-chain fatty acids in each group of samples. The method is as follows: First, freeze-dry 40 mg of the feces of the mice, weigh it, and add 600 μL of normal saline (85%). Then place the sample in a shaker and stir for 5 minutes, mix well and centrifuge (8000 RPM, 5 minutes). Take 200 μL of the supernatant, add 100 μL of sulfuric acid (50%), and then add 400 μL of n-hexane to extract SCFAs. Finally, pass the solution through an organic membrane and transfer it to a vial. Analyze the concentration of SCFAs, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid, by gas chromatography-mass spectrometry (GCMS).

[0148] (3) Experimental results of the Lactobacillus fermentum GLF-217 prevention group

[0149] The results of diversity analysis showed that compared with the Control group, under the same modeling conditions, the Shannon index of the mice in the Model group decreased significantly, and the Shannon index of the Pro group was higher (as Figure 15 shown).

[0150] Figure 16 This is the PCoA analysis of the intestinal flora of mice. The results of β-diversity showed that the Model group and the Control group were significantly separated, the Pro group was significantly separated from the Model group, there were significant differences in the abundance of bacterial species, and the distance between the Lactobacillus fermentum GLF-217 prevention group and the Control group was closer.

[0151] Figure 17 Results of the species-level analysis of the mouse gut microbiota. As Figure 17 shown, the potentially colitis-causing pathogens in the Model group, such as Escherichia coli, Aeromonas caviae, Clostridium perfringens, and Enterococcus faecalis, were significantly increased, while they were decreased in the Pro group; the potentially beneficial bacteria, such as Bifidobacterium pseudolongum, Akkermansia muciniphila, and Anaerotruncus sp G3 2012, were significantly decreased in the Model group and significantly increased in the Pro group.

[0152] In the analysis of the diversity of the mouse gut microbiota, it was found that the abundance of the bacterial species capable of producing short-chain fatty acids (SCFAs) increased in the Pro group. Figure 18 Schematic diagram of the differential metabolic pathways in the mice of the Lactobacillus fermentum GLF-217 prevention group. As shown in the figure, further analysis of the metabolic pathways found that the abundances of 7 microbial metabolic pathways related to the production of SCFAs decreased in the Model group and increased in the Pro group, including superpathway III of acetyl-CoA biosynthesis, pyruvate fermentation to acetate and lactate II, pyruvate fermentation to acetate and S-lactate I, glycolysis III (from glucose), fatty acid biosynthesis, and acetyl-CoA biosynthesis I and acetyl-CoA biosynthesis II.

[0153] To confirm the above results, we further detected the contents of SCFAs in each group by gas chromatography-mass spectrometry (GC-MS). Figure 19 Short-chain fatty acid levels in the mice of each group. The results showed that, compared with the Control group, the SCFAs (including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid) in the Model group were significantly decreased. Compared with the Model group, after the intake of Lactobacillus fermentum GLF-217, the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid increased extremely significantly (P<0.01). Based on the above results, we speculated that Lactobacillus fermentum GLF-217 increased the content of SCFAs by affecting the abundance of the microorganisms capable of producing SCFAs and the metabolic pathways of the microorganisms capable of producing SCFAs.

[0154] In order to further understand the role of SCFAs, we performed Pearson correlation analysis. The results showed that Clostridium perfringens and Escherichia coli in the Model group increased significantly, and were strongly negatively correlated with acetic acid, propionic acid, and butyric acid. Anaerotruncus sp G3 2012 and Lactobacillus johnsonii increased significantly in the Lactobacillus fermentation GLF-217 prevention group, and were strongly positively correlated with acetic acid, propionic acid, and butyric acid. At the same time, a correlation analysis of SCFAs and cellular immune factors was performed, and the results showed that SCFAs including acetic acid, propionic acid, and butyric acid were negatively correlated with pro-inflammatory factors TNF-α, IL-1β, IFN-γ, IL-6, and IL-17A, and positively correlated with anti-inflammatory factors IL-10 ( Figure 20 ).

[0155] SCFAs are important products of intestinal flora metabolism, have certain anti-inflammatory effects, and play an important role in maintaining the morphology and function of the intestinal mucosa. Combined with the improvement results of the above indicators, we speculate that Lactobacillus fermentum GLF-217 may regulate SCFAs to relieve ulcerative colitis through the following mechanisms.

[0156] That is, after taking Lactobacillus fermentum GLF-217, the abundance of intestinal microorganisms that produce SCFAs increased, upregulated the metabolic pathways that produce SCFAs, and promoted the production of SCFAs. SCFAs have the effect of promoting the secretion of anti-inflammatory cytokines and inhibiting the secretion of inflammatory factors. The changes in immune cytokines affected the physiological indicators of mice, increased the weight, colon length, water intake and food intake of mice, and reduced the disease activity index score and immune organ index. At the same time, the tissue pathology score was reduced and the content of ZO-1 and MUC-2 immunofluorescent proteins was increased.

[0157] After confirming that probiotics help alleviate ulcerative colitis, we explored their potential mechanisms of action. Pearson correlation analysis was used to explore the correlation and interaction between Lactobacillus fermentum GLF-217 and the commensal intestinal microbiota of mice. Lactobacillus fermentum GLF-217 was positively correlated with Anaerotruncus sp G3 2012, Bacteroides vulgatus, and Lactobacillus johnsonii, and positively correlated with acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate, while SCFAs were negatively correlated with proinflammatory factors TNF-α, IL-1β, IFN-γ, IL-6, and IL-17A, and positively correlated with the anti-inflammatory cytokine IL-10 ( Figure 21), thus affecting the physiological indices of the mice, increasing the body weight, colon length, water intake and food intake of the mice, reducing the disease activity index score and immune organ index. At the same time, the histopathological score was reduced, and the contents of ZO-1 and MUC-2 immunofluorescent proteins were increased.

[0158] Example 7 Improvement effect of Lactobacillus fermentum GLF-217 treatment group on physiological indices and DAI of mice

[0159] The test method and DAI score criteria of the Lactobacillus fermentum GLF-217 treatment group were the same as those of the Lactobacillus fermentum GLF-217 prevention group in Example 3. The establishment of the control group and the model group in the Lactobacillus fermentum GLF-217 treatment group was the same as that in Example 2. The water intake, food intake, body weight and disease activity index of the mice were monitored daily to evaluate the severity of the ulcerative colitis model establishment.

[0160] Figures 22 - 24 They are the graphs of the daily body weight, water intake and food intake changes of the mice respectively. The results show that from the 1st day to the 10th day, except for the Control group, the water intake, food intake and body weight of the mice in each group gradually decreased, and the degree was similar, which may be because each group was under the same DSS model establishment conditions during this stage. Then, from the 10th to the 18th day, the water intake, food intake and body weight of the mice in the Model group continued to decrease, while the water intake, food intake and body weight of the mice in the Treat group gradually increased, and there were significant differences compared with the Model group (P<0.05).

[0161] Figure 25 They are the disease activity indices of the mice in each group. Compared with the Control group, the disease activity indices of the other groups have been significantly increased since the 3rd day. Compared with the Model group, the disease activity indices of the mice in the Treat group and the Drug group gradually decreased from the 12th day and finally showed significant differences compared with the Model group (P<0.05).

[0162] Figure 26 They are the immune organ indices of the mice in each group. The kidney and liver coefficients of the mice in the Model group were significantly reduced, but were alleviated after ingesting Lactobacillus fermentum GLF-217 and Drug; the spleen coefficient was significantly increased, and ingesting Lactobacillus fermentum GLF-217 and Drug could alleviate this situation.

[0163] Figure 27 They are the colon lengths of the mice in each group. Compared with the Control group, the colon length of the mice in the Model group was significantly shortened, while the colon lengths of the mice in the Treat group and the Drug group were significantly increased compared with the model group.

[0164] Effect of Lactobacillus fermentum GLF-217 treatment group on inflammatory cytokines in dextran sulfate sodium-induced ulcerative colitis mice

[0165] Determination of serum cytokines: Refer to the test method in Example 4.

[0166] Figure 28 are the serum immune cytokine levels of mice in each group. The results showed that compared with the Control group, the levels of pro-inflammatory cytokines TNF-a, IL-1β, IFN-γ, IL-6 and IL-17A in the serum of Model group mice were significantly increased (P<0.05), and the anti-inflammatory cytokine IL-10 was significantly decreased (P<0.05), while the opposite effects were observed in the serum of mice in the Treat group and Drug group.

[0167] Effect of Lactobacillus fermentum GLF-217 treatment group on pathological lesions of dextran sulfate sodium-induced colitis mice

[0168] The determination of immune organ index, pathological analysis of intestinal tissue, and determination and calculation of immunofluorescent proteins were carried out according to the methods in Example 5.

[0169] Figure 29 are H&E stained sections of mouse colon. The results showed that in the Control group of healthy mice, the structures of each layer of intestinal tissue were clear and the boundaries were obvious; the mucosa was intact, the cell morphology was normal, and there was no obvious inflammatory cell infiltration. In the Model group, the intestinal tissue was almost completely ulcerated, there was no obvious intestinal gland structure, the mucosal epithelium fell off, the intestinal glands completely necrosed and disappeared, the injury invaded the serosa, the connective tissue was arranged loosely, accompanied by more neutrophil and lymphocyte infiltration; a small amount of neutrophils, lymphocytes infiltration and proliferated connective tissue were visible in the muscular layer and serosa. In contrast, in the Treat group, focal ulcers were visible in the mouse colon, the mucosal epithelium at the injury site was intact, the cytoplasm was loose, a small amount of intestinal glands necrosed and disappeared, replaced by proliferated connective tissue, accompanied by punctate neutrophil infiltration; the injury invaded the submucosa, and punctate neutrophil infiltration was visible. In the Drug group, larger area ulcers were visible in the intestinal tissue of mice, the mucosal epithelium at the injury site fell off, more intestinal glands necrosed and disappeared, replaced by proliferated connective tissue, accompanied by more neutrophils and lymphocytes infiltration; the injury invaded the submucosa, and a small amount of neutrophils and lymphocytes infiltration was visible.

[0170] Figure 30 are the pathological scores of colon tissues of mice in each group. It can be seen from Figure 30 that there were significant differences between the Control group and the Model group, while the scores of the Treat group and the Drug group were closer to the Control group and there were significant differences from the Model group (P<0.01).

[0171] Figure 31Colonic ZO-1 immunofluorescence sections of mice in each group. The results of immunofluorescence analysis showed that the contents of MUC-2 protein and ZO-1 protein were abundant in the Control group. However, in the Model group, due to the damage of the intestinal mucosa, there were very few tight junction proteins left. In the sections of mice in the Treat group, the distribution of ZO-1 could be clearly seen, and the expression level was significantly higher than that in other groups. There was also partial expression in the Drug group.

[0172] Figure 32 Colonic MUC-2 immunofluorescence sections of mice in each group. For the tight junction protein MUC-2, there were huge differences between the Model group and the Control group. At the same time, in the Treat group, the extensive distribution of MUC-2 was significantly more than that in other groups. There was also relatively high expression in the Drug group.

[0173] Figure 33 The surface density of ZO-1 and MUC-2 in the colon of mice in each group. As Figure 33 shown, to further quantitatively analyze the expression of tight junction proteins ZO-1 and MUC-2, a scatter plot of their surface density was drawn. It could be seen that the trends of the surface density of ZO-1 and MUC-2 were similar. Compared with other groups, the Treat group was closer to the Control group and there were significant differences from the Model group (P<0.05).

[0174] Determination of intestinal flora-related indicators in the treatment group of Lactobacillus fermentum GLF-217 in Example 10

[0175] The determination of intestinal flora-related indicators referred to the method in Example 6.

[0176] The results of diversity analysis showed that compared with the Control group, under the same modeling conditions, the Shannon index in the Model and Drug groups decreased significantly, and the Shannon index in the Treat group was higher ( Figure 34 ). At the same time, the results of β-diversity showed that each group was significantly separated from the Control group (P<0.01). The Treat group and the Drug group were significantly separated from the Model group, with significant differences in the species abundance, and the distance between the Treat group and the Control group was closer ( Figure 35 ).

[0177] Figure 36 Results of the species-level analysis of the intestinal flora of mice. As Figure 36As shown, the potential colitis-causing bacteria in the Model group, such as Escherichia coli, Aeromonas caviae, Clostridium perfringens, and Enterococcus faecalis, increased significantly, while they decreased in the Treat group; the potential beneficial bacteria, such as Bifidobacterium pseudolongum, Akkermansia muciniphila, Anaerotruncus sp G3 2012, and Lactobacillus reuteri, decreased significantly in the Model group, while they increased significantly in the Treat group.

[0178] In the analysis of the diversity of the mouse gut microbiota, it was found that the abundance of the bacterial species capable of producing short-chain fatty acids (SCFAs) increased in the Treat group. Figure 37 It is a schematic diagram of the differential metabolic pathways of the mice in the Lactobacillus fermentum GLF-217 treatment group. Further analysis of the metabolic pathways found that the abundances of 7 microbial metabolic pathways related to the production of SCFAs decreased in the Model group and increased in the Treat group, namely pyruvate fermentation to acetate and lactate II, pyruvate fermentation to acetate and S-lactate I, glycolysis III (from glucose), glycolysis II, glycolysis I, fatty acid biosynthesis, and acetyl-CoA biosynthesis II.

[0179] To confirm the above results, we further detected the content of SCFAs in each group by gas chromatography-mass spectrometry (GC-MS). Figure 38 It is the short-chain fatty acid levels of the mice in each group. The results showed that compared with the Control group, the SCFAs (including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid) in the Model group decreased significantly. Compared with the Model group, after the intake of Lactobacillus fermentum GLF-217, the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid increased extremely significantly (P<0.01), approaching the levels of the Drug group. Based on the above results, we speculate that Lactobacillus fermentum GLF-217 increases the content of SCFAs by affecting the abundance of the microorganisms capable of producing SCFAs and the metabolic pathways of the microorganisms capable of producing SCFAs.

[0180] To further understand the role of SCFAs, we performed a Pearson correlation analysis. Figure 39Correlation analysis of short-chain fatty acids. As shown in the figure, Clostridium perfringens and Escherichia coli in the Model group increased significantly, and were strongly negatively correlated with acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and hexanoic acid. Aeromonas caviae and Enterococcus faecalis increased significantly, and were negatively correlated with acetic acid, propionic acid and butyric acid. Anaerotruncus sp G32012, Lactobacillus johnsonii and Lactobacillus reuteri increased significantly in the Treat group, and were strongly positively correlated with acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and hexanoic acid. At the same time, the correlation analysis of SCFAs and cellular immune factors showed that SCFAs including acetic acid, propionic acid and butyric acid were negatively correlated with pro-inflammatory factors TNF-α, IL-1β, IFN-γ, IL-6 and IL-17A, and positively correlated with anti-inflammatory factor IL-10.

[0181] SCFAs are important products of intestinal flora metabolism, have certain anti-inflammatory effects, and play an important role in maintaining the morphology and function of the intestinal mucosa. Combined with the improvement results of the above indicators, we speculate that the fermented Lactobacillus GLF-217 treatment group may regulate SCFAs to alleviate DSS-induced ulcerative colitis through the following mechanisms. That is, after the intake of fermented Lactobacillus GLF-217, the abundance of intestinal microorganisms that produce SCFAs increased, the metabolic pathways that produce SCFAs were upregulated, and the production of SCFAs was promoted. SCFAs have the effects of promoting the secretion of anti-inflammatory cytokines and inhibiting the secretion of inflammatory factors. The changes in immune cytokines affected the physiological indicators of mice, increased the weight, colon length, water intake and food intake of mice, and reduced the disease activity index score and immune organ index. At the same time, the histopathological score was reduced, and the content of ZO-1 and MUC-2 immunofluorescent proteins was increased.

[0182] Figure 40Correlation network analysis of Lactobacillus fermentum GLF-217. Through Pearson correlation analysis, it was found that Lactobacillus fermentum GLF-217 was positively correlated with Anaerotruncus sp G3 2012, Lactobacillus reuteri, and Lactobacillus johnsonii, and positively correlated with acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid. SCFAs were negatively correlated with pro-inflammatory factors TNF-α, IL-1β, IFN-γ, IL-6, and IL-17A, thus affecting the physiological indexes of mice, increasing the body weight, colon length, water intake, and food intake of mice, reducing the disease activity index score and immune organ index. At the same time, the histopathological score was reduced, and the content of ZO-1 and MUC-2 immunofluorescent proteins was increased.

[0183] Figure 41 To analyze the schematic diagram of gene distribution using colon transcriptome data. As shown by the results of the volcano plot, the intake of Lactobacillus fermentum GLF-217 significantly affected the gene expression distribution. To further explore the effects of these differentially expressed genes (DEGs), we analyzed the pathways related to the DEGs.

[0184] Figure 42 Results of gene ontology (GO) enrichment analysis. The results can be divided into three major categories, namely biological processes, cellular components, and molecular functions. The GO analysis results showed that the DEGs in the Model group and the Control group were mainly involved in cellular components such as cytoplasm, extracellular space, extracellular region, biological processes such as cell cycle, and molecular functions such as metal ion binding. The DEGs in the Treat group and the Drug group were mainly involved in biological processes such as immune response, immunoglobulin production, and positive regulation of RNA polymerase II promoter transcription, cellular components such as extracellular space, and molecular functions such as DNA binding and sequence-specific DNA binding in the proximal region of the RNA polymerase II core promoter.

[0185] Example 11 Study on the inhibition or alleviation of hyperlipidemia by Lactobacillus fermentum GLF-217

[0186] (1) Experimental animals and materials

[0187] Eight-week-old golden hamsters were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Standard control feed and high-fat feed were purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. Kits for serum TC, TG, HDL-C, and LDL-C of golden hamsters were purchased from Nanjing Jiancheng Bioengineering Institute; kits for ALT, AST, TNF-α, IL-6, and adiponectin of golden hamsters were purchased from Shanghai Xinyu Biotechnology Co., Ltd.

[0188] (2) Evaluation of the preventive and therapeutic effects of Lactobacillus fermentum GLF-217 on high-fat diet golden hamsters

[0189] ① Prevention experiment grouping and period: 30 golden hamsters at 8 weeks of age. After 1 week of adaptive feeding, they were randomly divided into 3 groups, namely: control group (control group, normal fat diet, 10 mice); model group (model group, high-fat diet, 10 mice); GLF-217 prevention group (pro group, high-fat diet + probiotic suspension. The probiotic suspension was Lactobacillus fermentum GLF-217 of 10 9 CFU resuspended in 300 μL of sterile physiological saline and administered by gavage, 10 mice). During this process, the control group and the model group were given an equal amount of physiological saline as a control. They were continuously fed for 6 weeks. Body weight was recorded weekly and feces were collected and stored at -80 °C. On the last day of the sixth week, the mice were fasted for 16 hours and then euthanized. The animal experiment grouping information is shown in Table 5 below.

[0190] Table 5

[0191]

[0192] ② Treatment experiment grouping and period: 40 golden hamsters at 8 weeks of age. After 1 week of adaptive feeding + 6 weeks of high-fat diet feeding, they were randomly divided into 4 groups: control group (control group, growth maintenance diet feeding for 12 weeks, 10 golden hamsters), model group (model group, high-fat diet feeding for 12 weeks, 10 golden hamsters), drug group (lov group, a mixed hyperlipidemia model was established by feeding a high-fat diet for 6 weeks, and then lovastatin 4 mg / kg / day was given while feeding the high-fat diet for 6 weeks, 10 golden hamsters), GLF-217 treatment group (treat group, a mixed hyperlipidemia model was established by feeding a high-fat diet for 6 weeks, and then GLF-217 was given while feeding the high-fat diet for 6 weeks, 10 golden hamsters). The model group was fed a high-fat diet for 6 weeks to establish a mixed hyperlipidemia model (in the golden hamsters of the model group, triglyceride, total cholesterol, and low-density lipoprotein in the serum were significantly increased (P < 0.05). By comparing with the control group, the model was determined to be established). The animal experiment grouping information is shown in Table 6 below.

[0193] Table 6

[0194]

[0195] ③ Determination of serum biochemical indexes

[0196] Before euthanasia, 6 golden hamsters were randomly selected from each group and blood was collected from the orbital venous plexus using a capillary tube. First, the blood samples were allowed to clot naturally for 30 min. Then, the blood samples were centrifuged at 3000 rpm for 20 min at 4 °C to separate and collect the serum. The levels of TC (total cholesterol), TG (triglyceride), LDL-C (low-density lipoprotein), HDL-C (high-density lipoprotein), adiponectin, AST (aspartate aminotransferase), and ALT (alanine aminotransferase) in the serum were measured according to the kit instructions. The atherosclerosis index was calculated using the following formula.

[0197] AI = (TC - HDL-C) / HDL-C

[0198] ④ Physiological and pathological indicators

[0199] During the experiment, food intake was measured daily and body weight was recorded weekly. After euthanasia of the golden hamsters, the liver weight of each group of golden hamsters was measured and the immune organ index was calculated: liver index = liver weight (mg) / body weight (g).

[0200] Part of the liver was taken for H&E staining sections and analyzed and evaluated by section analysis.

[0201] ⑤ Short-chain fatty acid determination

[0202] After freeze-drying the feces of golden hamsters, short-chain fatty acids (SCFAs) were extracted and the levels of short-chain fatty acids in each group of samples were quantitatively analyzed using a gas chromatograph.

[0203] At the end of the experiment, the colonic contents of 6 randomly selected golden hamsters from each group were used for SCFA determination. First, 40 mg of the freeze-dried colonic contents were weighed and 600 mL of normal saline (85%) was added. Then the samples were placed on a shaker for 5 min, thoroughly mixed and centrifuged (8000 rpm, 5 min). Then 200 mL of the supernatant was taken and 100 mL of sulfuric acid (50%) was added for acidification. 400 μL of n-hexane was added to extract the short-chain fatty acids. The solution was finally passed through an organic membrane and transferred to a vial. The concentrations of SCFAs, including acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid, were analyzed using gas chromatography-mass spectrometry (GCMS; Agilent-7890, Santa Clara, CA, USA). The concentrations of SCFAs were calculated by the external standard method and expressed as mmol / g dry sample according to the method described above (81).

[0204] ⑥ Fecal DNA extraction, metagenomic sequencing, and data quality control

[0205] At the end of the experiment, the feces of golden hamsters were collected. Total DNA of fecal samples was extracted according to the instructions of the fecal DNA extraction kit. After detecting the DNA purity and integrity by 0.8% agarose gel electrophoresis, shotgun metagenomic sequencing was performed and a sequencing library was prepared. The diversity and differences among the intestinal microorganisms of golden hamsters in each group, as well as the differential metabolic pathways, were analyzed qualitatively and quantitatively using databases.

[0206] ⑦Data statistics and analysis

[0207] In this study, GraphPad and R software were used for statistical analysis of the data. All data were expressed as mean ± standard deviation (SD). Wilcoxon was used for significance analysis. When the value was *, P < 0.05; **, P < 0.01; ***, P < 0.001, it was considered to have significance, strong significance, and extremely strong significance. The ggplot2 package was used for principal coordinate analysis (PCoA) and to draw box plots and bubble plots. The DESeq2 software package was used for P-value calculation and correction in the metabolic pathway bubble plots.

[0208] (3) Experimental results of Lactobacillus fermentum GLF-217 on preventing and treating golden hamsters with high-fat diet

[0209] ①Lactobacillus fermentum GLF-217 reduced the body weight of mice with high-fat diet

[0210] Figure 43 This is a graph showing the body weight changes of mice in the Lactobacillus fermentum GLF-217 prevention group. The results are as Figure 43 shown. In the in vivo experiment of mice, by the 6th week, there was a significant difference in the body weight between the model group and the control group. At the same time, there was also a significant difference between the model group and the pro group. This indicates that adding Lactobacillus fermentum GLF-217 to the high-fat diet can significantly slow down the weight gain of mice.

[0211] Figure 44 This is a graph showing the body weight changes of mice in the Lactobacillus fermentum GLF-217 treatment group. As Figure 44 shown, after 12 weeks, the body weight of mice in the model group was still significantly higher than that in the control group and the treat group. The lov group and the treat group of mice ended 6 weeks of high-fat diet and received a 6-week intervention with high-fat diet and probiotics or drugs. The results showed that there was a significant difference between the Lov group and the Model group. By comparing the body weight changes of each group at the 6th week and the 12th week, it was found that the weight gain of the lov group and the GLF-217 group was not obvious. These results indicate that the treat group can significantly slow down the weight gain under high-fat diet conditions.

[0212] ②Lactobacillus fermentum GLF-217 improved the blood lipid levels of mice with high-fat diet

[0213] During the occurrence of obesity, the lipid metabolism of the body is disordered, and the lipid level in the blood tends to rise. It has been shown that a long-term high-fat diet can affect the levels of triglyceride (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum of mice. An increase in LDL-C in the blood can lead to cardiovascular diseases such as atherosclerosis, and the ratio of LDL-C to HDL-C is an index used clinically to evaluate the risk of cardiovascular diseases.

[0214] Figure 45 and 46 are schematic diagrams of the contents of TC, TG, and LDL-C in the serum of mice in the Lactobacillus fermentum GLF-217 prevention group and treatment group, respectively.

[0215] Figure 47 and 48 are schematic diagrams of the contents of AI, ADP, ALT, AST, TNF-α, and IL-6 in mice in the Lactobacillus fermentum GLF-217 prevention group and treatment group, respectively.

[0216] The TC, TG, and LDL-C in the serum of the model group were significantly higher than those in the control group. This shows that after six weeks of high-fat diet feeding, the blood lipid levels of the mice increased significantly. The TC, TG, and LDL-C in the pro group were significantly lower than those in the model group, indicating that GLF-217 can effectively alleviate the increase in blood lipid levels. The formula for calculating the atherosclerosis index: AI = (TC - HDL-C) / HDL-C.

[0217] It can be seen from the results that the AI index of the Model group was significantly higher than that of the control group, while the AI index of the pro group was significantly lower than that of the model group, indicating that GLF-217 can alleviate and reduce atherosclerosis caused by high fat. Compared with the model group, the contents of TC, TG, and LDL-C in the lov group and treat group were significantly reduced. And there was no significant difference in the blood lipid contents between the lov group and the treat group, indicating that both lovastatin and GLF-217 have the ability to reduce the four blood lipid indicators.

[0218] Adiponectin is an endogenous bioactive polypeptide or protein secreted by adipocytes. In an experimental atherosclerosis model, the plasma adiponectin level is negatively correlated with triglyceride and low-density lipoprotein, and positively correlated with high-density lipoprotein. Administration of adiponectin treatment significantly reduces the contents of blood triglyceride and low-density lipoprotein, increases the content of high-density lipoprotein, and alleviates atherosclerotic lesions. Adiponectin can inhibit the production and release of TNF, has a certain anti-inflammatory effect, and has an important cytoprotective effect on alcoholic liver injury.

[0219] The experimental results showed that after 6 weeks of the prevention experiment, the ADP content in the serum of the Model group decreased significantly, while the ADP content in the pro group was significantly higher than that in the model group; after 12 weeks of the treatment experiment, the ADP content in the serum of the model group decreased significantly, while the ADP contents in the lov group and the treat group were significantly higher than that in the model group, and the ADP contents in the lov group and the treat group were even higher than that in the control group. This indicates that GLF-217 can maintain or stimulate adipocytes to secrete ADP. It shows that GLF-217 can maintain or stimulate the normal secretion of ADP by adipocytes, thereby alleviating and improving atherosclerosis.

[0220] ③ Lactobacillus fermentum GLF-217 alleviated the liver injury and inflammation caused by high-fat diet and lovastatin

[0221] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are two important indicators for judging liver injury, and an increase in their contents means that there are problems with liver function. Both AST and ALT in the model group were higher than those in the control group, while both AST and ALT in the pro group were lower than those in the model group and were at the same level as the control group. Both TNF-α and IL-6 in the model group were higher than those in the control group; the content of TNF-α in the serum of the pro group was lower than that in the model group; the content of IL-6 in the serum of the pro group was lower than that in the model group. Therefore, GLF-217 can reduce the increase in TNF-α and IL-6 caused by high-fat diet.

[0222] Both AST and ALT in the model group and the lov group were higher than those in the control group, and the AST content in the lov group was higher than that in the model group. It is speculated that this may be because taking the drug aggravated the damage to the liver, resulting in an increase in the AST content. Both AST and ALT in the treat group were lower than those in the model group and the lov group. Except that the TNF-α content in the treat group was lower than that in the lov group, there were no significant differences in other groups.

[0223] The IL-6 content in the lov group was significantly higher than that in other groups, while there was no obvious increase in the treat group. This indicates that lovastatin can stimulate the production of the inflammatory factor IL-6, while the treat group will not cause an increase in inflammatory factors.

[0224] ④ Lactobacillus fermentum GLF-217 regulated the contents of intestinal metabolites after high-fat diet

[0225] After freeze-drying the cecal contents of golden hamsters, short-chain fatty acids (SCFAs) were extracted, and the SCFA concentration was measured by a gas chromatograph. Figure 49 Schematic diagram of the short-chain fatty acid content in golden hamsters of the Lactobacillus fermentum GLF-217 prevention group, as Figure 49As shown, the SCFAs production in the model group was lower, and the acid production in the pro group was better than that in the model group. Among them, compared with the model group, the contents of acetic acid, propionic acid, isobutyric acid, isovaleric acid, and valeric acid in the pro group were significantly higher than those in the model group (P<0.05). From this, we can conclude that a high-fat diet can lead to a decrease in the level of intestinal SCFAs, and the intervention of GLF-217 can significantly improve the content of beneficial metabolites in the intestine.

[0226] Figure 50 Schematic diagram of the content of short-chain fatty acids in golden hamsters in the treatment group of Lactobacillus fermentum GLF-217. As Figure 50 shown, the concentration of SCFAs in the high-fat diet group was lower than that in the control group. Among them, the SCFAs content in the lov group was the lowest, and the contents of acetic acid, propionic acid, isobutyric acid, and isovaleric acid were significantly lower than those in the model group. Compared with the Model group, there was no significant difference in the production of short-chain fatty acids in the treat group. From this, it can be concluded that the intake of lovastatin will reduce the content of short-chain fatty acids in the intestine, while the intake of GLF-217 will not reduce the content of SCFAs.

[0227] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Lactobacillus fermentum GLF-217, characterized in that, The Lactobacillus fermentum GLF-217 was preserved in the China General Microbiological Culture Collection Center on September 4, 2023, with the preservation number of CGMCC No. 28336.

2. Use of the Lactobacillus fermentum GLF-217 or its bacterial suspension according to claim 1 in the preparation of a pharmaceutical composition or health product that helps to reduce the contents of triglyceride, total cholesterol and low-density lipoprotein in serum or has an auxiliary protective effect on reducing liver injury.

3. Use of the Lactobacillus fermentum GLF-217 or its bacterial suspension according to claim 1 in the preparation of a pharmaceutical composition for preventing and / or treating ulcerative colitis.

4. The application according to claim 3, characterized in that, The ulcerative colitis is dextran sulfate sodium-induced ulcerative colitis.

5. The application according to claim 3, wherein The prevention and / or treatment of ulcerative colitis includes at least one of the following: improving the intestinal mucosal barrier, alleviating the inflammatory response or regulating the intestinal microbiota structure.

6. The application according to claim 5, characterized in that, The alleviation of the inflammatory response is one or more of increasing the expression of IL-10, decreasing the expression of IL-1β, decreasing the expression of IL-6, decreasing the expression of IFN-γ or decreasing the expression of TNF-α.

7. The application according to claim 5, wherein The regulation of the intestinal microbiota structure is one or more of promoting the growth of probiotics in the intestinal flora, inhibiting the growth of pathogenic bacteria in the intestinal flora, increasing the abundance of short-chain fatty acid-producing species or increasing the intestinal flora diversity; the improvement of the intestinal mucosal barrier refers to promoting the expression of mucin 2 and tight junction protein-1.

8. A bacterial agent, characterized in that, Comprising the Lactobacillus fermentum GLF-217 and / or its bacterial suspension according to claim 1.

9. Use of the bacterial agent according to claim 8 in the preparation of a pharmaceutical composition or health product that helps to reduce the contents of triglyceride, total cholesterol and low-density lipoprotein in serum or has an auxiliary protective effect on reducing liver injury.

10. Use of the bacterial agent according to claim 8 in the preparation of a pharmaceutical composition for preventing and / or treating ulcerative colitis.

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