Bacillus natto and ginseng insoluble dietary fiber composition and application thereof in prevention or treatment of colitis

Abstract

The invention belongs to the field of pharmaceutical compositions, and particularly relates to a bacillus natto and ginseng insoluble dietary fiber composition and application thereof to prevention or treatment of colitis, and the preservation number of bacillus natto is CGMCC No. 27770; the volume mass ratio of the bacillus natto JLNA2301 bacterial liquid to the ginseng insoluble dietary fibers in the composition is (8 to 10) ml: (1.25 to 7.5) g; the composition can resist UC and regulate intestinal flora, dual intervention of prevention and treatment of UC is achieved, and side effects caused by medicine use are avoided.

Description

Technical Field

[0001] This invention belongs to the field of compositions, and specifically relates to a composition and its application for the prevention or treatment of ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a recurrent inflammatory disease affecting the colonic mucosa, with a continuously rising global incidence rate and currently no cure. Long-term use of traditional drugs such as aminosalicylic acids, glucocorticoids, and immunosuppressants can easily lead to adverse reactions such as increased risk of infection, bone marrow suppression, and metabolic disorders.

[0003] Probiotics have shown great potential in regulating gut microbiota and enhancing barrier function, but single strains are insufficient to comprehensively regulate the complex gut microbiota network and lack depth in regulating microbial metabolic pathways. Bacillus natto (Bacillus natto) Bacillus natto Bacillus subtilis, also known as natto bacteria, is a GRAS-recognized edible strain. The gut health potential of Bacillus subtilis remains to be explored. As a prebiotic, insoluble dietary fiber can be fermented by gut microbiota to produce short-chain fatty acids (such as butyric acid), which then combine with probiotics to form synbiotics, exerting a stronger health effect. However, current research focuses on insoluble dietary fiber from traditional grain sources such as soybean residue and wheat bran, while the interventional value of ginseng insoluble dietary fiber in colitis has not been explored.

[0004] Ginseng is a traditional and precious Chinese medicinal herb, and a nourishing food with medicinal properties. Ginseng residue, a byproduct of the extraction of ginseng's active ingredients, contains a large amount of dietary fiber and is often treated as waste during production and processing, resulting in significant resource waste. Ginseng insoluble dietary fiber (IDF) contains numerous active groups such as hydroxyl, carboxyl, and amide groups, exhibiting good water-oil retention, adsorption, and cation exchange capabilities. It can regulate blood sugar, lower cholesterol, and prevent obesity, but its therapeutic properties for colitis have not been reported. Current combinations of probiotics and insoluble dietary fiber often employ lactic acid bacteria with fruit and grain insoluble dietary fiber (such as orange peel fiber and soybean residue fiber), without specifically designed formulations tailored to the pathological characteristics of colitis (UC), and no combination can simultaneously achieve precise regulation during both the prevention and treatment phases. Summary of the Invention

[0005] The purpose of this invention is to provide a composition containing Bacillus natto and ginseng insoluble dietary fiber, which can combat UC and regulate intestinal flora, achieving dual intervention for the prevention and treatment of UC, and avoiding the side effects caused by drug use.

[0006] The objective of this invention is achieved through the following technical solution: A composition containing Bacillus natto and ginseng insoluble dietary fiber, wherein the Bacillus natto is named JLNA2301 and has the accession number CGMCC No. 27770.

[0007] As a more preferred technical solution of the present invention: the ginseng insoluble dietary fiber is obtained by simultaneously enzymatically hydrolyzing ginseng residue with heat-stable α-amylase, neutral protease and amyloglucosidase in a one-step process, at a pH of 6.0, a temperature of 60℃ and a time of 30 min; the precipitate is collected by centrifugation; the mass ratio of the heat-stable α-amylase, neutral protease and amyloglucosidase is 1:1:1-3.

[0008] As a more preferred technical solution of the present invention: the volume-to-mass ratio of Bacillus natto JLNA2301 bacterial solution to ginseng insoluble dietary fiber in the composition is (8-10) ml:(1.25-7.5) g, and the viable count of Bacillus natto JLNA2301 in the bacterial solution is 1×10⁻⁶. 8 -1×10 10 CFU / ml.

[0009] Another object of the present invention is to provide the application of the above-mentioned composition containing Bacillus natto and ginseng insoluble dietary fiber in the preparation of feed, food or health products for the prevention or treatment of ulcerative colitis.

[0010] As a preferred technical solution of the present invention: Bacillus natto JLNA2301 bacterial solution is administered to mice via gavage, and ginseng insoluble dietary fiber is added to the mouse feed.

[0011] As a more preferred technical solution of the present invention: the composition has at least one of the following effects in preventing ulcerative colitis: (1) Prevent abnormal expression levels of the animal colon tissue barrier proteins Occludin, Claudin1, and Claudin3; (2) Reduce the level of lipopolysaccharide (LPS) in animal serum and downregulate the expression levels of colonic inflammatory proteins TLR4 and NF-κB p65; (3) Reduce animal intestinal tract Streptococcus Increase bacterial abundance Parabacteroides Microbial abundance; (4) Regulate the abundance of genes that regulate linoleic acid metabolism, sphingolipid metabolism, arginine and proline metabolism in animal gut microbiota.

[0012] As a more preferred technical solution of the present invention: the composition has at least one of the following effects in treating colitis: (1) Alleviate the abnormal expression of Occludin, Claudin1, and Claudin3 in the colonic tissue barrier proteins of animals with colitis; (2) Reduce serum lipopolysaccharide levels in animals with colitis and downregulate the expression levels of colonic inflammatory proteins TLR4 and NF-κB p65; (3) Reduces intestinal pressure in animals with colitis Allobaculum Microbial abundance; (4) Regulates the alanine metabolism, glutathione metabolism, butyrate metabolism and fatty acid metabolism pathways in the intestinal flora of animals with colitis.

[0013] The beneficial effects are as follows: The composition of this invention can be used for the prevention or treatment of ulcerative colitis, enabling oral administration and avoiding the side effects of traditional drugs. It also allows for phased intervention, improving the disease activity index and colon length in mice with dextran sulfate sodium-induced colitis from both preventative and therapeutic perspectives. This reduces serum lipopolysaccharide levels, upregulates the expression levels of colonic tight junction proteins Occludin, Claudin1, and Claudin3, maintains intestinal barrier integrity, inhibits goblet cell reduction and crypt loss, reduces serum inflammatory factors (LPS, IL-1β, IL-6, and TNF-α) levels in colitis mice, and simultaneously reduces colonic TLR4 and NF-κBp65 protein expression, inhibiting the LPS / TLR4 / NF-κB signaling pathway and alleviating colitis.

[0014] The composition of this invention improves the diversity and structure of the gut microbiota in colitis mice and reduces... Streptococcus and Allobaculum Increasing the abundance of harmful bacteria significantly upregulated the gene expression abundance of gut microbiota related to linoleic acid metabolism, glutathione metabolism, and amino acid metabolism pathways such as arginine and proline.

[0015] The compositions of the present invention have a significant ameliorative effect on colonic damage in colitis mice, indicating that they can be used to develop feed, food or health products for the prevention or treatment of ulcerative colitis. Attached Figure Description

[0016] Figure 1 Phylogenetic tree (A) and colony morphology (B) of Bacillus natto JLNA2301.

[0017] Figure 2 To investigate the effects of the composition on body weight (A), DAI score (B), and colon length (CD) in mice with colitis during the prevention process (comparison between the model group and the control group, #P<0.05, ##P<0.01, ###P<0.001; comparison between the model group and the composition group, ...), P < 0.05 P < 0.01; ns indicates no significant difference.

[0018] Figure 3To investigate the effects of the composition on HE staining (A, 200µm scale bar) and pathological score (B), aricin blue staining (C, 100µm scale bar), and the number of goblet cells (D) in colon tissue pathological sections of colitis mice during the prevention process (comparison between model group and control group, #P<0.05, ##P<0.01, ###P<0.001; comparison between model group and composition group, ... P < 0.05 P < 0.01).

[0019] Figure 4 To prevent abnormal levels of oxidative and inflammatory factors in colitis mice, the composition was used to: (A) serum SOD activity, (B) serum MDA level, and (CF) serum TNF-α, IL-1β, IL-6, and IL-10 levels (comparison between the model group and the control group, #P<0.05, ##P<0.01, ###P<0.001; comparison between the model group and the composition group, ...). P < 0.05 P < 0.01; ns indicates no significant difference.

[0020] Figure 5 To investigate the effects of the composition on the expression levels of colonic barrier proteins and inflammatory pathway proteins in colitis mice during the prevention process; Western blot analysis (WB) of colonic barrier proteins Occludin, Claudin1, and Claudin3 (A) and their gray-scale values ​​(BD); Western blot analysis (F) of serum lipopolysaccharide content (E) and TLR4 and NF-κB p65 proteins (GH) compared with protein band gray values ​​(model group vs. control group, #P < 0.05, ##P < 0.01, ###P < 0.001; model group vs. composition group, ... P < 0.05 P < 0.01).

[0021] Figure 6 Analysis of gut microbiota structure disorder in mice with the composition for preventing colitis; (A) α-diversity analysis (Chao1 and Shannon indices); (B) β-diversity principal coordinates analysis (PCoA) based on Bray-Crutis distance; (C) relative abundance (%) of taxonomic composition at the phylum and (D) genus levels; intergroup differences in species abundance at the phylum (E) and genus (FH) levels (comparison between model group and control group, △P<0.05, △△P<0.01, △△△P<0.001; comparison between model group and composition group, P<0.05, P<0.01, P < 0.001; ns indicates no significant difference.

[0022] Figure 7 The effects of the composition on intestinal marker species in colitis mice during prevention (A, B) and its correlation with colitis markers (C) were analyzed. P<0.05, P<0.01, P<0.001).

[0023] Figure 8 To investigate the statistical analysis of metabolic pathways that showed significant differences in the expression of functional genes in the gut microbiota among groups during the prevention process (AC, 95% confidence interval) and their Spearman correlation analysis with enteritis markers (D).

[0024] Figure 9 The effects of the composition on body weight (A), DAI score (B), and colon length (CD) in mice with colitis during treatment were investigated (comparison between the model group and the control group, #P<0.05, ##P<0.01, ###P<0.001; comparison between the model group and the composition group, ...). P < 0.05 P < 0.01; ns indicates no significant difference.

[0025] Figure 10 The effects of the treatment composition on HE staining (A, 200µm scale bar) and pathological score (B) of colon tissue pathological sections in colitis mice, aricin blue staining (C, 100µm scale bar) and the number of goblet cells (D) were investigated. (Comparison between the model group and the control group: #P<0.05, ##P<0.01, ###P<0.001; comparison between the model group and the composition group:) P < 0.05 P < 0.01).

[0026] Figure 11 To improve the levels of oxidative and inflammatory factors in colitis mice, the composition was used to measure: (A) serum SOD activity, (B) serum MDA level, and (CF) serum TNF-α, IL-1β, IL-6, and IL-10 levels (comparison between the model group and the control group, #P<0.05, ##P<0.01, ###P<0.001; comparison between the model group and the composition group, ...). P < 0.05 P < 0.01; ns indicates no significant difference.

[0027] Figure 12The effects of the treatment composition on the expression levels of colonic barrier proteins and inflammatory pathway proteins in colitis mice; Western blot analysis of colonic barrier proteins Occludin, Claudin1, and Claudin3 (A) and their gray-scale bar charts (BD); serum lipopolysaccharide content (E); and TLR4 protein and NF-κB p65 value bar charts; comparison of protein band gray-scale values ​​(GH) between the model group and the control group. P < 0.05 P < 0.01, P < 0.001; compared with the composition group, the model group, P < 0.05 P < 0.01; ns indicates no significant difference.

[0028] Figure 13 Analysis of gut microbiota structure in mice protected by the composition; (A) α-diversity analysis (Chao1 and Shannon indices); (B) Principal coordinate analysis of Beta diversity based on Bray-Crutis distance (PCoA); (C) relative abundance (%) of taxonomic composition at the phylum and (D) genus levels; intergroup differences in species abundance at the phylum (E) and genus (FH) levels (comparison between model group and control group, △P<0.05, △△P<0.01, △△△P<0.001; comparison between model group and composition group, P<0.05, P<0.01, P < 0.001; ns indicates no significant difference.

[0029] Figure 14 The effects of the composition on intestinal marker species in colitis mice during treatment (A, B) and its correlation with colitis markers (C) P<0.05, P<0.01, P<0.001).

[0030] Figure 15 Statistical analysis of metabolic pathways showing significant differences in gene expression of gut microbiota function among treatment groups (AC, 95% confidence interval) and Spearman correlation analysis (D) between them and enteritis markers. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, which can be obtained commercially by those skilled in the art.

[0032] The strains, animals, materials, and reagents used in the examples are as follows: Bacillus natto JLNA2301 was isolated from rice straw in paddy fields in Heilinzi Town, Gongzhuling City, Jilin Province. It was identified and preserved by the Food Microbiology Team of Jilin Academy of Agricultural Sciences and deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No: 27770.

[0033] Seven-week-old specific pathogen-free (SPF) male C57BL / 6J mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., Animal Production License No.: SCXK (Liaoning) 2020-0001. Maintenance feed was purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0034] Ginseng insoluble dietary fiber feed: Ginseng insoluble dietary fiber is added to the maintenance diet of mice at a mass fraction of 1.25%, 2.5%, and 5%. Both the maintenance diet and the ginseng insoluble dietary fiber feed are customized by Beijing Keao Xieli Feed Co., Ltd., and the products comply with the national standard GB13078-2017 "Feed Hygiene Standard".

[0035] The 5-year-old ginseng root was purchased from Jinjuyi Ginseng and Deer Antler Co., Ltd. in Fusong County, Jilin Province.

[0036] Example 1: Isolation, identification, preservation and preparation of Bacillus natto JLNA2301 bacterial culture.

[0037] (1) Isolation of Bacillus natto JLNA2301 The bacterial strain was collected from paddy fields in Heilinzi Town, Gongzhuling City, Jilin Province. 50g of rice straw was cut into short pieces, incubated in a 90℃ water bath for 5 minutes, and then placed in 200mL of sterile physiological saline. The mixture was shaken at 37℃ for 2 hours. The supernatant was inoculated into LB liquid medium and incubated at 37℃ for 1-2 days, during which the culture medium became turbid. The bacterial suspension was then serially diluted and incubated at 37℃ for 1-2 days. Typical single colonies were selected based on colony morphology and purified. A strain with round or irregular colonies, milky white in color, and slightly thin with wrinkles was obtained. Preliminary identification under microscopy revealed it to be Bacillus subtilis. Figure 1As shown, it is named JLNA2301. Furthermore, this embodiment demonstrates through experiments that the optimal culture temperature for strain JLNA2301 is 30~40℃, and the maximum tolerable temperature is 90℃.

[0038] (2) Identification of Bacillus natto JLNA2301 The physiological and biochemical identification of strain JLNA2301 was carried out with reference to the "Manual of Systematic Identification of Common Bacteria". The results are shown in Table 1 below.

[0039] Table 1

[0040] Molecular biological identification of strain JLNA2301: Healthy strain JLNA2301 was inoculated into LB liquid medium and cultured at 37℃ and 180 rpm for 48 h to obtain bacterial culture. After centrifugation, the mycelium was collected in EP tubes, washed twice with sterile water, and genomic DNA was extracted from strain JLNA2301 using a bacterial genomic DNA extraction kit. Using the extracted DNA product as a template, forward primer 341F (SEQ ID NO: 5) was used... ’ -CCTAYGGGRBGCASCAG-3 ’ ) and reverse primer 806R (SEQ ID NO: 5) ’ --3 ’ PCR amplification was performed, and the amplified products were sequenced to obtain the 16S rDNA V3-V4 gene sequences of strain JLNA2301, as shown in SEQ ID NO.3. Homology analysis of the 16S rDNA V3-V4 sequences was performed in the GenBank library using the BLAST program. The results showed that strain JLNA2301 was homologous to... Bacillus subtilis strain HBUAS68749 (GenBank: OP009859) shows 100% homology. See the phylogenetic tree below. Figure 1 .

[0041] (3) Preservation of Bacillus natto JLNA2301 Based on the colony characteristics, physiological and biochemical characteristics, and molecular biological identification results of strain JLNA2301 on petri dishes, strain JLNA2301 isolated in this invention was identified as Bacillus natto (Bacillus). Bacillus nattoBacillus natto JLNA2301 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27770, on July 3, 2023. The depositary address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0042] (4) Preparation of Bacillus natto JLNA2301 bacterial culture Strain activation: The bacterial culture stored in glycerol tubes at -80ºC was inoculated at a 2% inoculum into LB liquid medium (10% peptone, 3% glucose, 3% yeast extract, 5% sodium chloride, pH 6.2-6.5) for the first activation, and incubated at 37ºC for 12 h at 175 rpm. For the second activation, a 3% inoculum was inoculated into Erlenmeyer flasks containing 100 mL of LB liquid medium and incubated at 37ºC for 6-8 h. Viable cell counts were determined using the dilution plating method, reaching 1 × 10⁻⁶. 10 Use at CFU / mL.

[0043] Cell preparation: Pour the cultured bacterial solution into a 50 mL centrifuge tube, centrifuge at 4ºC, 4000 rpm for 10 min, discard the supernatant, and retain the bacterial cells. Wash the bacterial cells three times with sterile phosphate-buffered saline (PBS, pH 7.4) to remove any residual culture medium, centrifuge at 4ºC, 4000 rpm for 10 min. Resuspend the bacterial cells in sterile physiological saline (with a viable count of at least 1 × 10⁻⁶ cells / mL). 8 (CFU / mL), used for subsequent gavage.

[0044] Example 2: Preparation of ginseng insoluble dietary fiber feed.

[0045] The residue from ginseng root, extracted twice by boiling in 10 times its volume of water (2 hours each time), was dried at 60°C to obtain ginseng residue. Heat-stable α-amylase (0.5%), neutral protease (0.5%), and amylase (0.5%, 1%, or 1.5%) were added to the ginseng residue. A one-step enzymatic hydrolysis was performed at pH 6.0, 60°C, for 30 minutes. After the reaction, the residue was centrifuged, filtered, washed, dried, and pulverized to obtain ginseng insoluble dietary fiber (IDF). In the following examples, ginseng insoluble dietary fiber (IDF) was prepared using a mass ratio of heat-stable α-amylase, neutral protease, and amylase of 1:1:2.

[0046] Preparation of ginseng insoluble dietary fiber feed: Low-dose group: Ginseng insoluble dietary fiber was added to the maintenance feed at a final mass fraction of 1.25%; Medium-dose group: Ginseng insoluble dietary fiber was added to the maintenance feed at a final mass fraction of 2.5%; High-dose group: Ginseng insoluble dietary fiber was added to the maintenance feed at a final mass fraction of 5%.

[0047] Example 3: Composition for the prevention of ulcerative colitis.

[0048] Seven-week-old SPF-grade male C57BL / 6J mice, weighing 20-25g, were housed and acclimatized for one week in a constant temperature, humidity, and light environment (temperature 20-26℃, humidity 40-70%, 12h light / 12h darkness cycle). Mice in each group had free access to food and water, and were then randomly assigned to different groups. Each mouse received 2-3g of food and 3mL of water per day, and was administered 200μL of Bacillus natto JLNA2301 bacterial suspension (BN) via gavage daily, with an effective viable count of 1×10⁶. 8 -1×10 10 CFU / mL.

[0049] 1. Animal experiment grouping and administration.

[0050] Preparation of 5% sodium dextran sulfate (DSS) solution: Add 5g DSS to a volumetric flask containing 100mL sterile water and mix thoroughly. Prepare fresh solution daily for each use.

[0051] The animal experiment design for preventing colitis is shown in Table 2. C57BL / 6J mice were randomly divided into 5 groups (6 mice in each group). From day 1 to 14, the mice were given preventive drinking water and gavage according to the design in the table. From day 15 to 25, the drinking water was changed to 5% DSS to establish a colitis disease model.

[0052] Table 2

[0053] 2. Measurement of body weight, disease activity index, and colon length.

[0054] During the experiment, mouse body weight, food intake, activity level, colonic bleeding, and stool viscosity were recorded daily at regular intervals. The Disease Activity Index (DAI) was calculated, and the DAI scoring criteria are shown in Table 3. After the modeling period, the mice were sacrificed, and the length of the entire colon was measured.

[0055] Table 3

[0056] The composition prevented the deterioration of pathological indicators in mice with colitis, alleviated weight loss and abnormally high DAI scores caused by colitis, and significantly improved colonic shortening. Figure 2 As shown.

[0057] 3. Hematoxylin-eosin staining and aricin blue staining.

[0058] One day before the end of the experiment, mouse feces were aseptically collected and stored at -80ºC for later testing. After modeling, mice were fasted for 12 hours but allowed free access to water. Blood was collected from the mice's eyes, and the serum was separated and stored at -80ºC for later testing. All mice were euthanized by dislocation, and 1 cm of the distal colon was removed and immediately placed in 4% paraformaldehyde solution. The remaining colon tissue and cecal contents were stored at -80ºC.

[0059] Colon samples fixed with 4% paraformaldehyde were stained with hematoxylin-eosin (H&E) and aricin blue. The colon tissue structure and goblet cell count were observed under a microscope and photographed. Tissue damage was scored for each group of colon tissue sections, and the scoring criteria are shown in Table 4.

[0060] Table 4

[0061] The composition prevents the deterioration of colonic tissue pathology in colitis mice, such as Figure 3 As shown, the colon in the model group exhibited a significant inflammatory response, characterized by extensive mucosal ulceration, crypt loss, reduced goblet cell lysis, and decreased muscle layer thickness. In contrast, the composition group showed improved intestinal epithelial cell integrity and tissue damage, reduced goblet cell lysis, and lessened crypt loss.

[0062] 4. Serum and colon markers were measured.

[0063] The composition showed the following effects in preventing oxidative stress and abnormal inflammatory markers in colitis mice: Figure 4 As shown. The composition reversed the abnormalities in serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in the model group, alleviated the abnormally elevated levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum of mice in the model group, and significantly increased the levels of the anti-inflammatory cytokine IL-10. P <0.01).

[0064] 5. Detection of expression levels of colonic barrier proteins and inflammatory pathway proteins.

[0065] An equal amount of protein (30 μg) from each colon sample was electrophoresed on an SDS-polyacrylamide gel pre-stained with protein labels. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane, blocked at room temperature for 1.5 h, and gently agitated. The membrane was incubated overnight with primary antibody at 4°C, washed, and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The membrane was washed 1–3 times and developed using a 1:1 developing solution, and images were taken using a chemiluminescence imaging system.

[0066] The composition prevents damage to colonic barrier proteins in colitis mice, such as Figure 5As shown in AD. The composition enhanced the expression levels of the barrier proteins Occludin, Claudin3, and Claudin1 in the model group to varying degrees. P <0.01 or P <0.05). The composition prevents damage to the expression of proteins related to inflammatory signaling pathways in colitis mice, such as... Figure 5 As shown in EH, the composition reduced serum lipopolysaccharide levels in the model group and reversed the abnormally elevated expression levels of TLR4 and NF-κB p65 proteins, thus preventing colitis by inhibiting the LPS / TLR4 / NF-κB signaling pathway. This demonstrates that the composition intervention can prevent damage to colonic barrier protein expression and abnormal expression levels of inflammatory pathway proteins in colitis mice.

[0067] 6. Structural analysis and functional prediction of gut microbial diversity The gut microbiota structure and composition were analyzed using 16S rRNA sequencing technology. The hypervariable V3-V4 series of the bacterial 16S rRNA gene were amplified using PCR, with the universal bacterial forward primer 341F (5... ’ -CCTAYGGGRBGCASCAG-3 ’ ) and reverse primer 806R (5 ’ -GGACTCNNGGGTATCTAAT-3 ’ The purified amplified products were used to construct libraries on the Illumina-NovaSeq-6000 platform (Illumina, San Diego, USA) according to standard operating procedures, with the assistance of Shanghai Paisennong Biotechnology Co., Ltd. The expression of functional genes of gut microbiota in each group was analyzed using the KEGG signaling pathway database (https: / / www.kegg.jp / ).

[0068] (1) The combination intervention can prevent the reduction of intestinal flora diversity and structural damage in colitis mice.

[0069] like Figure 6 The composition shown prevented the decrease in gut microbiota diversity in colitis mice. Compared with the control group, the α-diversity index, which is related to both microbiota richness (Chao1) and diversity (Simpson), was decreased in the model group. Compared with the model group, the composition group showed a slight increase in the Simpson index (2.02-3.04%). Figure 6 A). Although α-diversity did not change significantly, β-diversity results showed ( Figure 6 B) Significant differences in community structure were observed among the control group, model group, and composition group, with the composition group being more similar to the control group, suggesting that the composition can effectively improve the intestinal microbial community structure of colitis mice.

[0070] Phylogenetic composition analysis revealed ( Figure 6 In the model group (C, 6E), the abundance of Firmicutes in the gut microbiota was abnormally increased, while the abundance of Bacteroidetes decreased significantly, and the F / B ratio increased significantly. P <0.01). The composition significantly altered the aforementioned abundance changes at the phylum level of the gut microbiota in colitis-affected mice. At the genus level, the taxonomic composition ( Figure 6 D, 6F-H), compared with the control group, the model group Prevotella The abundance of the bacterial community was significantly reduced. Streptococcus and Bacteroides The abundance of gut microbiota was significantly increased. After intervention with the composition, the abundance of these microbiota recovered to near-normal levels. In addition, the low-dose group also showed a significant increase in beneficial bacteria. Bifidobacterium Abundance in the gut of UC mice ( P <0.01). The above description indicates that the composition prevents intestinal flora imbalance in colitis mice.

[0071] (2) The combination intervention can prevent abnormalities in intestinal flora marker species and enteritis indicators in colitis mice.

[0072] like Figure 7 The composition shown can prevent dysbiosis of gut microbiota species composition in colitis mice. A heatmap of genus-level species composition shows that in the model group... Oscillospira , Streptococcus , Desulfovibrio The bacterial flora abundance was high. In the low-dose group... Bifidobacterium The bacterial flora abundance was high. In the medium-dose group... Prevotella The abundance of bacterial flora was relatively high in the high-dose group. Ruminococcus , Coprococcus , Parabacteroides High bacterial abundance ( Figure 7 A). Further analysis was conducted using a random forest model to identify marker species with high abundance among groups ( Figure 7 B). Odoribacter It is the most representative species of the microbial community structure in the model group; Bifidobacterium It is the most representative species of the gut microbiota structure in the low-dose group; Prevotella It is the most representative marker species of the gut microbiota structure in the medium-dose group; Parabacteroides It is the most representative species of the bacterial community structure in the high-dose group.

[0073] Spearman correlation analysis was used to further explore the relationship between gut microbiota and enteritis markers. Figure 7 C). Iconic species in the model group StreptococcusSignificantly negatively correlated with colon length, SOD, and IL-10 levels, and significantly positively correlated with lipopolysaccharide, MDA, IL-1β, IL-6, and TNF-α levels, all of which were inhibited in all groups of the composition. This indicates that the composition can regulate colon length and antioxidant levels, and reduce lipopolysaccharide and inflammation levels by altering the abundance of specific bacterial genera in the gut microbiota. These results suggest that the composition can prevent colitis by regulating the gut microbiota structure.

[0074] (3) Combination intervention can prevent metabolic dysfunction of fecal intestinal flora.

[0075] The expression of functional genes of gut microbiota in each group was analyzed using the KEGG signaling pathway database (https: / / www.kegg.jp / ).

[0076] The PICSTR method was used to predict changes in gut microbiota function caused by colitis, such as... Figure 8 The composition shown can prevent intestinal flora dysfunction in colitis mice. The overall gene expression abundance levels in each group under the primary signaling pathway were relatively similar. In the secondary signaling pathway, metabolic-related pathways showed significant changes. Further analysis of metabolic-related differential pathways revealed that, compared with the model group, the low-dose group increased the abundance of genes related to sphingolipid metabolism, fatty acid metabolism, and arginine and proline metabolism in the intestinal flora. Figure 8 A), the medium-dose group increased the abundance of genes related to fatty acid metabolism, linoleic acid metabolism, and sphingolipid metabolism in the gut microbiota. Figure 8 B), the high-dose group increased the abundance of genes related to linoleic acid metabolism in the gut microbiota (B). Figure 8 C).

[0077] Spearman correlation analysis was used to further explore the relationship between the above metabolic pathways and enteritis indicators. Figure 8 D). The linoleic acid metabolism enriched in the composition was positively correlated with colon length, SOD and IL-10 levels, and significantly negatively correlated with lipopolysaccharide, IL-1β, IL-6 and TNF-α levels. This suggests that the composition may increase colon length and antioxidant levels, reduce lipopolysaccharide content and inflammation levels, and ultimately alleviate colitis symptoms in mice by regulating the linoleic acid metabolism level of the gut microbiota.

[0078] Example 4: Composition for the treatment of ulcerative colitis.

[0079] 1. Animal experiment grouping and administration.

[0080] The animal experimental design for the treatment of colitis with the composition is shown in Table 5. C57BL / 6J mice were randomly divided into 5 groups (6 mice in each group), namely control group, model group, low-dose group, medium-dose group and high-dose group of composition. The drugs were administered continuously for 8 days to establish a disease model of ulcerative colitis. BN was Bacillus natto JLNA2301 and IDF was ginseng insoluble dietary fiber.

[0081] Table 5

[0082] 2. Measurement of body weight, disease activity index, and colon length.

[0083] The composition improved the body weight and pathological indicators of mice with colitis, such as Figure 9 As shown. The composition alleviated weight loss and abnormally high DAI scores caused by colitis, and significantly improved colonic shortening.

[0084] 3. Hematoxylin-eosin staining and aricin blue staining.

[0085] The composition protects the colonic tissue structure in mice during the active phase of colitis. Figure 10 As shown, the colon in the model group exhibited a significant inflammatory response, with extensive mucosal ulceration, crypt loss, reduced goblet cell lysis, and decreased muscle layer thickness. In contrast, the composition group showed improved intestinal epithelial cell integrity and tissue damage, significantly reduced goblet cell lysis, and lessened crypt loss.

[0086] 4. Serum and colon markers were measured.

[0087] The composition improves oxidative stress and inflammatory markers in colitis mice, such as Figure 11 As shown, the composition alleviated the abnormalities in serum SOD activity and MDA content in the model group mice, reversed the abnormally elevated levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in mouse serum, and significantly increased the content of the anti-inflammatory cytokine IL-10. P <0.01).

[0088] 5. Detection of expression levels of colonic barrier proteins and inflammatory pathway proteins.

[0089] The composition alleviates colonic barrier protein damage in colitis mice, such as Figure 12 As shown in AD, the composition enhanced the expression levels of barrier proteins Occludin, Claudin3, and Claudin1 in the model group to varying degrees. P <0.01 or P<0.05). The composition alleviates the impairment of expression of proteins related to inflammatory signaling pathways in colitis mice, such as... Figure 12 As shown in EH, the composition reduced serum lipopolysaccharide levels in the model group and reversed the abnormally elevated expression levels of TLR4 and NF-κB p65 proteins, thus alleviating colitis by inhibiting the LPS / TLR4 / NF-κB signaling pathway. This indicates that the composition improves the impaired expression of colonic barrier proteins and the abnormal expression levels of inflammatory pathway proteins in colitis mice.

[0090] 6. Structural analysis and functional prediction of gut microbial diversity (1) The composition improves the reduced diversity and structural disorder of the intestinal flora in mice with colitis.

[0091] like Figure 13 The composition shown protects the stability of the intestinal flora structure in colitis mice. Compared with the control group, the bacterial abundance (Chao1) and diversity (Simpson) indices in α-diversity were significantly reduced in the model group. P <0.01). Compared with the model group, the Simpson index of the combined groups was significantly increased ( P <0.01)( Figure 13 A). β-diversity analysis showed that the control group, model group, and composition group were separated from each other, exhibiting significant inter-group structural differences ( Figure 13 B). At the phylum level, the combined intervention altered the gut microbiota composition in colitis mice, particularly Firmicutes and Bacteroidetes (B). Figure 13 C). At the genus level, compared with the control group, the model group Allobaculum The abundance of the bacterial community increased significantly, and its abundance decreased after intervention with the composition. Figure 13 D). Compared with the control group, the Firmicutes / Bacteroidetes ratio in the model group was significantly increased ( P <0.01), the F / B ratio decreased significantly after the composition intervention ( Figure 13 E). Furthermore, the low-dose group significantly promoted the beneficial gut microbiota. Bifidobacterium and Lactobacillus proliferation ( Figure 12 F, 12G, P <0.01). The above description indicates that the composition protects the intestinal flora structure of colitis mice.

[0092] (2) The composition alleviated abnormalities in intestinal flora marker species and enteritis indicators in mice with colitis.

[0093] like Figure 14 The composition shown alleviates the abnormal species composition structure of the gut microbiota in mice with colitis, as indicated by the genus-level species composition heatmap. Figure 14 A), in the model group Ruminococcus, Allobaculum The bacterial flora abundance was high. In the low-dose group... Bifidobacterium The abundance of bacterial flora was relatively high in the medium-dose group. Bacteroides The abundance of microbial communities was relatively high. Further analysis using a random forest model was conducted to identify marker species for inter-group differences in abundance (…). Figure 14 B). Allobaculum It is the most representative species of the microbial community structure in the model group; Bifidobacterium It is the most representative species of the gut microbiota structure in the low-dose group; Bacteroides It is the most representative species of the bacterial community structure in the medium-dose group.

[0094] Spearman correlation analysis was used to further explore the relationship between gut microbiota and enteritis markers. Figure 14 C). Atypically elevated marker species in the model group Allobaculum Significantly negative correlations were observed with colon length, SOD, and IL-10 levels, while significant positive correlations were observed with lipopolysaccharide, MDA, IL-1β, IL-6, and TNF-α levels. These correlations were reversed in the composition group. This indicates that the composition can regulate colon length and antioxidant levels, and reduce lipopolysaccharide and inflammation levels by altering the abundance of specific genera of gut microbiota. These results suggest that the composition can alleviate colitis by regulating gut microbiota structure.

[0095] (3) The composition protects the metabolic function of fecal intestinal flora.

[0096] The PICSTR method was used to predict changes in gut microbiota function caused by ulcerative colitis, such as... Figure 15The composition shown protected the function of the gut microbiota in colitis mice. The overall gene expression levels in each group were relatively similar under the primary signaling pathway. In the secondary signaling pathway, metabolic-related pathways showed significant changes. Compared with the model group, the low-dose group increased the abundance of genes related to D-Alanine metabolism, glutathione metabolism, and butanoate metabolism in the gut microbiota; the medium-dose group increased the abundance of genes related to glutathione metabolism, D-Alanine metabolism, fatty acid metabolism, and butanoate metabolism; and the high-dose group significantly increased the abundance of genes related to glutathione metabolism, D-Alanine metabolism, fatty acid metabolism, and butanoate metabolism. These metabolic pathways may be the main mechanisms by which the composition protects the intestinal function of colitis mice.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition containing Bacillus natto and ginseng insoluble dietary fiber, characterized in that, The Bacillus natto strain was named JLNA2301 and its accession number is CGMCC No. 27770.

2. The composition according to claim 1, characterized in that, The ginseng insoluble dietary fiber mentioned above is produced by simultaneously enzymatically hydrolyzing ginseng residue with heat-stable α-amylase, neutral protease, and amyloglucosidase in a one-step process at pH 6.0, temperature 60℃, and time 30 min; the precipitate is collected by centrifugation; the mass ratio of the three enzymes is 1:1:(1-3).

3. The composition according to claim 1, characterized in that, The volume-to-mass ratio of Bacillus natto JLNA2301 bacterial suspension to ginseng insoluble dietary fiber in the composition is (8-10) ml:(1.25-7.5) g, and the viable count of Bacillus natto JLNA2301 in the bacterial suspension is 1×10⁻⁶. 8 -1×10 10 CFU / ml.

4. The use of the composition according to claim 1 in the preparation of feed, food or health products for the prevention or treatment of ulcerative colitis.

5. The application according to claim 4, characterized in that: Bacillus natto JLNA2301 bacterial solution was administered to mice via gavage, and ginseng insoluble dietary fiber was added to the mouse feed.

6. The application according to claim 4, characterized in that, The composition is effective in preventing ulcerative colitis in at least one of the following ways: (1) Prevent abnormal expression levels of the animal colon tissue barrier proteins Occludin, Claudin1, and Claudin3; (2) Reduce the level of lipopolysaccharide (LPS) in animal serum and downregulate the expression levels of colonic inflammatory proteins TLR4 and NF-κB p65; (3) Reduce animal intestinal tract Streptococcus Increase bacterial abundance Parabacteroides Microbial abundance; (4) Regulate the abundance of genes that regulate linoleic acid metabolism, sphingolipid metabolism, arginine and proline metabolism in animal gut microbiota.

7. The application according to claim 4, characterized in that, The composition is effective in treating colitis in at least one of the following ways: (1) Alleviate the abnormal expression of Occludin, Claudin1, and Claudin3 in the colonic tissue barrier proteins of animals with colitis; (2) Reduce serum lipopolysaccharide levels in animals with colitis and downregulate the expression levels of colonic inflammatory proteins TLR4 and NF-κB p65; (3) Reduces intestinal pressure in animals with colitis Allobaculum Microbial abundance; (4) Regulates the alanine metabolism, glutathione metabolism, butyrate metabolism and fatty acid metabolism pathways in the intestinal flora of animals with colitis.

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