Probiotic composition for relieving inflammatory bowel disease as well as preparation method and application thereof

Abstract

The invention discloses a probiotic composition for relieving inflammatory bowel disease as well as a preparation method and application thereof, and belongs to the technical field of microbial compositions. The probiotic composition is prepared from lactobacillus salivarius and a protective agent solution. The preparation method comprises the following steps: S1, inoculating lactobacillus salivarius GZLC1 into an MRS culture medium for culturing; transferring the strain into a fermentation tank for amplification culture to prepare a bacterial suspension; s2, centrifuging the cultured bacterial suspension, collecting thalli in precipitates, washing the thalli with sterile normal saline, and removing residual culture medium and other impurities to obtain concentrated bacterial liquid; and S3, fully mixing the concentrated bacterial liquid with the protective agent solution to prepare the probiotic composition. The probiotic composition provided by the invention comprehensively intervenes the occurrence and development of the inflammatory bowel disease from three aspects of oxidation resistance, inflammation resistance and flora regulation through a multi-target synergistic effect, and provides a new thought and method for preventing and treating the inflammatory bowel disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial compositions, and in particular relates to a probiotic composition for alleviating inflammatory bowel disease, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a type of chronic nonspecific intestinal inflammatory disease whose etiology is not yet fully understood. It is mainly manifested by diarrhea, abdominal pain, and bloody stools in severe cases. IBD mainly includes ulcerative colitis (UC) and Crohn's disease (CD). Among them, UC is a chronic nonspecific inflammatory bowel disease, which is mainly characterized by recurrent diarrhea, mucus, pus and blood in the stool, abdominal pain, and associated symptoms such as anemia and malnutrition. Its lesions usually start in the rectum and can continuously extend to the proximal colon, resulting in significant changes in stool consistency and characteristics; CD is a transmural inflammatory disease that can affect the entire digestive tract. The lesions are segmentally distributed and are commonly found in the terminal ileum and colon. Compared with UC, CD has a wider range of lesions, and the inflammation can penetrate the entire layer of the intestinal wall, often accompanied by complications such as fistulas and abscesses.

[0003] At present, the treatment of IBD mainly includes drug therapy, diet and nutritional support, and surgical treatment. Drug therapy is mainly based on 5-aminosalicylic acid drugs, glucocorticoids, immunosuppressants and biological agents, with the goal of controlling acute attacks, maintaining remission and preventing recurrence. However, traditional drugs have limited efficacy in some patients, and long-term use may cause side effects. For example, glucocorticoids may cause osteoporosis, diabetes, etc., and immunosuppressants may increase the risk of infection and tumors. Biological agents are expensive and not effective for all patients. Therefore, IBD has become a chronic inflammatory disease that seriously threatens human health.

[0004] Probiotics have shown significant potential in the treatment of IBD. Currently, probiotics cover a variety of naturally occurring microorganisms as well as genetically engineered strains designed to enhance specific functions. Commonly used probiotics in clinical and research include Bifidobacterium, Enterococcus, Saccharomyces cerevisiae, Lactobacillus salivarius, and Escherichia coli. These probiotics exert their therapeutic effects through a variety of mechanisms, such as promoting the expression of tight junction proteins occludin and zonula occludens-1 (ZO-1), enhancing the barrier function of intestinal epithelial cells, thereby effectively reducing intestinal permeability and preventing bacterial and endotoxin translocation. In addition, in terms of immune regulation, probiotics can regulate the balance of the intestinal immune system, inhibit the release of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β, and promote the production of anti-inflammatory cytokines such as IL-10 and TGF-β, thereby alleviating intestinal inflammatory responses, providing new ideas and methods for the treatment of colitis.

[0005] Therefore, in the process of preventing or treating IBD, it is particularly important to develop a more targeted, safer and more effective drug. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention discloses a probiotic composition for alleviating inflammatory bowel disease, and a preparation method and application thereof. The probiotic composition provided by the present invention provides a new strategy for the treatment of inflammatory bowel disease, especially ulcerative colitis, by exerting multiple effects of anti-oxidative stress, anti-inflammation and regulating intestinal flora, which can effectively alleviate inflammatory reactions and improve the intestinal microecological environment.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a probiotic composition for alleviating inflammatory bowel disease. The probiotic composition is prepared from Lactobacillus salivarius and a protective agent solution.

[0009] Furthermore, the salivarius Lactobacillus is classified and named as Lactobacillus salivarius GZLC1, and is preserved in the China Center for Type Culture Collection, with the preservation number: CCTCC NO: M 2023735. The address of the preservation unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation time is May 11, 2023.

[0010] Furthermore, the protective agent solution is prepared by uniformly mixing the protective agent and sterile water in a mass volume ratio of (5-20) g:100 mL.

[0011] Furthermore, the protective agent is composed of one or a combination of lactose, starch, microcrystalline cellulose, mannitol, gelatin, and magnesium stearate.

[0012] The present invention also provides a method for preparing the above-mentioned probiotic composition for alleviating inflammatory bowel disease, characterized in that it comprises the following steps:

[0013] S1. The frozen Lactobacillus salivarius GZLC1 was inoculated into MRS medium, the pH of which was 6.5-7.0, and cultured under anaerobic conditions at 37°C for 18-24 hours; then the strain was transferred to a fermenter, and cultured for 18-24 hours at a temperature of 37°C and a pH of 6.0-6.5, and the OD600 value of the bacterial solution was measured using a spectrophotometer. When the OD600 value of the bacterial solution reached 0.5-1.0, the culture was stopped to obtain a bacterial suspension;

[0014] S2. The bacterial suspension cultured in step S1 was centrifuged at 4000 rpm and 4°C for 10 min, the bacterial cells in the precipitate were collected, and the bacterial cells were washed twice with 0.85% sterile saline to remove the residual culture medium and other impurities to obtain a concentrated bacterial solution;

[0015] S3. The concentrated bacterial solution obtained in step S2 is fully mixed with the protective agent solution to obtain a mixed solution, that is, a probiotic composition.

[0016] Furthermore, the number of viable Lactobacillus salivarius GZLC1 in the probiotic composition in step S3 is 1×10 6 ~1×10 12 CFU / mL.

[0017] Furthermore, in step S3, the volume ratio of the concentrated bacterial solution to the protective agent solution is 1:(1-3).

[0018] The present invention also provides the use of the probiotic composition in preparing a pharmaceutical product for preventing or treating inflammatory bowel disease.

[0019] Furthermore, the dosage form of the drug product is an oral preparation, enteric-coated capsule, tablet, granule, suspension or enema.

[0020] Furthermore, the inflammatory bowel disease is ulcerative colitis induced by dextran sulfate sodium.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The probiotic composition provided by the present invention exhibits significant pleiotropic effects in improving enteritis-related pathological conditions, as follows: (i) By increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px), it effectively removes free radicals in the body, reduces the level of malondialdehyde (MDA), and reduces oxidative stress damage, thereby protecting colon tissue from free radical damage and maintaining the health and function of intestinal cells. (ii) By down-regulating the expression of pro-inflammatory factors (such as TNF-α, IL-6) and up-regulating the expression of anti-inflammatory factors (such as IL-10), it balances the inflammatory response of the intestinal immune system, effectively inhibits excessive inflammatory response, promotes the repair and regeneration of intestinal tissue, and provides an important guarantee for alleviating enteritis symptoms. (iii) It significantly changes the composition of intestinal flora, increases the abundance of beneficial bacteria (such as lactic acid bacteria, bifidobacteria, etc.), and significantly reduces the abundance of pathogenic bacteria and inflammation-related bacteria. This optimization of flora structure helps to restore the balance of intestinal microecology and further consolidates its role in promoting intestinal health.

[0023] In summary, the probiotic composition of the present invention comprehensively intervenes in the occurrence and development of inflammatory bowel disease from three aspects of anti-oxidation, anti-inflammatory and flora regulation through multi-target synergistic effects. It can not only effectively alleviate the pathological changes related to inflammatory bowel disease, but also fundamentally improve the intestinal microecological environment, providing new ideas and methods for the prevention and treatment of inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Effects of the probiotic combination provided by the present invention on clinical symptoms, tissue structure and serum-related indicators of mice with DSS-induced enteritis (a: DAI score, b: colon length, c: H&E staining, d: antioxidant capacity and inflammation level);

[0025] Figure 2 Effects of the probiotic composition provided by the present invention on the Venn diagram and Alpha diversity analysis of the intestinal flora of mice with DSS-induced enteritis (a: Venn diagram, b: index, c: rarefaction curve, d: rank abundance curve;

[0026] Figure 3 Beta diversity analysis of intestinal flora of mice with DSS-induced enteritis and structural analysis of intestinal flora at different classification levels (a: PCoA and NMDS, b: intestinal microbiota at different classification levels, c: Firmicutes vs. Bacteroidetes) by the probiotic composition provided by the present invention;

[0027] Figure 4 The effect of the probiotic composition provided by the present invention on the differential flora of intestinal microorganisms in mice with DSS-induced enteritis;

[0028] Figure 5 The present invention provides an effect of the probiotic composition provided by the present invention on the KEGG function of intestinal microorganisms in mice with DSS-induced enteritis. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] The invention provides a probiotic composition for alleviating inflammatory bowel disease. The probiotic composition is prepared from Lactobacillus salivarius and a protective agent solution.

[0033] Among them, the above-mentioned Lactobacillus salivarius is classified and named as Lactobacillus salivarius GZLC1, which is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2023735, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit time is May 11, 2023. The protective agent solution is prepared by mixing the protective agent and sterile water in a mass volume ratio of (5 to 20) g: 100 mL, and the protective agent is composed of one or a combination of lactose, starch, microcrystalline cellulose, mannitol, gelatin, and magnesium stearate.

[0034] The present invention also provides a method for preparing the above-mentioned probiotic composition for alleviating inflammatory bowel disease, and the specific steps are as follows:

[0035] S1. The frozen Lactobacillus salivarius GZLC1 was inoculated into MRS medium with a pH value of 6.5-7.0 and cultured at 37°C under anaerobic conditions for 18-24 hours; then the bacteria were transferred to a fermentation tank and cultured for 18-24 hours at a temperature of 37°C and a pH value of 6.0-6.5. The OD600 value of the bacterial solution was measured using a spectrophotometer. When the OD600 value of the bacterial solution reached 0.5-1.0, the culture was stopped to obtain a bacterial suspension.

[0036] S2. Centrifuge the bacterial suspension cultured in step S1 at 4000 rpm and 4°C for 10 min, collect the bacterial cells in the precipitate, and wash the bacterial cells twice with 0.85% sterile saline to remove residual culture medium and other impurities to obtain a concentrated bacterial solution.

[0037] S3. The concentrated bacterial solution obtained in step S2 is fully mixed with the above-mentioned protective agent solution in a volume ratio of 1:(1-3) to obtain a mixed solution, that is, a probiotic composition is prepared.

[0038] The viable number of Lactobacillus salivarius GZLC1 in the prepared probiotic composition was 1×10 6 ~1×10 12 CFU / mL.

[0039] The present invention also provides the use of the probiotic composition in preparing a pharmaceutical product for preventing or treating inflammatory bowel disease.

[0040] The dosage form of the above-mentioned drug product is oral preparation, enteric-coated capsule, tablet, granule, suspension or enema. In addition, the above-mentioned inflammatory bowel disease can be dextran sulfate sodium induced (DSS) ulcerative colitis.

[0041] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments:

[0042] Example 1

[0043] The probiotic composition for alleviating inflammatory bowel disease in this embodiment comprises the following specific steps:

[0044] S1. The frozen Lactobacillus salivarius GZLC1 was inoculated into MRS medium with a pH value of 6.5 and cultured under anaerobic conditions at 37°C for 24 hours; then the strain was transferred to a fermenter and cultured for 24 hours at a temperature of 37°C and a pH value of 6.0 to obtain a bacterial suspension.

[0045] S2. Centrifuge the bacterial suspension cultured in step S1 at 4000 rpm and 4°C for 10 min, collect the bacterial cells in the precipitate, and wash the bacterial cells twice with 0.85% sterile saline to remove residual culture medium and other impurities to obtain a concentrated bacterial solution.

[0046] S3. The concentrated bacterial solution obtained in step S2 is fully mixed with the protective agent solution in a volume ratio of 1:2 to obtain a mixed solution, that is, a probiotic composition.

[0047] The protective agent is composed of lactose, starch, microcrystalline cellulose, mannitol and gelatin in a mass ratio of 1:2:1:2:1. The protective agent solution contains sterile protective agent and water in a mass volume ratio of 7g:100mL.

[0048] The viable number of Lactobacillus salivarius GZLC1 in the probiotic composition prepared above was 1×10 9 CFU / mL.

[0049] Example 2

[0050] This example is an experiment on the effect of the probiotic composition prepared in Example 1 on the clinical symptoms, tissue structure and serum-related indicators of mice with colitis induced by dextran sulfate sodium (DSS).

[0051] Thirty ICR male mice produced by Yangzhou University with a body weight of 18 g ± 2 g were randomly divided into three groups, namely, a control group (CD), a model group (DD) and a probiotic composition treatment group (TD), with 10 mice in each group. The experimental period was 28 days. On the 1st to 7th day of the experiment, the control group (CD) and the model group (DD) were gavaged with an equal amount of sterile water, and the probiotic composition treatment group (TD) was gavaged with 0.2 mL of the probiotic composition prepared in Example 1 (containing 2×10 8CFU of live Lactobacillus salivarius GZLC1); on the 8th to 14th day of the experiment, the control group (CD) continued to drink sterile water every day, and the model group (DD) and the probiotic composition treatment group (TD) drank 2.5% DSS solution every day; on the 15th to 21st day, all groups resumed drinking sterile water; on the 22nd to 28th day, the model group (DD) and the probiotic composition treatment group (TD) drank 2.5% DSS solution again, and the colitis model was repeatedly induced until the end of the experiment. The specific detection and analysis are as follows.

[0052] (1) During the modeling period, the weight changes and fecal characteristics of the mice were recorded every day, and the fecal occult blood was detected by fecal occult blood test strips according to the following formula:

[0053]

[0054] The disease activity index (DAI) was calculated.

[0055] The typical clinical symptoms of DSS-induced colitis in mice include diarrhea, weight loss, and fecal occult blood. The severity of the disease in each group of mice was quantitatively evaluated by the disease activity index (DAI) score. Figure 1 As shown in (a), it can be seen from the figure: the DAI score of the control group (CD) was 0.60±0.17, indicating that the mice did not show obvious symptoms of colitis and were in good health; the DAI score of the model group (DD) was 3.99±0.57, which was significantly higher than that of the control group, indicating that DSS successfully induced severe colitis symptoms; the DAI score of the probiotic composition treatment group (TD) was 1.75±0.40, which was higher than that of the control group (CD), but significantly lower than that of the model group (DD), indicating that the probiotic composition provided by the present invention has a significant alleviating effect on DSS-induced colitis in mice.

[0056] (2) At the end of the experiment, the mice in the above groups were euthanized by ether inhalation anesthesia, and the colon was immediately removed, the length was measured, and the colon was fixed in 4% paraformaldehyde solution for 48 h; the fixed colon specimens were sent to Wuhan Pinnuofei Biotechnology Co., Ltd. for hematoxylin and eosin (HE) staining, which included washing, dehydrating, and cleaning the tissues and embedding them in paraffin to prepare sections with a thickness of 4 μm for subsequent microscopic observation and pathological analysis.

[0057] Figure 1(b) is the measurement result of the colon length of each group of mice. It can be seen from the figure that the average colon length of the control group (CD) is 10.05±1.06cm, indicating that the colon structure of normal mice is intact and not damaged; the average colon length of the model group (DD) is significantly shortened to 6.81±1.73cm, indicating that DSS-induced colitis leads to severe colon tissue damage and inflammatory response; the average colon length of the probiotic composition treatment group (TD) is 8.62±0.87cm, which is significantly longer than that of the model group (DD), but still slightly shorter than that of the control group (CD), indicating that the probiotic composition provided by the present invention can partially alleviate the colon damage caused by DSS after treatment.

[0058] Figure 1 (c) is the pathological changes of the colon tissue of each group of mice. It can be seen from the figure that the colon structure of the control group (CD) is intact, the villi are arranged neatly and in the same shape, the goblet cells are scattered between the epithelial cells, the lymphocytes and plasma cells are very few, and there is no sign of inflammation or fibrosis; the colon villi of the model group (DD) are disordered, some villi are atrophied, fused or even missing, the number of goblet cells is significantly reduced, and the inflammatory cells in the lamina propria are widely infiltrated, showing typical pathological characteristics of colitis; the colon villi structure of the probiotic composition treatment group (TD) is partially restored, although it is still slightly deformed or shortened compared with the control group (CD), but the arrangement is more orderly than the model group (DD), and the number of goblet cells is increased compared with the model group (DD), indicating that the mucosal secretion function is partially restored; the inflammatory cell infiltration in the lamina propria is significantly reduced, and only a small number of lymphocytes and plasma cells are seen. This shows that the inflammatory response of mice is significantly weakened after treatment with the probiotic composition provided by the invention, and the tissue structure is gradually restored.

[0059] (3) At the end of the experiment, the mice in each group were euthanized by ether inhalation anesthesia, and blood was collected from the eyeballs and the serum was separated for the evaluation of immune and antioxidant capacity.

[0060] Figure 1(d) is the results of the analysis of serum antioxidant capacity and inflammatory factors in each group of mice. It can be seen from the figure that the serum antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels of the normal control group (CD) are maintained within the normal range; compared with the control group (CD), the above antioxidant indexes of the model group (DD) are significantly reduced, indicating that DSS-induced colitis leads to a decrease in the body's antioxidant capacity; after the probiotic composition treatment group (TD) is intervened by the probiotic composition provided by the present invention, the levels of T-AOC, GSH-Px and SOD are significantly improved, close to normal levels, indicating that the probiotic composition provided by the present invention can effectively enhance the body's antioxidant capacity. In addition, among the lipid peroxidation indicators, the malondialdehyde (MDA) level is significantly increased in the model group (DD), and is on a downward trend in the probiotic composition treatment group (TD), indicating that the probiotic composition provided by the present invention can reduce oxidative stress damage. At the same time, analysis of inflammatory factors showed that the levels of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the model group (DD) were significantly increased, while these levels in the probiotic composition treatment group (TD) returned to the normal range, indicating that the probiotic composition provided by the present invention can effectively inhibit the overexpression of proinflammatory factors; the content of serum anti-inflammatory cytokine interleukin-10 (IL-10) in the model group (DD) was low, and the expression of IL-10 in the serum of the Lactobacillus salivarius treatment group (TD) was significantly increased, indicating that the probiotic composition provided by the present invention can regulate immune balance by enhancing the secretion of anti-inflammatory factors.

[0061] Example 3

[0062] In this example, 16S rRNA high-throughput sequencing analysis was performed on the mouse colon tissue collected in Example 2 to evaluate the effect of the probiotic composition provided by the present invention on the structure and diversity of intestinal flora.

[0063] The fecal total DNA extraction kit (Solarbio) was used to extract sample DNA, and specific primers were used for PCR amplification of the V3-V4 variable region of the bacterial 16SrRNA gene. The amplified products were purified, quantified, and homogenized to construct a sequencing library. The raw sequencing data were filtered by Trimmomatic v0.33 software, and the sequences were de-noised and purified using the DADA2 algorithm. The Alpha diversity (including Feature, ACE, Chao1, Simpson, Shannon, PD_whole_tree, and Coverage indexes) and Beta diversity (including principal coordinate analysis PCoA and non-metric multidimensional scaling analysis NMDS) of the samples were comprehensively evaluated using QIIME22020.6 software. To further analyze the differences in the microbial community, the variance analysis method was used to identify the microbial groups with significant changes. Variance analysis was used to analyze the differences in the microbial community, and PICRUSt2 software was used to predict the functional pathways of sample differences. The specific test results are analyzed as follows.

[0064] Figure 2 The effect of the probiotic composition provided by the present invention on the intestinal flora of mice with DSS-induced colitis Venn diagram and Alpha diversity analysis. Through the analysis of 15 samples, a total of 5445 Amplicon Sequence Variants (ASVs) were identified (such as Figure 2 a), of which 271 ASVs were present in the control group (CD), model group (DD) and probiotic composition treatment group (TD); the number of unique ASVs in each group was: 1654 in the CD group, 1597 in the DD group, and 1566 in the TD group; in addition, the overlap of ASVs between different groups was as follows: there were 111 overlapping ASVs between the CD group and the DD group, 126 overlapping ASVs between the CD group and the TD group, and 120 overlapping ASVs between the DD group and the TD group. The species richness of the samples was evaluated by alpha diversity indices such as Chao1, ACE, Shannon, Simpson and PD_whole_tree, and the results showed that there were no significant differences among the samples in each group (such as Figure 2 b); In addition, the rarefaction curve (such as Figure 2 c) and abundance level curves (e.g. Figure 2 d) all tended to be flat, indicating that the current sequencing depth is sufficient to comprehensively reflect the microbial composition in the samples.

[0065] Figure 3 The probiotic composition provided by the present invention is used for Beta diversity analysis of intestinal flora in DSS-induced colitis mice and structural analysis of intestinal flora at different classification levels.

[0066] (i) By evaluating the differences in microbial community structure among the three groups of samples (CD, DD, and TD) using β-diversity analysis, principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS), we revealed significant differences in microbial composition among the three groups (e.g. Figure 3 a).

[0067] (ii) Figure 3 (b) shows that at the phylum level, Firmicutes (CD = 39.11%, DD = 50.86%, TD = 38.19%) and Bacteroidetes (CD = 46.86%, DD = 26.14%, TD = 37.76%) were the two most abundant bacterial groups in the three groups; among them, the proportion of Firmicutes was the highest in the DD group (50.86%) and the lowest in the TD group (38.19%); the proportion of Bacteroidetes was the highest in the CD group (46.86%) and the lowest in the DD group (26.14%). In addition, Actinobacteria, Proteobacteria, and Campylobacteria also showed high abundance in specific groups, such as Actinobacteria in the CD group (8.78%), Proteobacteria in the DD group (10.76%), and Campylobacter in the TD group (11.10%).

[0068] Further analysis at the taxonomic level showed that Bacteroidia (CD=46.86%, DD=26.14%, TD=37.76%), Bacilli (CD=24.94%, DD=25.17%, TD=25.18%) and Clostridia (CD=14.15%, DD=25.68%, TD=10.48%) accounted for a high proportion in the three groups, but Campylobacteria accounted for a higher proportion in the TD group than Clostridia (10.48%).

[0069] At the genus level, unclassified_Muribaculaceae was most abundant in the CD (28.12%) and TD (20.13%) groups, and was also distributed in the DD (9.78%) group. The genera Dubosiella and Muribaculum were more abundant in the CD group (15.89%) and less abundant in the TD and DD groups (3.57%). Bacteroides (8.26%) and Parabacteroides (8.05%) accounted for a significant proportion in the DD group. It is worth noting that in the TD group, the abundance of Helicobacter pylori (11.10%) and Bacteroides (8.87%) was relatively high.

[0070] (iii) If Figure 3(c) shows that the ratio of Firmicutes to Bacteroidetes (abbreviated as F / B ratio) observed in the model group (DD) was significantly increased. Firmicutes are important producers of short-chain fatty acids (SCFAs), including butyrate, acetate, and propionate. The increase in the F / B ratio may lead to the overproduction of these SCFAs, thereby affecting the intestinal microenvironment. Although SCFAs generally have anti-inflammatory properties, under certain pathological conditions, excessive SCFAs may change the intestinal pH and inhibit the growth of Bacteroidetes, thereby exacerbating the imbalance of the microbiota. In addition, the elevated F / B ratio may promote the expression of proinflammatory cytokines such as TNF-α and IL-6 by activating the toll-like receptor (TLR) signaling pathway; at the same time, the reduction in the number of Bacteroidetes will lead to a decrease in the production of anti-inflammatory cytokines such as IL-10, further disrupting the balance of the immune system. The increase in the F / B ratio may also increase the production of intestinal reactive oxygen species (ROS) while reducing antioxidant capacity, thereby exacerbating oxidative damage to colonic tissue.

[0071] In summary, an increase in the F / B ratio may not only disrupt the structure of the intestinal microbial community, but may also affect the intestinal immune response and physical barrier function through multiple mechanisms, ultimately leading to the deterioration of intestinal health.

[0072] Figure 4 The effect of the probiotic composition provided by the present invention on the differential flora of intestinal microorganisms in mice with DSS-induced colitis. Through a systematic analysis of the relative abundance of the colonic intestinal flora of the above mice, it was found that there were significant differences in the control group (CD), model group (DD) and probiotic composition treatment group (TD) at the phylum level and genus level, as follows:

[0073] (i) phylum level analysis, such as Figure 4 As shown in (a): compared with the CD group, the relative abundance of Actinobacteria in the TD group was significantly reduced, indicating that Lactobacillus salivarius intervention may have an inhibitory effect on the growth of Actinobacteria; the Bacteroidetes maintained a high abundance in both the CD group and the TD group, but decreased significantly in the DD group, indicating that DSS-induced colitis may lead to a decrease in the Bacteroidetes, and its abundance can be partially restored after treatment with the probiotic composition provided by the present invention; the change trend of the Proteobacteria is opposite to that of the Bacteroidetes, which increased significantly in the DD group and showed a decreasing trend in the TD group, indicating that the probiotic composition provided by the present invention can improve the intestinal microecological balance by inhibiting the excessive growth of the Proteobacteria.

[0074] (ii) Genus level analysis, such as Figure 4(b) shows: changes in pathogen abundance. In the DD group, the abundance of potential pathogens such as Alistipes, Candidatus_Soleaferrea, Frisingicoccus, Romboutsia, Streptococcus and Olsenella increased significantly, while the abundance of these genera was lower in the CD and TD groups, indicating that DSS-induced colitis may promote the overgrowth of pathogens, and the probiotic composition provided by the present invention can effectively inhibit the proliferation of these pathogens. Changes in probiotic abundance. Probiotics such as Alloprevotella, Anaerotruncus, Candidatus_Arthromitus, Christensenellaceae_R_7_group, Herbinix, unclassified_Rs_E47_termite_group and Rikenella decreased significantly in the DD group, while they increased in the TD group. The probiotic composition provided by the present invention can improve intestinal health by promoting the growth of these beneficial bacteria.

[0075] Figure 5 The effect of the probiotic composition provided by the present invention on the KEGG function of intestinal microorganisms in mice with DSS-induced enteritis. Through comparative analysis of the metabolic pathways of microorganisms in the above different groups, the DSS-induced enteritis model group (DD group) and the normal control group (CD group) and the probiotic composition treatment group (TD) had significant differences in metabolic function. The specific results are as follows: the relative abundance of the Global and Overview metabolic pathways in the DD group was significantly lower than that in the CD group, indicating that DSS-induced enteritis may have inhibited the overall metabolic activity of the intestinal microbiota; compared with the CD group, the relative abundance of metabolic pathways related to cell motility, signal transduction and membrane transport in the DD group was significantly increased, and this change may reflect the adaptive response of intestinal microorganisms to environmental pressure under enteritis, such as responding to inflammatory stimuli by enhancing cell motility and signal transduction; compared with the TD group, the relative abundance of the carbohydrate metabolic pathway in the DD group was significantly increased, suggesting that DSS-induced colitis may cause the intestinal microbiota to tend to use carbohydrates as an energy source, and this trend was alleviated after the probiotic composition provided by the present invention, indicating that the probiotic composition provided by the present invention may improve the intestinal microecological balance by regulating the carbohydrate metabolic pathway; compared with the DD group, the relative abundance of the cell motility pathway in the TD group was significantly increased, indicating that the intervention of the probiotic composition promoted the motility of beneficial bacteria, thereby improving intestinal health.

[0076] The above is a detailed introduction to a probiotic composition for inflammatory bowel disease disclosed in the present invention, as well as its preparation method and application. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A probiotic composition for alleviating inflammatory bowel disease, characterized in that: The probiotic composition is prepared from Lactobacillus salivarius and a protective agent solution.

2. The probiotic composition for alleviating inflammatory bowel disease according to claim 1, characterized in that: The salivarius Lactobacillus is classified and named as Lactobacillus salivarius GZLC1, and is preserved in the China Center for Type Culture Collection with the preservation number: CCTCC NO: M 2023735. The address of the preservation unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation time is May 11, 2023.

3. The probiotic composition for alleviating inflammatory bowel disease according to claim 1, characterized in that: The protective agent solution is prepared by uniformly mixing the protective agent and sterile water in a mass volume ratio of (5-20) g:100 mL.

4. The probiotic composition for alleviating inflammatory bowel disease according to claim 3, characterized in that: The protective agent comprises one or a combination of lactose, starch, microcrystalline cellulose, mannitol, gelatin and magnesium stearate.

5. The method for preparing the probiotic composition for alleviating inflammatory bowel disease according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. The frozen Lactobacillus salivarius GZLC1 was inoculated into MRS medium, the pH of which was 6.5-7.0, and cultured under anaerobic conditions at 37°C for 18-24 hours; then the strain was transferred to a fermenter, and cultured for 18-24 hours at a temperature of 37°C and a pH of 6.0-6.5, and the OD600 value of the bacterial solution was measured using a spectrophotometer. When the OD600 value of the bacterial solution reached 0.5-1.0, the culture was stopped to obtain a bacterial suspension; S2. The bacterial suspension cultured in step S1 was centrifuged at 4000 rpm and 4°C for 10 min, the bacterial cells in the precipitate were collected, and the bacterial cells were washed twice with 0.85% sterile saline to remove the residual culture medium and other impurities to obtain a concentrated bacterial solution; S3. The concentrated bacterial solution obtained in step S2 is fully mixed with the protective agent solution to obtain a mixed solution, that is, a probiotic composition.

6. The method for preparing a probiotic composition for alleviating inflammatory bowel disease according to claim 5, characterized in that: The number of viable Lactobacillus salivarius GZLC1 in the probiotic composition in step S3 is 1×10 6 ~1×10 12 CFU / mL.

7. The method for preparing a probiotic composition for alleviating inflammatory bowel disease according to claim 5, characterized in that: In step S3, the volume ratio of the concentrated bacterial solution to the protective agent solution is 1:(1-3).

8. Use of the probiotic composition according to any one of claims 1 to 4 in the preparation of a pharmaceutical product for preventing or treating inflammatory bowel disease.

9. The use according to claim 8, characterized in that: The dosage form of the drug product is oral preparation, enteric-coated capsule, tablet, granule, suspension or enema.

10. The use according to claim 8, characterized in that: The inflammatory bowel disease is ulcerative colitis induced by dextran sulfate sodium.

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