Application of probiotic metagen and organic acid in improvement of liver health and colitis

Abstract

The invention discloses an application of probiotic metagen and organic acid in improvement of liver health and colitis, and relates to the technical field of metagen. The invention discovers that both malic acid and / or sebacic acid and acellular supernatant of K56 inactivated bacteria can significantly inhibit the expression of proinflammatory factors, and both have good anti-inflammatory activity. After intervention of K56 acellular supernatant, malic acid and sebacic acid, abnormal liver weight gain of a subject caused by high-fat diet can be relieved, and liver injury caused by the high-fat diet is relieved. Therefore, development of sebacic acid, malic acid or mixed organic acid of malic acid and sebacic acid has a good application prospect in preparation of products for improving liver health. In addition, after intervention of malic acid or sebacic acid, the basic phenotype of the DSS-induced colitis of the test body is relieved. The invention has a good application prospect for developing and preparing products for preventing or treating acute colitis or ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of postbiotic technology, and more specifically, to the application of probiotic postbiotics and organic acids in improving liver health and colitis. Background Technology

[0002] In 2021, the International Scientific Association for Probiotics and Prebiotics (ISAPP) defined metabiotics as preparations of non-living microorganisms and / or their components that are beneficial to the health of the host. Metabiotics can be non-living, intact cells or structural fragments such as cell walls. Many metabiotics also include soluble metabolites, which may be secreted by living bacteria or released into the host environment after bacterial lysis, such as short-chain fatty acids / medium- and long-chain fatty acids, polysaccharides, teichoic acid, vitamins / cofactors, organic acids, and peptides.

[0003] Studies have shown that heat-killed Weissella JW15 reduces LPS-induced pro-inflammatory function in RAW264.7 cells by inhibiting NF-κB activation, thus exhibiting anti-inflammatory effects

[14] . Heat-killed LGG bacteria can reduce LPS-induced pro-inflammatory factors and increase anti-inflammatory mediators

[15] . Peptidoglycan from Lactobacillus reuteri has been shown to inhibit the production of pro-inflammatory factors in RAW 264.7 cells induced by Porphyromonas gingivalis lipopolysaccharide

[17] . Metagenic components may provide better anti-inflammatory functions through their own or combined effects with live bacteria, thereby helping to improve the host's inflammatory response. Bárbara Ivana Layús et al. demonstrated that cell-free supernatant from Lactobacillus plantarum CRL 759 can significantly reduce the secretion of inflammatory parameters, infiltration of inflammatory cells, and clinical symptoms of inflammation in a lipopolysaccharide (EIU)-induced uveitis model

[21] . In summary, epigenetics exhibits good anti-inflammatory activity in both in vivo and in vitro studies, which helps to make epigenetics a potential tool for the treatment and prevention of inflammation-related diseases.

[0004] While epibiotics offer unique advantages over live bacteria and other drugs in alleviating colitis, their application still faces several challenges. First, due to the complex composition of epibiotics, most domestic and international research focuses on mixtures to study their functional activity, without analyzing their specific material composition or identifying the active ingredient within the mixture. Consequently, the main mechanisms by which epibiotics interact with the host, maintain host health, and prevent disease remain unclear. Further research is needed to elucidate and clarify the material composition of epibiotics, which may contribute to understanding their mechanisms of action. Second, the diverse manufacturing processes of epibiotics, such as heat inactivation, irradiation, high pressure, and ultrasound, contribute to a degree of ambiguity in their definition. Different preparation processes affect the material composition of epibiotics differently, thus impacting their bioactivity. Although epibiotics are considered safe and effective, a comprehensive system for effectively assessing their safety is currently lacking. More clinical studies are needed to determine the reliability and safety of epibiotics during long-term use.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of probiotic postbiotics and organic acids in improving liver health and colitis to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides the use of malic acid or a mixture of malic acid and sebacic acid in the preparation of products that improve liver health.

[0009] Secondly, the present invention provides the use of malic acid in the preparation of products for the prevention or treatment of acute colitis or ulcerative colitis.

[0010] Thirdly, the present invention provides an application of probiotic postbiotic products in the preparation of products that improve liver health, characterized in that the probiotic postbiotic products are obtained from the cell-free supernatant of inactivated probiotics; the probiotics include Lactobacillus paracasei K56, whose accession number is CGMCC No. 15139 or DSM27447, and Lactobacillus paracasei K56 is disclosed and recorded in Chinese invention patent CN201710009845.0.

[0011] Fourthly, the present invention provides the application of sebacic acid in the preparation of products that improve liver health.

[0012] Fifthly, the present invention provides the use of sebacic acid or a mixture of sebacic acid and malic acid in the preparation of products for the prevention or treatment of acute colitis or ulcerative colitis.

[0013] The present invention has the following beneficial effects:

[0014] This invention utilizes non-targeted metabolomics to analyze the cell-free supernatant components of *Lactobacillus paracasei* K56, revealing the presence of malic acid and sebacic acid in the supernatant. Subsequently, the anti-inflammatory activity of the screened post-biotic components was evaluated in vivo and in vitro using a RAW264.7 macrophage model, a zebrafish inflammation model, and a DSS-induced UC mouse model, elucidating the mechanism of action of the active components.

[0015] This invention reveals that malic acid and / or sebacic acid, as well as cell-free supernatant from K56 inactivated bacteria, can significantly inhibit the expression of pro-inflammatory factors and exhibit good anti-inflammatory activity. Compared to other organic acids, malic acid and / or sebacic acid show stronger anti-inflammatory activity. Intervention with K56 cell-free supernatant, malic acid, and sebacic acid can alleviate abnormal liver weight gain in subjects induced by a high-fat diet and reduce liver damage caused by a high-fat diet. Simultaneously, malic acid and sebacic acid intervention regulate the expression of intestinal inflammatory factors such as TNF-α, IL-1β, and IL-10, and significantly upregulate the expression of tight junction proteins ZO-1 and Occludinb. Therefore, malic acid and / or sebacic acid can improve liver health. Thus, this invention has promising applications in developing products that improve liver health using sebacic acid, malic acid, or mixed organic acids of malic acid and sebacic acid.

[0016] After intervention with malic acid or sebacic acid, the basic phenotype of DSS-induced colitis in subjects was alleviated, including restoration of body weight and normal colonic morphology, decrease in DAI index and MPO activity, and increase in colonic length. Therefore, the present invention has promising application prospects for the development and preparation of products for the prevention or treatment of acute colitis or ulcerative colitis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 For technology roadmap;

[0019] Figure 2SEM image of cell morphology after inactivation of Lactobacillus paracasei K56 (Note: White arrows represent cell rupture, and red circles represent intracellular lysed substances).

[0020] Figure 3 Effects of K56 inactivated bacteria and cell-free supernatant on TNF-α production in LPS-stimulated RAW264.7 cells (A) Inactivated bacteria; (B) Cell-free supernatant (Note: Compared with Control, ####P<0.0001; Compared with LPS group, ***P<0.001, ****P<0.0001);

[0021] Figure 4 Untargeted metabolomics results of cell-free supernatant metabolites from Lactobacillus paracasei; (A) Pie chart of cell-free supernatant metabolite composition; (B) Heatmap of the top 30 organic acids by peak area;

[0022] Figure 5 The effect of organic acids on TNF-α production in LPS-stimulated RAW264.7 cells (Note: Compared with Control, ####P<0.0001; Compared with LPS group, *P<0.05,**P<0.01,***P<0.001; MA: malic acid, DA: sebacic acid, 6-HA: 6-hydroxyhexanoic acid, HA: 3-(4-hydroxyphenyl)lactic acid, PA: phenyllactic acid);

[0023] Figure 6 Effects of malic acid (MA) and sebacic acid (DA) on TNF-α production in LPS-stimulated RAW264.7 cells (Compared with Control, ####P<0.0001; Compared with LPS group, *P<0.05,**P<0.01,***P<0.001; MA: malic acid, DA: sebacic acid);

[0024] Figure 7 The effect of K56 post-biotic components on liver weight and liver weight index in zebrafish (Note: HFD+K121: cell-free supernatant at 121℃, HFD+MA: malic acid, HFD+DA: sebacic acid);

[0025] Figure 8 The effect of K56 post-biotic components on the histopathology of zebrafish liver tissue (Note: yellow arrows represent lipid droplet accumulation, HFD+K121: cell-free supernatant, HFD+MA: malic acid, HFD+DA: sebacic acid);

[0026] Figure 9The effect of K56 postbiotic components on inflammatory factors in zebrafish liver (Note: Compared with Control, #P<0.05, ##P<0.01, ####P<0.0001; Compared with HFD group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; HFD+K121: cell-free supernatant at 121℃, HFD+MA: malic acid, HFD+DA: sebacic acid);

[0027] Figure 10 Effects of K56 postbiotic components on the intestinal barrier of zebrafish ((A) HE staining of zebrafish intestine; (B) mRNA expression of tight junction proteins). Note: White arrows represent goblet cells, and red arrows represent inflammatory infiltration. Compared with Control, #P<0.05, ###P<0.001; compared with HFD group, *P<0.05, ***P<0.001, ****P<0.0001; HFD+K121: cell-free supernatant at 121℃, HFD+MA: malic acid, HFD+DA: sebacic acid.

[0028] Figure 11 The effect of K56 postbiotic components on intestinal inflammatory factors in zebrafish (Note: Compared with the Control group, #P<0.05, ##P<0.01, ####P<0.0001; compared with the HFD group, *P<0.05,

[0029] **P<0.01,***P<0.001,****P<0.0001; HFD+K121: cell-free supernatant at 121℃, HFD+MA: malic acid, HFD+DA: sebacic acid);

[0030] Figure 12 The effect of K56 postbiotic components on the β diversity of gut microbiota in zebrafish (HFD+K121: cell-free supernatant at 121℃, HFD+MA: malic acid, HFD+DA: sebacic acid);

[0031] Figure 13 The effects of K56 postbiotic components on the gut microbiota of zebrafish at the phylum level were shown in the bar charts: (A) phylum-level species abundance (Bar plot); (B) phylum-level dominant species abundance (Histogram). Compared with the Control group, #P<0.05, ##P<0.01; compared with the HFD group, *P<0.05; HFD+K121:

[0032] 121℃ cell-free supernatant, HFD+MA: malic acid, HFD+DA: sebacic acid);

[0033] Figure 14The effect of K56 postbiotic components on the gut microbiota of zebrafish at the genus level ((A) Species composition abundance Bar plot at the genus level; (B) LEfSe multilevel species discrimination analysis (HFD+K121: cell-free supernatant at 121℃, HFD+MA: malic acid, HFD+DA: sebacic acid)).

[0034] Figure 15 To illustrate the species differences in gut microbiota among zebrafish groups at the genus level ((A) HFD vs HFD + cell-free supernatant; (B) HFD vs HFD + malic acid; (C) HFD vs HFD + sebacic acid);

[0035] Figure 16 The effect of K56 postbiotic components on the rate of change in body weight in DSS-induced colitis mice ((A) cell-free supernatant group; (B) malic acid group; (C) sebacic acid group; compared with DSS, ####P<0.0001; compared with DSS group, low-dose group, **P<0.01, ***P<0.001, ****P<0.0001; compared with DSS group, high-dose group, &P<0.05, &&P<0.01; DSS+K121-L / DSS+K121-H: low-dose / high-dose cell-free supernatant, DSS+MA-L / DSS+MA-H: low-dose / high-dose malic acid, DSS+DA-L / DSS+DA-H: low-dose / high-dose sebacic acid).

[0036] Figure 17 The effect of K56 post-biotic components on DAI scores in DSS-induced colitis mice (compared to Control, ####P<0.0001; low-dose group compared to DSS group, ***P<0.001, ****P<0.0001; high-dose group compared to DSS group, &P<0.05, &&P<0.01, &&&P<0.001; DSS+K121-L / DSS+K121-H: low-dose / high-dose cell-free supernatant, DSS+MA-L / DSS+MA-H: low-dose / high-dose malic acid, DSS+DA-L / DSS+DA-H: low-dose / high-dose sebacic acid);

[0037] Figure 18 Effects of K56 postbiotic components on colon length in DSS-induced colitis mice (compared to Control, ####P<0.0001; compared to DSS group, **P<0.01, ***P<0.001, ****P<0.0001; DSS+K121-L / DSS+K121-H: low / high dose cell-free supernatant, DSS+MA-L / DSS+MA-H: low / high dose malic acid, DSS+DA-L / DSS+DA-H: low / high dose sebacic acid);

[0038] Figure 19 Effects of K56 postbiotic components on colonic MPO in DSS-induced colitis mice (compared to Control, ####P<0.0001; compared to DSS group, *P<0.05, **P<0.01, ***P<0.001; DSS+K121-L / DSS+K121-H: low / high dose cell-free supernatant, DSS+MA-L / DSS+MA-H: low / high dose malic acid, DSS+DA-L / DSS+DA-H: low / high dose sebacic acid);

[0039] Figure 20 Effects of K56 post-biotic components on colonic histopathology in DSS-induced colitis mice ((A) HE staining of colonic tissue sections (10x, 100 μm); (B) Pathological scores; Compared with Control, ####P<0.0001; Compared with DSS group, ****P<0.0001; DSS+K121-L / DSS+K121-H: low / high dose cell-free supernatant, DSS+MA-L / DSS+MA-H: low / high dose malic acid, DSS+DA-L / DSS+DA-H: low / high dose sebacic acid);

[0040] Figure 21 The effect of K56 postbiotic components on the mRNA expression of colonic inflammatory factors (compared with Control, ##P<0.01, ###P<0.001, ####P<0.0001; compared with DSS group, *P<0.05, **P<0.01, ***P<0.001; DSS+K121-L / DSS+K121-H: low / high dose cell-free supernatant, DSS+MA-L / DSS+MA-H: low / high dose malic acid, DSS+DA-L / DSS+DA-H: low / high dose sebacic acid);

[0041] Figure 22 Volcano plots of differentially expressed genes in the colon ((A) DSS vs Control; (B) DSS+K121-L vs DSS; (C) DSS+MA-L vs DSS; (D) DSS+DA-L vs DSS);

[0042] Figure 23 KEGG enrichment analysis of differentially expressed genes ((A) DSS vs Control; (B) DSS+K121-L vs DSS);

[0043] Figure 24KEGG enrichment analysis of differentially expressed genes ((C)DSS+MA-L vs DSS; (D)DSS+DA-L vs DSS);

[0044] Figure 25 Differential gene clustering analysis (heatmap of differentially expressed genes in inflammatory pathways; compared with Control, ##P<0.01, ###P<0.001, ####P<0.0001; compared with DSS group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; DSS+K121-L: low-dose cell-free supernatant, DSS+MA-L: low-dose malic acid, DSS+DA-L: low-dose sebacic acid);

[0045] Figure 26 The results of differential gene qPCR are shown in the figure (compared with Control, ##P<0.01, ###P<0.001, ####P<0.0001; compared with DSS group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; DSS+K121-L: low-dose cell-free supernatant, DSS+MA-L: low-dose malic acid, DSS+DA-L: low-dose sebacic acid). Detailed Implementation

[0046] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0047] In a first aspect, the present invention provides the use of malic acid or a mixture of malic acid and sebacic acid in the preparation of products that improve liver health.

[0048] Experiments have shown that intervention with malic acid and a mixture of malic acid and sebacic acid can alleviate abnormal liver weight gain in subjects induced by a high-fat diet and reduce liver damage caused by a high-fat diet. Therefore, malic acid and a mixture of malic acid and sebacic acid hold promise for the preparation of products that improve liver health.

[0049] In a preferred embodiment of the present invention, improving liver health includes:

[0050] (1) It has an auxiliary protective effect against liver damage;

[0051] (2) Treatment of liver injury.

[0052] In a preferred embodiment of the present invention, when preparing a product that has an auxiliary protective effect against liver injury, the product is a drug, food, or health product; when preparing a product for treating liver injury, the product is a drug.

[0053] In a preferred embodiment of the present invention, the dosage form of the health product is capsule, tablet, powder, granule, pill, or oral liquid.

[0054] In a preferred embodiment of the invention, the food is selected from beverages, meat, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, electrolyte drinks, drinking water, chewing gum, capsules, tea, and vitamin complexes. Electrolyte drinks include, but are not limited to, solid beverages. Chewing gum and candy can be chewable tablets.

[0055] In a preferred embodiment of the present invention, the dosage form of the drug is selected from: oral liquid, tablet, powder, granule, capsule, enema or gastric enema.

[0056] In a preferred embodiment of the present invention, the product has at least one use from the group consisting of:

[0057] (1) Decreased liver weight or a decreasing trend in liver weight;

[0058] (2) The liver weight index is reduced or the liver weight index shows a decreasing trend;

[0059] (3) Improve the neatness and / or density of hepatocyte arrangement;

[0060] (4) The number of nucleoli in hepatocytes is increased or shows a trend of increasing;

[0061] (5) Cytoplasmic vacuoles shrink or show a tendency to shrink;

[0062] (6) The hepatic steatosis has decreased or shown a decreasing trend;

[0063] (7) Reduces liver inflammation.

[0064] In a preferred embodiment of the present invention, alleviating liver inflammation includes: reducing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors;

[0065] In a preferred embodiment of the present invention, the pro-inflammatory factor is selected from at least one of TNF-α, IL-6 and IL-1β;

[0066] In a preferred embodiment of the present invention, the anti-inflammatory factor is selected from at least one of TGF-β and IL-10.

[0067] In a preferred embodiment of the present invention, liver injury is selected from any one of acute liver failure or acute liver injury, subacute liver failure, acute-on-chronic liver failure, chronic liver failure or chronic liver injury, drug-induced liver injury, endotoxin-induced liver injury, liver injury with endotoxemia, and liver failure.

[0068] In a preferred embodiment of the present invention, chronic liver injury is induced by a high-fat diet;

[0069] In a preferred embodiment of the present invention, the drug is an oral drug or an injectable drug.

[0070] In a preferred embodiment of the present invention, the drug further includes a pharmaceutically acceptable carrier or excipient. The carrier or excipient is selected from at least one of the following: protectants, excipients, binders, disintegrants, lubricants, fragrances, preservatives, stabilizers, suspending agents, dispersants, and diluents.

[0071] Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylene pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, hydrolyzed starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, hydroxypropyl starch, sodium glycolate starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, talc, and sodium lauryl sulfate; and flavorings such as citric acid, menthol, and glycine. Orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (e.g., methylparaben, ethylparaben, propylparaben, butylparaben); stabilizers, such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0072] Secondly, the present invention also provides the use of malic acid in the preparation of products for the prevention or treatment of acute colitis or ulcerative colitis.

[0073] Experiments have shown that malic acid intervention significantly inhibits the expression of pro-inflammatory factors and exhibits strong anti-inflammatory activity. After malic acid intervention, the basic phenotype of DSS-induced colitis in subjects (e.g., mice) was alleviated, including restoration of mouse body weight and normal colon morphology, decreased DAI index and MPO activity, and increased colon length. Malic acid exerts its anti-inflammatory effect by inhibiting the mRNA expression of IL-6, IL-1β, and TNF-α.

[0074] The above-mentioned product is a pharmaceutical product. In a preferred embodiment of the present invention, the pharmaceutical product has at least one of the following uses:

[0075] (1) Reduce intestinal barrier damage;

[0076] (2) Relieves intestinal inflammation;

[0077] (3) Reduce the relative abundance of intestinal actinomycetes;

[0078] (4) Increase the relative abundance of the genera *Bosea* and *Cetobacterium* in the gut;

[0079] (5) Reduce the relative abundance of Vibrio and Shewanella;

[0080] (6) Increase the abundance of unclassified_f_Kineosporiaceae and norank_f_JG30-KF-CM45;

[0081] (7) Reduce the abundance of Paraclostridium;

[0082] (8) Restore the subject's weight;

[0083] (9) Reduced the subject's disease activity index score;

[0084] (10) Improves colonic shortening caused by acute colitis or ulcerative colitis;

[0085] (11) Reduces colonic myeloperoxidase activity;

[0086] (12) Restore the colonic crypt structure of the subjects;

[0087] (13) Alleviating DSS-induced acute colitis or ulcerative colitis by inhibiting the IL-17 signaling pathway.

[0088] In a preferred embodiment of the present invention, reducing intestinal barrier damage includes: reducing damage to the intestinal mucosa, submucosa, muscularis propria, and serosa; increasing the number of goblet cells; and reducing inflammatory cell infiltration at least one of the following:

[0089] In a preferred embodiment of the present invention, alleviating intestinal inflammation includes: reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors;

[0090] In a preferred embodiment of the present invention, inhibiting the IL-17 signaling pathway includes inhibiting the mRNA expression of at least one of the genes S100a8, S100a9, Cxcl2, Cxcl5, Lcn2 and IL-11 in the IL-17 signaling pathway.

[0091] Thirdly, the present invention also provides the application of probiotic postbiotic products in the preparation of products that improve liver health. The probiotic postbiotic products are prepared from the cell-free supernatant of inactivated probiotics. The probiotics include Lactobacillus paracasei K56, whose preservation number is CGMCC No. 15139 or DSM27447.

[0092] Experiments have shown that K56 cell-free supernatant intervention can alleviate abnormal liver weight gain in zebrafish caused by a high-fat diet and reduce liver damage caused by a high-fat diet. It also reduces liver pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increases anti-inflammatory factors IL-10 and TGF-β.

[0093] Preservation information for strain K56 is based on the strain information disclosed in patent CN107916236B.

[0094] In a preferred embodiment of the present invention, the probiotic postbiotic product further includes: a carrier and / or auxiliary materials;

[0095] In a preferred embodiment of the present invention, the probiotic postbiotic product is in the form of liquid, solid or semi-solid.

[0096] In a preferred embodiment of the present invention, the inactivated bacteria are the fermentation products of probiotics; the sterilization conditions are: 70℃-121℃, treatment for 5min-15min.

[0097] In other embodiments, the above-mentioned inactivated bacteria can also be inactivated by ultrasonic inactivation, lysozyme inactivation, and pulsed electric field inactivation. As long as biologically active metagenes can be obtained, other inactivation methods besides thermal inactivation are also within the protection scope of this invention.

[0098] Inactivation conditions can be 70-120℃, 70-115℃, 70-110℃, 70-105℃, 70-100℃, 75-121℃, 75-115℃, 75-110℃, 75-100℃, 75-90℃, 80-90℃, 85-90℃, 110-121℃, 90-121℃, or any value within the above ranges. Treatment time is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, or any value between 5 and 15 min.

[0099] In a preferred embodiment of the present invention, improving liver health includes:

[0100] (1) It has an auxiliary protective effect against liver damage;

[0101] (2) Treatment of liver injury.

[0102] In a preferred embodiment of the present invention, when preparing a product that has an auxiliary protective effect against liver injury, the product is a drug, food, or health product; when preparing a product for treating liver injury, the product is a drug.

[0103] In a preferred embodiment of the present invention, the dosage form of the health product is capsule, tablet, powder, granule, pill, or oral liquid.

[0104] In a preferred embodiment of the invention, the food is selected from beverages, meat, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, electrolyte drinks, drinking water, chewing gum, capsules, tea, and vitamin complexes. Electrolyte drinks include, but are not limited to, solid beverages. Chewing gum and candy can be chewable tablets.

[0105] In a preferred embodiment of the present invention, the dosage form of the drug is selected from: oral liquid, tablet, powder, granule, capsule, enema or gastric enema.

[0106] In a preferred embodiment of the present invention, the product has at least one use from the group consisting of:

[0107] (1) Decreased liver weight or a decreasing trend in liver weight;

[0108] (2) The liver weight index is reduced or the liver weight index shows a decreasing trend;

[0109] (3) Improve the neatness and / or density of hepatocyte arrangement;

[0110] (4) The number of nucleoli in hepatocytes is increased or shows a trend of increasing;

[0111] (5) Cytoplasmic vacuoles shrink or show a tendency to shrink;

[0112] (6) The hepatic steatosis has decreased or shown a decreasing trend;

[0113] (7) Reduces liver inflammation.

[0114] In a preferred embodiment of the present invention, alleviating liver inflammation includes: reducing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors;

[0115] In a preferred embodiment of the present invention, the pro-inflammatory factor is selected from at least one of TNF-α, IL-6 and IL-1β;

[0116] In a preferred embodiment of the present invention, the anti-inflammatory factor is selected from at least one of TGF-β and IL-10;

[0117] In a preferred embodiment of the present invention, liver injury is selected from any one of acute liver failure or acute liver injury, subacute liver failure, acute-on-chronic liver failure, chronic liver failure or chronic liver injury, drug-induced liver injury, endotoxin-induced liver injury, liver injury with endotoxemia, and liver failure.

[0118] In a preferred embodiment of the present invention, chronic liver injury is induced by a high-fat diet;

[0119] In a preferred embodiment of the present invention, the drug is an oral drug or an injectable drug.

[0120] Fourthly, the present invention also provides the application of sebacic acid in the preparation of products that improve liver health.

[0121] Experiments have shown that sebacic acid intervention can alleviate abnormal liver weight gain in subjects induced by a high-fat diet and reduce liver damage caused by such a diet. Therefore, sebacic acid holds promise for use in the preparation of products that improve liver health.

[0122] In a preferred embodiment of the present invention, improving liver health includes:

[0123] (1) It has an auxiliary protective effect against liver damage;

[0124] (2) Treatment of liver injury.

[0125] In a preferred embodiment of the present invention, when preparing a product that has an auxiliary protective effect against liver injury, the product is a drug, food, or health product; when preparing a product for treating liver injury, the product is a drug.

[0126] In a preferred embodiment of the present invention, the dosage form of the health product is capsule, tablet, powder, granule, pill, or oral liquid.

[0127] In a preferred embodiment of the invention, the food is selected from beverages, meat, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, electrolyte drinks, drinking water, chewing gum, capsules, tea, and vitamin complexes. Electrolyte drinks include, but are not limited to, solid beverages. Chewing gum and candy can be chewable tablets.

[0128] In a preferred embodiment of the present invention, the dosage form of the drug is selected from: oral liquid, tablet, powder, granule, capsule, enema or gastric enema.

[0129] In a preferred embodiment of the present invention, the product has at least one use from the group consisting of:

[0130] (1) Decreased liver weight or a decreasing trend in liver weight;

[0131] (2) The liver weight index is reduced or the liver weight index shows a decreasing trend;

[0132] (3) Improve the neatness and / or density of hepatocyte arrangement;

[0133] (4) The number of nucleoli in hepatocytes is increased or shows a trend of increasing;

[0134] (5) Cytoplasmic vacuoles shrink or show a tendency to shrink;

[0135] (6) The hepatic steatosis has decreased or shown a decreasing trend;

[0136] (7) Reduces liver inflammation.

[0137] In a preferred embodiment of the present invention, alleviating liver inflammation includes: reducing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors;

[0138] In a preferred embodiment of the present invention, the pro-inflammatory factor is selected from at least one of TNF-α, IL-6 and IL-1β;

[0139] In a preferred embodiment of the present invention, the anti-inflammatory factor is selected from at least one of TGF-β and IL-10;

[0140] In a preferred embodiment of the present invention, liver injury is selected from any one of acute liver failure or acute liver injury, subacute liver failure, acute-on-chronic liver failure, chronic liver failure or chronic liver injury, drug-induced liver injury, endotoxin-induced liver injury, liver injury with endotoxemia, and liver failure.

[0141] In a preferred embodiment of the present invention, chronic liver injury is induced by a high-fat diet;

[0142] In a preferred embodiment of the present invention, the drug is an oral drug or an injectable drug.

[0143] Fifthly, the present invention also provides the use of sebacic acid or a mixture of sebacic acid and malic acid in the preparation of products for the prevention or treatment of acute colitis or ulcerative colitis.

[0144] Experiments have shown that sebacic acid or a mixture of sebacic acid and malic acid significantly inhibits the expression of pro-inflammatory factors and exhibits strong anti-inflammatory activity. After intervention with sebacic acid or a mixture of sebacic acid and malic acid, the basic phenotype of DSS-induced colitis in subjects (e.g., mice) was alleviated, including restoration of mouse body weight and normal colon morphology, decreased DAI index and MPO activity, and increased colon length. Sebacic acid exerts its anti-inflammatory effect by inhibiting the mRNA expression of IL-6, IL-1β, and TNF-α, and the expression of tight junction proteins ZO-1 and Occludinb is also significantly upregulated. Furthermore, the gut microbiota of zebrafish underwent significant changes under the intervention of malic acid and sebacic acid. The relative abundance of beneficial bacteria such as *Bosea*, *Cetobacter*, and *Aeromonas* increased, while the relative abundance of *Vibrio* and *Shewanella* decreased.

[0145] The above-mentioned product is a pharmaceutical product. In a preferred embodiment of the present invention, the pharmaceutical product has at least one of the following uses:

[0146] (1) Reduce intestinal barrier damage;

[0147] (2) Relieves intestinal inflammation;

[0148] (3) Reduce the relative abundance of intestinal actinomycetes;

[0149] (4) Increase the relative abundance of the genera *Bosea* and *Cetobacterium* in the gut;

[0150] (5) Reduce the relative abundance of Vibrio and Shewanella;

[0151] (6) Increase the abundance of unclassified_f_Kineosporiaceae and norank_f_JG30-KF-CM45;

[0152] (7) Reduce the abundance of Paraclostridium;

[0153] (8) Restore the subject's weight;

[0154] (9) Reduced the subject's disease activity index score;

[0155] (10) Improves colonic shortening caused by acute colitis or ulcerative colitis;

[0156] (11) Reduces colonic myeloperoxidase activity;

[0157] (12) Restore the colonic crypt structure of the subjects;

[0158] (13) Alleviating DSS-induced acute colitis or ulcerative colitis by inhibiting the IL-17 signaling pathway;

[0159] In a preferred embodiment of the present invention, reducing intestinal barrier damage includes: reducing damage to the intestinal mucosa, submucosa, muscularis propria, and serosa; increasing the number of goblet cells; and reducing inflammatory cell infiltration at least one of the following:

[0160] In a preferred embodiment of the present invention, alleviating intestinal inflammation includes: reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors;

[0161] In a preferred embodiment of the present invention, inhibiting the IL-17 signaling pathway includes inhibiting the mRNA expression of at least one of the genes S100a8, S100a9, Cxcl2, Cxcl5, Lcn2 and IL-11 in the IL-17 signaling pathway.

[0162] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0163] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0164] Example 1

[0165] This embodiment screened and validated the anti-inflammatory components of *Lactobacillus paracasei* K56 postbiotics. Specifically, non-targeted metabolomics was used to explore potential anti-inflammatory substances in the cell-free supernatant of K56, and the effect of K56 postbiotic components on TNF-α production in LPS-stimulated RAW264.7 cells was verified to demonstrate the anti-inflammatory activity of K56 postbiotics. (Technical roadmap referenced.) Figure 1 As shown.

[0166] 1. Experimental reagents and instruments

[0167] 1.1 Experimental Materials

[0168] The strain, Lacticaseibacillus paracasei K56 (K56), was derived from Inner Mongolia Dairy Technology Research Institute Co., Ltd.

[0169] Cells: RAW264.7 macrophages were donated by the Department of Nutrition and Health, China Agricultural University.

[0170] 1.2 Experimental Reagents

[0171] Table 1 Experimental Reagents

[0172]

[0173] Anhydrous ethanol, chloroform, isopropanol, and cysteine ​​hydrochloride were all of analytical grade, and water was distilled water and ultrapure water.

[0174] 1.3 Experimental Apparatus

[0175] Table 2 Experimental Instruments

[0176]

[0177] 2. Experimental Methods

[0178] 2.1 Preparation of main reagents

[0179] DMEM complete medium: Add 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin mixture (P / S) to DMEM high glucose medium, mix well and store at 4°C.

[0180] LPS solution: Dissolve 1 mg of LPS in 1 mL of DMEM high-glucose medium and mix thoroughly to obtain a 1 mg / mL LPS solution. Aliquot and store at -20°C. Dilute to the required concentration with medium before experimentation.

[0181] Cell cryopreservation solution: Mix fetal bovine serum and DMSO in a 9:1 ratio. Prepare fresh before use.

[0182] Organic acid solution: Dissolve 1 mg of organic acid in 1 mL of pure water, heat and mix well, the concentration of the stock solution is 1 mg / mL. Dilute to the required concentration with culture medium before the experiment.

[0183] 2.2 Strain Culture

[0184] Lactobacillus paracasei K56 was cultured overnight at 37°C in MRS broth. Prior to the experiment, K56 was stored at -80°C. The cultured Lactobacillus paracasei K56 was centrifuged at 4500×g for 10 minutes, followed by washing three times with sterile water. Bacterial counts were determined by plate counting, and the bacterial concentration was adjusted to 1×10⁻⁶ using sterile water. 10 CFU / mL. A new sample needs to be prepared before each experiment.

[0185] 2.3 Preparation of K56 heat-inactivated bacteria and cell-free supernatant

[0186] The bacterial suspension, after adjusting the concentration, was inactivated at 121°C for 10 minutes. The inactivated bacterial solution is the heat-inactivated bacteria. Then, it was centrifuged at 12000 rpm for 10 minutes. After centrifugation, the supernatant was filtered through a 0.22 μm sterile filter membrane. The resulting liquid is the cell-free supernatant. After sample preparation, it was stored at -80°C for later use.

[0187] 2.4 SEM observation of K56 cell morphology after inactivation

[0188] Referring to reference [1], the morphological changes of live and heat-inactivated bacteria were observed by SEM. Live or inactivated cells were fixed in 2.5% glutaraldehyde fixative at 4℃, and then subjected to gradient dehydration treatment with different concentrations of anhydrous ethanol (30%, 50%, 70%, 80%, 90%, 100%) for 30 minutes. After dehydration, the samples were dried in an oven at 37℃ for 24 hours. The samples were then subjected to critical point drying and gold sputtering, and observed under a scanning electron microscope (Hitachi, S-4800, Japan).

[0189] Literature [1] SUN Z, ZHAO Z, FANG B, et al. Effect of thermal inactivation onantioxidant, anti-inflammatory activities and chemical profile of postbiotics[J]. Foods, 2023, 12(19).

[0190] 2.5 Cell-free supernatant LC-MS / MS assay

[0191] In this embodiment, K56 was used to identify substances in cell-free supernatant using non-targeted metabolomics. Sample preparation was performed according to our previous study [1]. The samples were analyzed by LC-MS / MS using a Thermo UHPLC-Q Exactive HF-X mass spectrometer. After separation by an HSS T3 column, the samples were analyzed by MS / MS for compound identification. The operating parameters of the Q Exactive HF-X mass spectrometer were as follows: negative mode -3500V, positive mode 3500V, capillary temperature set to 325℃, heater temperature set to 425℃, MS resolution set to 60,000, and MS / MS resolution set to 7500. The raw LC-MS / MS data were preprocessed using Progenesis QI software (Waters Corporation, USA) to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity. Metabolites were retrieved and identified using the HMDB database (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ), and the data were uploaded to the Majorbio cloud platform (https: / / cloud.majorbio.com) for analysis. At least 80% of the metabolite characteristics detected in any sample were retained. After filtering, the minimum metabolite value in a specific sample (where the metabolite level is below the limit of quantitation) was estimated, and each metabolic characteristic was summed and normalized. To reduce errors caused by sample preparation and instrument instability, a summation normalization method was used to process the response intensity of the sample mass spectrometry peaks to obtain a normalized data matrix. Simultaneously, variables with a relative standard deviation (RSD) > 30% in the quality control samples were removed, and a log10 transformation was performed to obtain the final data matrix for subsequent analysis.

[0192] 2.6 Culture of RAW264.7 macrophages

[0193] Cell resuscitation: After removing the cells from liquid nitrogen, they were rapidly thawed in a 37°C water bath. They were then transferred to centrifuge tubes containing 9 mL of complete culture medium and centrifuged at 1000 rpm for 5 min. The cryopreservation solution was discarded, and 3 mL of complete culture medium was added and mixed thoroughly before transferring to culture dishes, with an appropriate amount of culture medium added. The cells were cultured in a CO2 incubator, with the culture medium changed every other day to ensure complete removal of DMSO.

[0194] Cell passage: When the cell density reaches 80%-90%, proceed with passage culture. First, discard the old culture medium and wash three times with sterile PBS buffer. After adding 3 mL of culture medium, gently scrape off cells in the same direction using a cell scraper and collect them in a centrifuge tube. Centrifuge at 1000 rpm for 5 min and discard the culture medium. Add an appropriate amount of culture medium to adjust the cell concentration to a suitable seeding density and evenly seed the cells in culture dishes. Passage the cells every two days to maintain their growth status.

[0195] Cell cryopreservation: Following cell passage methods, centrifuge and discard the culture medium, then add the prepared cryopreservation solution and mix thoroughly. Add 1 mL of cell suspension to a cryovial and label it. Place it in a gradient cryopreservation box.

[0196] It was stored overnight in an ultra-low temperature freezer at -80℃ and then transferred to liquid nitrogen for long-term storage.

[0197] 2.7 Establishment of an in vitro cellular inflammation model

[0198] RAW264.7 macrophages (1×10⁶ cells / mL) were seeded in 12-well plates and stimulated with 1 μg / mL LPS to induce cellular inflammation for 24 h. The treatments for each group were as follows:

[0199] (1) Control group: DMEM complete medium was added to the well plate, and no other treatment was performed. The plate was incubated for 24 hours.

[0200] (2) Model group (LPS): In addition to adding DMEM complete medium, 1 μg / mL LPS was added for stimulation for 24 hours;

[0201] (3) Treatment group (LPS + cell-free supernatant / organic acid): After stimulating and inducing inflammation with 1 μg / mL LPS, different concentrations of intervention drugs were added and cultured for 24 hours.

[0202] 2.8 Effects of different concentrations of inactivated bacteria on macrophage viability

[0203] (1) Collect macrophages in the logarithmic growth phase, seed 100 μL per well in a 96-well plate at a seeding density of 5 × 10⁵ cells / mL, and incubate in a cell culture incubator for 24 hours;

[0204] (2) Add a final concentration of 1×10 to each well. 7 5×10 7 1×10 8 5×10 8 1×10 9 100 μL of CFU / mL inactivated bacteria was incubated at 37℃ for 24 hours, with 6 replicates per group.

[0205] (3) Discard the old culture medium, add 100 μL of DMEM culture medium (without FBS) containing 10 μL LCK-8 to each well, and incubate in an incubator for 30 minutes;

[0206] (4) The absorbance (OD value) was measured at a wavelength of 450 nm using an ELISA reader. Cell viability (%) = (OD value of treatment group - OD value of blank group / OD value of normal group - OD value of blank group) × 100%, where the blank group has no cells; the normal group has only DMEM cells without any treatment.

[0207] 2.9 Effects of different concentrations of organic acids on macrophage viability

[0208] Collect macrophages in the logarithmic growth phase, at a ratio of 5 × 10⁻⁶. 5 At a seeding density of 100 μL / mL, 100 μL was seeded into each well of a 96-well plate and incubated in a cell culture incubator for 24 hours.

[0209] Add 100 μL of organic acid (malic acid, sebacic acid) to each well at a final concentration of 0, 5, 10, 20, or 50 μg / mL, and incubate at 37°C for 24 hours. Each group is repeated 6 times.

[0210] Refer to (3) and (4) of 2.8 for the remaining steps.

[0211] 2.10 Cell RNA extraction and RT-qPCR detection

[0212] 2.10.1 Cell RNA Extraction

[0213] (1) Discard the old culture medium and wash the cells twice with pre-cooled PBS. Add 500 μL of Trizol to each well, incubate on ice for 5 minutes, and mix thoroughly to ensure complete cell lysis. Transfer to 1.5 mL EP tubes and label them.

[0214] (2) Add 100 μL of ice-cold chloroform and mix well. Let stand for 2 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Carefully pipette 200 μL of the supernatant into a new EP tube and label it.

[0215] (3) Add an equal volume of pre-cooled isopropanol to the EP tube, mix well, and let stand at room temperature for 10 minutes. Centrifuge at 10,000 rpm for 10 minutes at 4°C, and you can see white RNA precipitate at the bottom of the tube;

[0216] (4) Discard the supernatant, add 500 μL of 75% ethanol (anhydrous ethanol prepared with DEPC water), mix thoroughly, centrifuge at 7500 rpm for 5 minutes at 4°C, and carefully discard the supernatant.

[0217] (5) Centrifuge at 7500 rpm for 2 minutes at 4℃, and use a 10 μL pipette tip to remove any residual ethanol from the tube wall to shorten the drying time. Dry at room temperature for 2 minutes;

[0218] (6) Resuspend the precipitated RNA in 20 μL of DEPC water. Determine the concentration and purity of the RNA using a Nanodrop 2000. Store at -80°C for later use.

[0219] 2.10.2 RNA Reverse Transcription

[0220] Dilute the RNA to an appropriate concentration and perform reverse transcription using the All-In-One 5×RT MasterMix kit. The reaction volume is as follows:

[0221] Table 3 Reverse Transcription Reaction System

[0222]

[0223] After preparing the reaction mixture, vortex it thoroughly. After gentle centrifugation, place the sample into a gene amplification instrument for reverse transcription. The reaction procedure is as follows:

[0224] Table 4 Reverse Transcription Procedure

[0225]

[0226] The obtained cDNA was stored at -20°C for later use.

[0227] 2.10.3 Real-time quantitative PCR detection

[0228] The experiment used TB Premix Ex Taq™ (Tli RNaseH Plus) was used with the ROX plus kit. The 20 μL reaction mixture is as follows:

[0229] Table 5 qPCR system

[0230]

[0231] The two-step PCR amplification system is as follows:

[0232] Table 6 qPCR reaction procedures

[0233]

[0234] The experiment used GADPH as an internal reference gene, and the relative expression level of each target gene was determined using the 2-ΔΔCt method. The primer sequences are as follows:

[0235] Table 7 Primer sequences

[0236]

[0237] 2.11 Data Statistical Analysis

[0238] Data are expressed as mean ± standard deviation, and all experiments were repeated at least three times. For comparisons between groups, analysis of variance (ANOVA) with two-tailed Student's t-test and Dunnett's multiple comparison test was used. Data analysis was performed using GraphPad Prism 9.0.0. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0239] 3. Experimental Results:

[0240] 3.1 Scanning electron microscopy observation of Lactobacillus paracasei K56 cell morphology after inactivation

[0241] The morphological changes of K56 after inactivation at different temperatures were observed using scanning electron microscopy (SEM). Figure 2 It can be seen that the K56 live bacterial cells have smooth, regular surfaces and intact cell membranes. However, the bacterial cells treated at 80℃ showed significant morphological changes, with cell rupture and the release of small amounts of intracellular material. As the temperature continued to rise, the bacterial cells continued to shrink and rupture, accompanied by white clusters of granules and leakage of intracellular contents. These results indicate that metabiotics are a mixed state comprising intact dead cells, cell walls and cell membranes, and intracellular exudates.

[0242] 3.2 Effects of K56 inactivated bacteria and cell-free supernatant on TNF-α production in RAW264.7 cells.

[0243] In the pathogenesis of chronic diseases, TNF-α production induces an excessive inflammatory response in the body. To investigate the anti-inflammatory effects of K56 inactivated bacteria and cell-free supernatant, TNF-α mRNA levels were assessed by RT-qPCR. Compared with the control group, LPS stimulation of RAW264.7 cells significantly increased TNF-α mRNA expression (P<0.0001). After K56 inactivation treatment at different temperatures, LPS-induced TNF-α production in RAW264.7 macrophages was significantly reduced (P<0.0001). Inactivation at 121℃ showed better inhibitory effects. Figure 3 (A) Similarly, compared with the LPS group, K56 cell-free supernatant at different temperatures significantly downregulated TNF-α mRNA expression (P<0.001), and the inhibitory effect of cell-free supernatant at 121℃ was better ( Figure 3 (B in the text). These results preliminarily suggest that K56 inactivated bacteria and cell-free supernatant can inhibit LPS-induced inflammatory responses.

[0244] 3.3 Non-targeted metabolomics analysis of cell-free supernatant metabolites

[0245] To investigate potential anti-inflammatory substances in the cell-free supernatant of *Lactobacillus paracasei*, a detailed analysis of the cell-free supernatant of three *Lactobacillus paracasei* strains with potential anti-inflammatory activity was performed using non-targeted metabolomics. The results showed that a large number of organic substances were identified in the cell-free supernatant, including lipids, amino acids and peptides, organic acids, nucleotides, and sugars. Lipids were the most abundant, followed by amino acids and peptides and organic acids. Figure 4 (A). The cell-free supernatant of K56 contains 2831 substances, including 783 lipids, 736 amino acids and peptides, and 363 organic acids, accounting for 66.5% in total.

[0246] In summary, this embodiment demonstrates using an LPS-stimulated RAW264.7 cell model that inactivated Lactobacillus paracasei K56 bacteria and cell-free supernatant at different inactivation temperatures can significantly reduce the expression of the pro-inflammatory factor TNF-α (P<0.05), indicating that both inactivated K56 bacteria and cell-free supernatant have good anti-inflammatory activity and can be used for subsequent experiments.

[0247] Scanning electron microscopy revealed that upon heating, *Lactobacillus paracasei* K56 exhibited cellular rupture and the release of intracellular substances. Non-target metabolomics analysis showed that the cell-free supernatant of *Lactobacillus paracasei* K56 was primarily composed of lipids, amino acids and peptides, organic acids, nucleotides, and sugars.

[0248] Example 2

[0249] This embodiment investigates the ameliorative effect of K56 postbiotic components on high-fat diet-induced inflammation in zebrafish. Zebrafish, due to their significant genetic similarity to humans, rapid development, high reproductive capacity, and ease of gene editing, have been proven to be an emerging animal model for studying inflammatory diseases, and can be used to promptly screen compounds affecting inflammation. In this embodiment, a high-fat diet induced inflammation in the zebrafish gut and liver. First, HE staining was used to observe the ameliorative effects of the K56 postbiotic components malic acid and sebacic acid on liver and gut damage caused by a high-fat diet. RT-qPCR was used to detect the expression of inflammatory factors in the liver and intestines, thus demonstrating the anti-inflammatory effects of malic acid and sebacic acid. Finally, 16S sequencing was used to analyze the zebrafish gut microbiota to explore whether the two organic acids could alleviate intestinal inflammation by restoring gut microbiota dysbiosis.

[0250] 1. Experimental reagents and instruments

[0251] 1.1 Laboratory Animals

[0252] This animal experiment was approved by the Institutional Animal Care Committee of China Agricultural University (approval number AW21903202-5-2) and conducted in accordance with animal welfare and ethical guidelines. The experimental fish were wild-type male zebrafish of the AB strain, purchased from Beijing Yigao Yongsheng Trading Co., Ltd. The zebrafish were acclimatized for two weeks prior to the experiment.

[0253] 1.2 Experimental Reagents

[0254] Table 8 Experimental Reagents

[0255]

[0256] 1.3 Experimental Apparatus

[0257] Table 9 Experimental Instruments

[0258]

[0259] 2 Experimental Methods

[0260] 2.1 Grouping and rearing of zebrafish

[0261] Four-month-old zebrafish (n=240, average initial weight=350mg) were randomly divided into 8 groups, with 5 replicates per group and 6 fish per replicate. The initial weight of each fish was recorded. During the rearing period, the system supplied circulating water at a temperature of 26±1℃, a pH of 7.0-7.7, and an ammonia nitrogen concentration of <0.1mg / L. Fish were fed twice daily at 9:00 AM and 4:00 PM, at a rate of 3% of the average initial weight per tank, for 4 weeks.

[0262] 2.2 Feed Preparation

[0263] The experimental diets were divided into 5 groups: control diet, high-fat diet (HFD), high-fat diet + K56 121℃ cell-free supernatant (HFD+K121), and high-fat diet + organic acids (HFD+malic acid MA / HFD+sediic acid DA). The control diet was provided by Beijing Sibefu Biotechnology Co., Ltd., and the high-fat diet was made by adding 20% ​​(w / w) lard to the control diet. The nutritional composition of the experimental diets is shown in Table 10. The feed preparation method was based on the method of Zhang et al. [2], and the feeds were stored at -20℃ before use. The K56 cell-free supernatant was added to the high-fat diet at a concentration of 1×10¹⁰ CFU / mL. The amount of malic acid and sediic acid added was calculated based on their relative content in the cell-free supernatant. The relative content of malic acid and sediic acid in the 121℃ cell-free supernatant is shown in Table 11. Using L-phenylalanine-d5 (0.003 mg / mL) as an internal standard, the calculated value of 40 mL of cell-free supernatant (1 × 10⁻⁶ mg / mL) was [calculated / calculated]. 9The bacterial culture concentration (CFU / mL) contained 0.2 mg of malic acid and 0.02 mg of sebacic acid. Therefore, malic acid was added at 40 mg / kg and sebacic acid at 4 mg / kg to 50 g of high-fat feed.

[0264] Literature[2]ZHANG FL,YANG YL,ZHANG Z,et al.Surface-displayed Amuc_1100from Akkermansia muciniphila on Lactococcus lactis ZHY1 improves hepaticsteatosis and intestinal health in high-fat-fed zebrafish[J].Frontiers inNutrition,2021,8.

[0265] Table 10 Feed ingredients and composition

[0266]

[0267] Note: ① Multivitamins (g / kg diet): Thiamine 0.438g; Riboflavin 0.632g; Vitamin B6 0.908g; D-Pantothenic Acid 1.724g; Niacin 4.583g; Biotin 0.211g; Folic Acid 0.549g; Vitamin B12 0.001g; Inositol 21.053g; Vitamin K3 0.889g; Vitamin A Acetate 0.677g; Vitamin D3 0.116g; Vitamin E Acetate 12.632g;

[0268] ② Multiminerals (g / kg diet): CoCl2·6H2O (1%) 0.074g; CuSO4·5H2O 2.5g; FeSO4·7H2O 73.2g; NaCl 40.0g; MgSO4·7H2O 284.0g; MnSO4·H2O 6.50g; KI 0.68g; Na2SeO3 0.10g; ZnSO4·7H2O 131.93g; cellulose 501.09g.

[0269] Table 11 Relative concentrations (mg / mL) of malic acid and sebacic acid

[0270]

[0271] 2.3 Sample Collection

[0272] After the feeding experiment, the experimental fish in each tank were anesthetized with an ice bath. Each fish was then weighed, the liver weight was recorded, and the HSI (HSI = 100% × liver weight / final body weight) was calculated. The liver and intestines were collected and immediately frozen in liquid nitrogen. The liver used for HE sectioning was fixed with 4% paraformaldehyde. Other tissues were stored at -80°C.

[0273] 2.4 Liver HE staining

[0274] After the experiment, fresh livers from three fish in each treatment group were taken, fixed with 4% paraformaldehyde for 24 hours, and then embedded in paraffin before sectioning. The sections were stained with hematoxylin and eosin (HE). After the sections were returned, morphological changes in the histopathological sections were observed under a fluorescence microscope.

[0275] 2.5 RNA extraction from relevant tissues and RT-qPCR detection

[0276] 2.5.1 Total RNA extraction from liver and intestine

[0277] Liver and intestine samples were cryopreserved in liquid nitrogen for gene expression analysis of liver and intestinal inflammatory factors. Three liver samples were pooled as a single sample, with six replicates. One intestinal sample was also used as a single sample, with six replicates.

[0278] (1) Remove the liver and intestinal samples stored at -80℃ and place them on an ice box. Add 1 mL of Trizol and homogenize at low temperature. Place the homogenized samples at 4℃ for 5 min.

[0279] (2) Centrifuge at 4℃ and 12000rpm for 10min, transfer the supernatant to a new sterile enzyme-free EP tube and label it.

[0280] (3) The subsequent steps are the same as step 2.10.1 in Example 1, except that 200 μL of chloroform and 1 mL of 75% anhydrous ethanol are added, and the other conditions remain unchanged.

[0281] 2.5.2 RNA reverse transcription refers to step 2.10.2 in Example 1.

[0282] 2.5.3 Real-time quantitative PCR

[0283] The experiment used TB Premix Ex Taq™ (Tli RNaseH Plus) and ROX plus kit were used. The experiment used a 10 μL reaction volume, following the qPCR reaction and amplification system described in section 2.3.10.3.

[0284] The experiment used Rps11 as an internal reference gene, and the relative expression level of each target gene was determined using the 2-ΔΔCt method. The primer sequences are as follows:

[0285] Table 12 Primer sequences

[0286]

[0287] 2.6 Intestinal 16S Sequencing and Analysis

[0288] According to the manufacturer's instructions, genomic DNA of the gut microbiota was extracted from zebrafish using the PF Mag-Bind Stool DNA Kit. 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-

[0289] PCR amplification of the variable regions of the 16S ribosomal RNA gene V3-V4 was performed using specific primers (GGACTACHVGGGTWTCTAAT-3′). After amplification, the product was recovered and purified using a 2% agarose gel. Paired-end sequencing was performed using an Illumina MiSeq PE300 / PE250 sequencing platform. The raw sequencing sequences were quality controlled using FASTP, and assembly was performed using FLASH. After quality filtering, chimeric sequences were removed, and OTU clustering of the optimized sequences was performed using UPARSE, with a similarity of 97%. Species annotation (confidence threshold: 70%) was performed on representative OTU sequences using the 16S rRNA gene database Silva (v138).

[0290] 2.7 Data Statistical Analysis

[0291] Data are expressed as mean ± standard deviation, and all experiments were repeated at least three times. For comparisons between groups, analysis of variance (ANOVA) with two-tailed Student's t-test and Dunnett's multiple comparison test was used. Data analysis was performed using GraphPad Prism 9.0.0. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0292] All 16s sequencing data analyses were performed on the MajorBio cloud platform (https: / / cloud.majorbio.com). PCoA analysis based on the Bray-Curtis distance algorithm was used to compare the β-diversity of the bacterial communities among the groups. The Wilcoxon rank-sum test was used to analyze the differences in relative abundance at the genus level between the two groups. LEfSe analysis (LDA>3, P<0.05) was used to identify species with significant differences at the genus level among the different groups.

[0293] 3. Experimental Results

[0294] 3.1 Effects of K56 post-biotic components on liver weight and liver weight index in zebrafish

[0295] After 4 weeks of rearing, the liver weight and liver weight index of the zebrafish were measured, and the results were as follows: Figure 7 As shown in the figure, compared with the control group, the liver weight of zebrafish fed a high-fat diet tended to increase. Compared with the HFD group, the liver weight tended to decrease after intervention with 121℃ cell-free supernatant (HFD+K121), malic acid (HFD+MA), and sebacic acid (HFD+DA). Similarly, the liver weight index of zebrafish in the HFD group also tended to increase compared with the control group. The liver weight index tended to decrease after intervention with 121℃ cell-free supernatant (HFD+K121), malic acid (HFD+MA), and sebacic acid (HFD+DA). Among them, the intervention with malic acid (HFD+MA) was more effective. Therefore, malic acid and sebacic acid can alleviate abnormal liver weight gain caused by a high-fat diet.

[0296] 3.2K56 postbiotic components reduce liver pathological damage.

[0297] To investigate the effects of K56 supplementation on zebrafish liver health, hematoxylin and eosin (HE) staining was used to observe liver damage in zebrafish. Figure 8 In the control group, zebrafish livers were dense and structurally intact, with hepatocyte nuclei located centrally. In the HFD group, numerous hepatocytes showed lateral nuclear shift, and compared to the control group, the HFD group had an increased number of hepatocytes with vacuoles. Intervention with K56 cell-free supernatant (HFD+K121) and organic acids (HFD+MA, HFD+DA) resulted in more regular and compact hepatocyte arrangement, increased nucleoli, and smaller cytoplasmic vacuoles, alleviating hepatic steatosis in HFD-fed zebrafish. Therefore, malic acid and sebacic acid can alleviate liver damage induced by a high-fat diet.

[0298] 3.3K56 post-biotic components alleviate liver inflammation in zebrafish induced by a high-fat diet.

[0299] RT-qPCR was used to detect the mRNA expression of inflammatory factors in zebrafish liver to evaluate the alleviating effect of K56 post-biotic intervention on liver inflammation. The results are as follows: Figure 9Compared with the control group, after HFD modeling, the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β were significantly increased (P<0.0001), while the levels of anti-inflammatory factors TGF-β and IL-10 were significantly decreased (P<0.05). After K56 postbiotic intervention, the expression of TNF-α, IL-6, and IL-1β was significantly decreased compared with the HFD group (P<0.01). Malic acid (HFD+MA) had a more significant inhibitory effect on pro-inflammatory factors. Cell-free supernatant (HFD+K121) and organic acids (HFD+MA, HFD+DA) significantly upregulated the mRNA expression of the anti-inflammatory factor TGF-β (P<0.05), while malic acid (HFD+MA) significantly increased the expression of IL-10 mRNA (P<0.05). Therefore, K56 cell-free supernatant, malic acid, and sebacic acid can alleviate liver inflammation induced by a high-fat diet by reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors.

[0300] 3.4K56 postbiotic components reduce intestinal barrier damage.

[0301] from Figure 10 As shown in Figure A, zebrafish fed with HFD exhibited signs of intestinal damage. The HFD group showed mild disruption of the intestinal mucosa, submucosa, muscularis propria, and serosa, with reduced goblet cells and inflammatory cell infiltration. Compared to the HFD group, K56 postbiotic treatment restored the intestinal damage induced by HFD in zebrafish. Further validation of gene expression levels related to intestinal barrier function was conducted. RT-qPCR results showed that, compared to the Control group, HFD significantly reduced the expression levels of intestinal tight junction proteins (P<0.05), including Occludin and Zonula occlusionns-1 (ZO-1). Figure 10 (Middle B). As we expected, K56 supplementation significantly increased the mRNA expression of ZO-1 and Occludinb (P<0.05), indicating that some intestinal barrier-related functions were restored.

[0302] The 3.5K56 postbiotic component alleviates intestinal inflammation in zebrafish caused by a high-fat diet.

[0303] The expression of intestinal inflammatory factor mRNA in zebrafish was detected to evaluate the alleviating effect of K56 postbiotic intervention on intestinal inflammation. The results are as follows: Figure 11Compared with the control group, HFD significantly increased the expression of pro-inflammatory factors TNF-α and IL-1β (P<0.05) and significantly inhibited the expression of anti-inflammatory factor IL-10 (P<0.0001). Compared with the HFD group, sebacic acid (HFD+DA) significantly inhibited the expression of IL-1β (P<0.05), and the K56 postbiotic component significantly increased the expression of anti-inflammatory factor IL-10 (P<0.01). Malic acid (HFD+MA) and sebacic acid (HFD+DA) significantly downregulated the expression of pro-inflammatory factor TNF-α (P<0.01). Therefore, K56 cell-free supernatant, malic acid, and sebacic acid can also alleviate intestinal inflammation induced by a high-fat diet by reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors.

[0304] 3.6K56 postbiotic components alter the gut microbiota composition of zebrafish.

[0305] 3.6.1 Effects of K56 postbiotic components on β-diversity of zebrafish gut microbiota.

[0306] To investigate the effects of K56 postbiotic on the gut microbiota of zebrafish, changes in the gut microbiota were analyzed using 16S rRNA gene sequencing. Results are shown below. Figure 12 PCoA analysis, represented by the relative abundance distance of the Bray-Curtis genus, showed changes of 34.9% and 19.29% for PC1 and PC2, respectively. The Control group and the HFD group were significantly different and clustered independently. These results indicate a significant difference in gut microbiota between the Control and HFD groups, with HFD causing changes in the zebrafish gut microbiota. After K56 postbiotic intervention, the gut microbiota was completely separated from the Control group, but there was varying degrees of overlap among the treatment groups. Therefore, K56 postbiotic intervention alters the zebrafish gut microbiota, with malic acid (HFD+MA) and sebacic acid (HFD+DA) gut microbiota showing some similarity in structure.

[0307] 3.6.2 Changes in the gut microbiota of zebrafish at the phylum level

[0308] This embodiment evaluated the composition of the top 10 microorganisms by relative abundance at the phylum level in each group. Figure 13 It is evident that the gut microbiota in each group are mainly composed of Proteobacteria, Actinobacteriota, Firmicutes, and Verrucomicrobiota. Figure 13(A) Compared with the Control group, the relative abundance of Proteobacteria was significantly reduced in the HFD group (P<0.01), while the relative abundance of Actinobacteria and Firmicutes increased, and the relative abundance of Verrucomicrobia remained unchanged. The K56 postbiotic reduced the relative abundance of Actinobacteria but had no effect on the relative abundance of species at other phyla. Specifically, in the malic acid group (HFD+MA), the relative abundance of Actinobacteria was significantly decreased (P<0.05). Figure 13 (B)

[0309] 3.6.3 Changes in zebrafish gut microbiota at the genus level.

[0310] Further analysis of the composition of the gut microbiota at the genus level was conducted, and the results are shown in [the table below]. Figure 14 In group A, compared with the control group, HFD led to an increase in the relative abundance of *Vibrio*, *Legionella*, and *Shewanella*, while decreasing the relative abundance of *Ancylobacter*, *Aeromonas*, *Bosea*, *Xanthobacter*, and *Cetobacterium*. Compared with the HFD group, the abundance of *Aeromonas* and *Cetobacterium* increased in the cell-free supernatant group (HFD+K121), while the abundance of *Shewanella* decreased; malic acid (HFD+MA) and sebacic acid (HFD+DA) interventions increased the relative abundance of *Bosea* and *Cetobacterium*, and decreased the relative abundance of *Vibrio* and *Shewanella*.

[0311] Subsequently, we performed LEfSe analysis to assess the different eigengroups among the groups. Figure 14 (B) Based on the results of LDA scores ≥3, Ancylobacter, Flavobacterium, and ZOR0006 were significantly enriched in the Control group; Shewanella and Paracoccus were significantly enriched in the HFD group; Chlamydia were significantly enriched after intervention with cell-free supernatant (HFD+K121); Legionella were significantly enriched in the malic acid group (HFD+MA); and GKS98_freshwater_group was significantly enriched in the sebacic acid group (HFD+DA).

[0312] 3.6.4 Species differences in gut microbiota at the genus level among zebrafish groups.

[0313] The study analyzed the differences between the HFD group and the intervention group at the genus level, such as Figure 15Compared to the HFD group, *Shewanella*, *Lactobacillus*, and *Paracoccus* were significantly reduced in the cell-free supernatant group (HFD+K121), while *norank_f_cvE6* was significantly increased. After malate (HFD+MA) intervention, the abundance of *unclassified_f_Kineosporiaceae* and *norank_f_JG30-KF-CM45* significantly increased, while the abundance of *Paraclostridium* significantly decreased. Compared to the HFD group, sebacic acid (HFD+DA) significantly increased the relative abundance of *Plesiomonas*, *GKS98_freshwater_group*, and *Roseomonas*.

[0314] In summary, intervention with K56 cell-free supernatant, malic acid, and sebacic acid alleviated abnormal liver weight gain in zebrafish induced by a high-fat diet and reduced liver damage caused by a high-fat diet. It also reduced the levels of pro-inflammatory liver factors TNF-α, IL-6, and IL-1β, while increasing the levels of anti-inflammatory factors IL-10 and TGF-β. Furthermore, malic acid and sebacic acid intervention regulated the expression of intestinal inflammatory factors TNF-α, IL-1β, and IL-10, and significantly upregulated the expression of tight junction proteins ZO-1 and Occludinb.

[0315] Furthermore, the gut microbiota of zebrafish underwent significant changes under the intervention of malic acid and sebacic acid. The relative abundance of beneficial bacteria such as *Bosea*, *Cetobacter*, and *Aeromonas* increased, while the relative abundance of *Vibrio* and *Shewanella* decreased.

[0316] Example 3

[0317] This study investigated the ameliorative effect of K56 post-biotic components on DSS-induced colitis in mice. Transcriptomics was used to further explore the mechanism of action of K56 post-biotic components in improving acute colitis. Acute colitis in mice was induced with 3% DSS, a widely used animal model. First, mouse body weight and disease activity index were monitored daily from the start of modeling, and the mice's condition was observed. HE staining and qPCR were used to detect the ameliorative effects of malic acid and sebacic acid on colonic injury and inflammation. Finally, RNA-seq technology was used to screen differentially expressed genes and the KEGG pathway, preliminarily exploring the signaling pathways by which the K56 post-biotic components malic acid and sebacic acid improve colitis. However, further validation of these signaling pathways is needed to provide more effective information for subsequent research on anti-inflammatory mechanisms.

[0318] 1. Experimental reagents and instruments

[0319] 1.1 Laboratory Animals

[0320] This animal experiment was approved by the Institutional Animal Care Committee of China Agricultural University (approval number: AW31903202-5-2) and strictly adhered to animal welfare and ethical guidelines. Eighty SPF-grade male C57BL / 6J mice (6 weeks old), weighing 22±2g, were purchased from Beijing Speford Biotechnology Co., Ltd., and housed on the SPF-grade animal housing platform at China Agricultural University. During the experiment, the housing platform maintained a constant temperature of 22±2℃ and a constant humidity of 50%-60%, with diurnal alternation. Free access to food and water was ensured for all experimental animals.

[0321] 1.2 Experimental Reagents

[0322] Table 13 Experimental Reagents

[0323]

[0324] 1.3 Experimental Apparatus

[0325] Table 14 Experimental Instruments

[0326]

[0327] 2. Experimental Methods

[0328] 2.1 Establishment of an acute colitis model

[0329] C57BL / 6J mice were randomly divided into 8 groups of 10 mice each, and the experiment began after one week of acclimatization. An acute colitis model was induced in mice using 3% (w / v) DSS. The experiment lasted 19 days, with the following groupings (200 μL / mouse administered by gavage daily):

[0330] (1) Control group: Normal drinking water was given for 19 consecutive days, while pure water was administered by gavage.

[0331] (2) DSS group (DSS): Normal drinking water was given for the first 7 days. Starting from the 8th day, 3% DSS solution was given for 7 consecutive days. Normal drinking water was resumed for the last 5 days. Pure water was administered by gavage every day.

[0332] (3) Cell-free supernatant group (DSS+K121-L / DSS+K121-H): Normal drinking water was given for the first 7 days. Starting from the 8th day, 3% DSS solution was given for 7 consecutive days. Normal drinking water was resumed for the last 5 days. The high-dose group was given 2×10¹⁰ CFU / day of cell-free supernatant at 121℃ by gavage, and the low-dose group was given 10⁹ CFU / day of cell-free supernatant at 121℃ by gavage.

[0333] (4) The amount of malic acid and sebacic acid added was calculated based on their relative content in the cell-free supernatant. The relative content of malic acid and sebacic acid in the cell-free supernatant at 121℃ is shown in Table 15. Using L-phenylalanine-d5 (0.003 mg / mL) as an internal standard, the amount of malic acid and sebacic acid added in 40 mL of cell-free supernatant (1×10⁻⁶ mg / mL) was calculated. 9 The relative content of malic acid (CFU / mL bacterial culture concentration) is 0.2 mg, and the relative content of sebacic acid is 0.02 mg.

[0334] (5) Organic acid groups (DSS+MA-L / DSS+MA-H, DSS+DA-L / DSS+DA-H): Normal drinking water was given for the first 7 days. Starting from the 8th day, 3% DSS solution was given for 7 consecutive days. Normal drinking water was resumed for the last 5 days. The high-dose group was given malic acid 4 mg / day by gavage, and the low-dose group was given malic acid 0.2 mg / day by gavage. The high-dose group was given sebacic acid 0.4 mg / day by gavage, and the low-dose group was given sebacic acid 0.02 mg / day by gavage.

[0335] Table 15 Relative concentrations (mg / mL) of malic acid and sebacic acid

[0336]

[0337] 2.2 Weight and DAI detection

[0338] During the modeling period, the mice's weight was recorded daily, and their fecal characteristics and the presence of fecal blood were observed and recorded. Simultaneously, the Disease Activity Index (DAI) was scored on the mice, based on the rate of weight loss, fecal characteristics, and the presence of fecal occult blood. Specific scoring criteria are shown in Table 16. DAI = (Rate of weight loss + Fecal characteristics + Fecal occult blood) / 3.

[0339] Table 16 DAI Scores

[0340]

[0341] 2.3 Sampling and Colon Length Measurement

[0342] After the experiment, the mice's eyeballs were removed and blood was collected. They were then dissected, and the colon was removed and its length measured. The colonic tissue was fixed in 4% paraformaldehyde for HE staining. The contents of the small intestine, colon, and cecum were collected, flash-frozen in liquid nitrogen, and stored at -80°C.

[0343] 2.4 Detection of colonic myeloperoxidase (MPO)

[0344] Cleanse the contents of the colon thoroughly, absorb excess water with absorbent paper, and weigh accurately. Homogenize the tissue with the prepared homogenization medium at a 1:19 weight-to-volume ratio; centrifugation is not required. Perform subsequent measurements according to the instructions of the Nanjing Jiancheng MPO assay kit.

[0345] 2.5 HE staining of colon.

[0346] The colon was washed with pre-cooled PBS buffer and fixed by immersion in 4% paraformaldehyde. The fixed colon was then dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Finally, the stained sections were examined under a fluorescence microscope to observe the extent of damage to the colon tissue. Experienced researchers performed randomized, double-blind histological scoring of the colon sections using the following criteria:

[0347] Table 17 HE Pathology Scoring Details

[0348]

[0349] 2.6 RNA extraction from relevant tissues and RT-qPCR detection

[0350] 2.6.1 Total RNA extraction from the colon

[0351] (1) Take out the colon sample stored at -80℃ and place it on an ice box. Weigh 50mg of colon and add 1mL of Trizol. Use sterile scissors to cut the tissue into small pieces and homogenize it at low temperature. Place the homogenized sample at 4℃ for 5min.

[0352] (2) The subsequent steps are as described in 2.5.1 of Example 2. After washing the supernatant with 75% anhydrous ethanol, add 1 mL of LiCl to wash the RNA precipitate to prevent DSS from affecting the PCR amplification results. Mix well at 4°C and centrifuge at 7500 rpm for 5 minutes.

[0353] (3) Use a 10μL pipette tip to remove any remaining LiCl from the tube wall to shorten the drying time. Dry at room temperature for 2 minutes.

[0354] 2.6.2 RNA reverse transcription refers to 2.5.2 in Example 2.

[0355] 2.6.3 Real-time quantitative PCR was performed as described in 2.5.3 of Example 2. β-actin was used as an internal reference gene, and the relative expression levels of each target gene were determined using the 2-ΔΔCt method. The primer sequences are as follows:

[0356] Table 18 Primer Sequences

[0357]

[0358] 2.7 Colonic Transcriptomics Sequencing and Analysis

[0359] 2.7.1 RNA extraction and sequencing:

[0360] Referring to the method in Example 2.6, using Total RNA was extracted from the colon using reagents. RNA concentration and purity were determined using Nanodrop 2000, and agarose gel electrophoresis was used to evaluate its integrity. High-quality RNA samples were selected to construct libraries (OD260 / 280 = 1.8-2.2, OD260 / 230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, RNA volume > 1 μg). RNA purification, reverse transcription, library construction, and sequencing were performed by Shanghai Meiji Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer Illumina's instructions (San Diego, CA). The colon RNA-seq transcriptome library was obtained using... streedmrnaprep, ligation of millumina (San Diego, CA), was prepared using 1 μg of total RNA. In short, mRNA was isolated from total RNA by base pairing with the 3' end of the mRNA using magnetic beads with Oligo(dT) primers. The mRNA was then randomly fragmented using fragment buffer. Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) and random hexamer primers (Illumina). The synthesized cDNA was then processed according to Illumina's library construction protocol. A library with a target fragment of 300 bp was selected on 2% Low Range Ultra agarose gel and amplified for 15 PCR cycles using Phusion DNA polymerase (NEB). After quantification using Qubit 4.0, the contra-end RNA-seq library was sequenced using a NovaSeq 6000 sequencer (2 × 150 bp reads).

[0361] 2.7.2 Transcriptome bioinformatics analysis.

[0362] First, raw paired-end reads were pruned and quality-controlled using fastp (https: / / github.com / OpenGene / fastp) with default parameters. Clean reads were aligned to a reference genome using HiSat2 (http: / / ccb.jhu.edu / software / hisat2 / index.shtml). Next, RSEM was used to quantify gene and transcript expression levels. The TPM (transcripts per million reads) method was used to calculate the expression level of each transcript to identify differentially expressed genes (DEGs) between different samples (where |log2FC|≧1 and FDR<0.05 are differentially expressed genes). Furthermore, KEGG pathway enrichment analysis of differentially expressed genes was performed using KOBAS (http: / / kobas.cbi.pku.edu.cn / home.do), with Fisher's exact test used to calculate the results and multiple tests performed using the FDR method. KEGG pathways satisfying P-value <0.05 were considered significantly enriched in differentially expressed genes.

[0363] 2.8 Data Statistical Analysis

[0364] Data are expressed as mean ± standard deviation, and all experiments were repeated at least three times. For comparisons between groups, analysis of variance (ANOVA) with two-tailed Student's t-test and Dunnett's multiple comparison test was used. Data analysis was performed using GraphPad Prism 9.0.0. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0365] 3. Experimental Results

[0366] 3.1 Effects of K56 postbiotic components on mouse body weight and condition.

[0367] After starting to drink DSS, the mice were observed and their daily weight was recorded. The rate of weight change in each group of mice was calculated, and the results are as follows: Figure 16During the modeling period, mice drinking 3% DSS showed poor mental state, slow movement, dull fur, and significant bloody and loose stools. Compared to the normal group, the DSS group mice showed a decreasing body weight trend. Even after DSS administration was discontinued on day 8, the body weight of the DSS group mice continued to decrease. During the 7 days of DSS administration, mice in all groups experienced weight loss after intervention with high and low doses of K56 postbiotics. After DSS withdrawal (days 8-12), the mental state of the mice in all intervention groups gradually recovered, and the bloody stools gradually subsided. On day 11, compared with the DSS group, the body weight of mice in the low-dose cell-free supernatant group (DSS+K121-L) and the high and low-dose malic acid groups (DSS+MA-L, DSS+MA-H) was significantly increased (P<0.01); the body weight of mice in the sebacic acid group (DSS+DA-L, DSS+DA-H) showed no significant change. On day 12, high and low doses of cell-free supernatant (DSS+K121-L, DSS+K121-H), malic acid (DSS+MA-L, DSS+MA-H), and high dose sebacic acid (DSS+DA-H) all restored mouse body weight, with statistically significant differences (P<0.05). In summary, malic acid (DSS+MA) and sebacic acid (DSS+DA) were effective in restoring mouse body weight.

[0368] 3.2 Effect of K56 postbiotic components on the disease activity index (DAI).

[0369] During the period of drinking DSS, the changes in the disease activity index (DAI) of mice in each group were as follows: Figure 17 As shown in the figure, the DAI score in the control group remained basically unchanged. Compared with the control group, the DAI of DSS mice increased significantly, and the increase was rapid starting from day 4. After DSS was removed on day 8, the DAI of DSS mice began to level off and showed a downward trend starting from day 11. Compared with the DSS group, the increase in DAI was slower after intervention with different doses of malic acid (DSS+MA) and sebacic acid (DSS+DA). Starting from day 10, the DAI of mice in each intervention group was significantly lower than that in the DSS group (P<0.05). The results indicate that both malic acid (DSS+MA) and sebacic acid (DSS+DA) can significantly reduce the DAI score.

[0370] 3.3 Effect of K56 postbiotic components on colon length in mice.

[0371] After dissection, the colon length of mice in each group was measured and statistically analyzed, and the results are as follows: Figure 18The average colon length in the control group was 5.85 cm, and in the DSS group it was 4.46 cm. Compared with the control group, the colon of DSS mice was significantly shortened (P<0.0001). Compared with the DSS group, the colon length of mice significantly increased after intervention with malic acid (DSS+MA) and sebacic acid (DSS+DA) (P<0.05). Therefore, malic acid (DSS+MA) and sebacic acid (DSS+DA) can improve the colon shortening induced by DSS.

[0372] 3.4 Effect of K56 postbiotic components on myeloperoxidase (MPO) activity in the colon.

[0373] To investigate the effect of K56 postbiotic on the inflammatory response of colonic tissue, the MPO content in the colon was measured.

[0374] ( Figure 19 Compared with the control group, the MPO activity in the DSS group was significantly increased (P<0.0001). K56 postbiotic treatment significantly decreased colonic MPO activity (P<0.05). High-dose malic acid (DSS+MA-H) had no significant effect on MPO activity. Therefore, malic acid (DSS+MA) and sebacic acid (DSS+DA) can reduce MPO activity in DSS mice.

[0375] The effect of 3.5K56 postbiotic components on colonic pathology.

[0376] To observe the effect of K56 postbiotic components on colonic injury, mouse colon tissue sections were stained with hematoxylin and eosin (HE). The results are as follows: Figure 20 In the control group, the colonic mucosa of mice was intact, with clearly visible crypt structures, intact goblet cells, and no obvious inflammatory cell infiltration. Compared with the control group, the DSS group showed severe mucosal damage, epithelial cell loss, reduced goblet cells, and inflammatory cell infiltration. The colonic injury score was significantly higher in the DSS group (P<0.05). Figure 20 (B) Malic acid (DSS+MA) and sebacic acid (DSS+DA) intervention reduced colonic epithelial damage, restored intestinal mucosa and crypt structures, and decreased inflammatory cell infiltration, thereby significantly reducing the pathological score of colonic tissue (P<0.05). The results indicate that malic acid (DSS+MA) and sebacic acid (DSS+DA) can improve DSS-induced colonic injury in mice and promote the recovery of damaged colons to normal morphology.

[0377] Effects of 3.6K56 postbiotic components on colonic inflammatory factor mRNA.

[0378] To investigate the alleviating effect of K56 postbiotic on colonic inflammation, the expression of colonic inflammatory factor mRNA was detected using RT-qPCR. Figure 21 Compared with the control group, DSS induction significantly upregulated the expression of TNF-α, IL-1β, and IL-6 mRNA in the mouse colon (P<0.05). High and low doses of K56 cell-free supernatant (DSS+K121-L, DSS+K121-H) significantly downregulated the expression of TNF-α, IL-1β, and IL-6 mRNA (P<0.05). High and low doses of malic acid (DSS+MA-L, DSS+MA-H) significantly reduced IL-1β and IL-6 mRNA in the mouse colon (P<0.05). Low-dose malic acid (DSS+MA-L) significantly inhibited TNF-α expression (P<0.05), but high-dose malic acid (DSS+MA-H) had no significant inhibitory effect. High and low doses of sebacic acid (DSS+DA-L, DSS+DA-H) treatment also reduced TNF-α, IL-1β, and IL-6 mRNA levels, but the differences were not statistically significant. Low-dose cell-free supernatant (DSS+K121-L) and malic acid (DSS+MA-L) showed more significant inhibitory effects on TNF-α and IL-6 than high-dose treatments. In conclusion, malic acid (DSS+MA) and sebacic acid (DSS+DA) can exert anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokine mRNA, thereby alleviating DSS-induced acute colitis in mice.

[0379] 3.7DSS-induced transcriptome analysis of mouse colon.

[0380] 3.7.1 Screening for differentially expressed genes

[0381] Based on the above results, RNA sequencing was used to investigate the changes in transcriptional levels in colonic tissue after intervention with K56 cell-free supernatant, malic acid, and sebacic acid, and to screen for differentially expressed genes between the intervention group and the DSS group. Gene expression in both groups was analyzed using DESeq2 software. The criteria for screening differentially expressed genes were: |log2FC|>2, padjust<0.05. The volcano plot of differentially expressed genes is shown below. Figure 22 As shown in the diagram. Red dots represent genes that are significantly upregulated, blue dots represent genes that are significantly downregulated, and gray dots represent genes that show no significant difference.

[0382] Compared with the Control group, the DSS group showed 319 upregulated genes and 98 downregulated genes. Compared with the DSS group, after low-dose cell-free supernatant (DSS+K121-L) intervention, 9 genes were significantly upregulated and 248 genes were significantly downregulated; in the low-dose malic acid group (DSS+MA-L), 19 genes were significantly upregulated and 336 genes were significantly downregulated; in the low-dose sebacic acid group (DSS+DA-L), 47 genes were significantly upregulated and 529 genes were significantly downregulated.

[0383] 3.7.2 KEGG pathway enrichment analysis of differentially expressed genes

[0384] After identifying differentially expressed genes between the two groups, KEGG pathway enrichment was performed on these genes, and the top 20 significantly enriched pathways were selected (padjust < 0.05 was considered a significantly enriched KEGG pathway). The study paid particular attention to signaling pathways related to inflammation and immune regulation. Figure 23 As shown in Figure A, compared with the Control group, the differentially regulated genes in the DSS group were enriched in the IL-17 signaling pathway, cytokine-cytokine receptor interaction, TNF signaling pathway, PI3K-Akt signaling pathway, and NF-kappa B signaling pathway; compared with the DSS group, the differentially regulated genes in the low-dose cell-free supernatant group (DSS+K121-L) were enriched in the cytokine-cytokine receptor interaction.

[0385] (Cytokine-cytokine receptor interaction), PI3K-Akt signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, JAK-STAT signaling pathway, and TNF signaling pathway Figure 23In the low-dose malic acid group (DSS+MA-L), downregulated genes were enriched in the IL-17 signaling pathway, cytokine-cytokine receptor interaction, TNF signaling pathway, PI3K-Akt signaling pathway, TGF-beta signaling pathway, and NF-kappa B signaling pathway. Figure 24 In the low-dose sebacic acid group (DSS+DA-L), downregulated genes were enriched in the PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, IL-17 signaling pathway, and TGF-beta signaling pathway. Figure 24 (D). The signaling pathways enriched by differentially expressed genes are closely related to inflammation and immune regulation. We found that genes downregulated by malic acid (DSS+MA-L) and sebacic acid (DSS+DA-L) intervention and differentially expressed genes upregulated by DSS were enriched in the same signaling pathways. Differentially expressed genes were highly enriched in the IL-17 signaling pathway. These results suggest that malic acid (DSS+MA-L) and sebacic acid (DSS+DA-L) may alleviate DSS-induced acute colitis by inhibiting the IL-17 signaling pathway.

[0386] 3.7.3 Differential gene clustering analysis of KEGG pathway enrichment.

[0387] Cluster analysis was performed on differentially expressed genes enriched in the IL-17 signaling pathway, such as... Figure 25 As shown in the figure. The results revealed significant fold changes in S100a8, S100a9, Cxcl5, Cxcl2, Lcn2, and IL-11 within the IL-17 signaling pathway, and these changes were highly correlated with inflammation. qPCR was used to validate these genes, and the results are shown in the figure. Figure 26 The experimental results showed that their expression levels were higher in the DSS group compared to the Control group. Malic acid and sebacic acid could significantly reduce their expression. These results are consistent with the transcriptome sequencing results, validating the reliability of the sequencing findings.

[0388] In summary, malic acid and sebacic acid intervention alleviated the basic phenotype of DSS-induced colitis in mice, including restoration of body weight and normal colonic morphology, decreased DAI index and MPO activity, and increased colonic length. Malic acid and sebacic acid exert their anti-inflammatory effects by inhibiting the mRNA expression of IL-6, IL-1β, and TNF-α.

[0389] Genes downregulated by malic acid and sebacic acid, along with differentially expressed genes upregulated by DSS, were highly enriched in the IL-17 signaling pathway. RT-qPCR results showed that malic acid and sebacic acid significantly reduced the mRNA expression of differentially expressed genes S100a8, S100a9, Cxcl2, Cxcl5, Lcn2, and IL-11 in the IL-17 signaling pathway. These results were consistent with the transcriptome sequencing results, validating the reliability of the sequencing findings.

[0390] Example 4

[0391] This embodiment compares the anti-inflammatory activities of the top 30 most abundant organic acids in cell-free supernatants, including 6-hydroxyhexanoic acid (6-HA), phenyllactic acid (PA), 3-(4-hydroxyphenyl)lactic acid (HA), malic acid (MA), and sebacic acid (DA).

[0392] 1. Verification of the anti-inflammatory activity of organic acids

[0393] The anti-inflammatory activity of the five organic acids screened above was verified. Figure 5 The results showed that malic acid (MA) at concentrations of 10 μg / mL and 50 μg / mL significantly reduced TNF-α expression (P<0.05), and sebacic acid (DA) at concentration of 10 μg / mL significantly reduced TNF-α expression (P<0.05); while 6-hydroxyhexanoic acid (6-HA) at concentration of 10 μg / mL and phenyllactic acid (PA) at concentration of 50 μg / mL significantly increased TNF-α expression (P<0.01). 3-(4-hydroxyphenyl)lactic acid (HA) had no effect on TNF-α expression. Therefore, it is further speculated that among the top 30 organic acids, malic acid (MA) and sebacic acid (DA) exhibit stronger anti-inflammatory activity compared to other organic acids.

[0394] 2. Effects of malic acid and sebacic acid on the viability of RAW264.7 cells

[0395] To determine the cytotoxic effects of malic acid (MA) and sebacic acid (DA) on RAW264.7 cells, a CCK-8 assay was performed. The results showed that the effects of different concentrations of organic acids on RAW264.7 cell viability were investigated. Compared to untreated cells, sebacic acid (DA) treatment increased cell viability with increasing concentration. Malic acid (MA) treatment decreased cell viability with increasing concentration, with the strongest cell viability observed at a concentration of 10 μg / mL. Overall, different doses of malic acid (MA) and sebacic acid (DA) (5–50 μg / mL) were non-toxic to the viability of RAW 264.7 cells.

[0396] Based on cytotoxicity results, the effects of malic acid (MA) and sebacic acid (DA) at concentrations of 10 μg / mL and 50 μg / mL were investigated on TNF-α production in RAW264.7 cells. Figure 6 LPS stimulation significantly increased the level of TNF-α mRNA in cells (P<0.0001). At 10 μg / mL and 50 μg / mL, malic acid (MA) and sebacic acid (DA) significantly reduced the level of TNF-α mRNA in LPS-induced RAW264.7 cells compared with the LPS group (P<0.05), with 10 μg / mL malic acid (MA) and sebacic acid (DA) showing better inhibitory effects on TNF-α.

[0397] This embodiment compares the anti-inflammatory activities of 6-hydroxyhexanoic acid (6-HA), phenyllactic acid (PA), 3-(4-hydroxyphenyl)lactic acid (HA), malic acid (MA), and sebacic acid (DA) among the top 30 most abundant organic acids in cell-free supernatants, demonstrating that malic acid (MA) and sebacic acid (DA) exhibit stronger anti-inflammatory activities.

[0398] Cell viability assays revealed that different concentrations of organic acids had no effect on cell viability. Using a RAW264.7 cell model, it was demonstrated that malic acid and sebacic acid at concentrations of 10 μg / ml and 50 μg / ml significantly inhibited TNF-α expression (P<0.05), with the 10 μg / mL concentration showing the best inhibitory effect.

[0399] In summary, this invention first utilizes an LPS-stimulated RAW264.7 cell model to evaluate the anti-inflammatory activity of inactivated *Lactobacillus paracasei* K56 and its cell-free supernatant, using the pro-inflammatory factor TNF-α as an indicator. Subsequently, non-targeted metabolomics analysis was used to analyze the metabolite composition of the cell-free supernatant of *Lactobacillus paracasei* K56. The top 30 most abundant organic acids were compared using the cell model, and organic acids with good anti-inflammatory activity were screened. A high-fat diet-induced zebrafish model was then constructed, and the anti-inflammatory activity of K56 postbiotic components in vivo was studied through changes in liver weight, liver and intestinal damage, changes in inflammatory factors, and intestinal flora composition. Finally, a DSS-induced mouse colitis model was used to investigate the alleviating effect of K56 postbiotic components on mouse colitis, using changes in body weight, DAI score, colon length, and colon damage as indicators. The inhibitory ability of K56 postbiotics on colonic inflammation was studied using MPO activity and inflammatory factor expression. The potential mechanism of action of K56 postbiotic components in improving mouse colitis was speculated using RNA-seq technology. The main results are as follows:

[0400] (1) The intact cell-free supernatant of K56 significantly downregulated the expression level of the pro-inflammatory factor TNF-α in RAW264.7 macrophages. Based on non-targeted metabolomics analysis, it was shown that the cell-free supernatant of K56 was mainly composed of organic acids, lipids, amino acids and peptides. The anti-inflammatory activities of the top 30 most abundant organic acids in the cell-free supernatant of K56, namely 6-hydroxyhexanoic acid, phenyllactic acid, 3-(4-hydroxyphenyl)lactic acid, malic acid and sebacic acid, were compared using a cell model. The results showed that malic acid and sebacic acid had significant anti-inflammatory activities. Both organic acids at 10 μg / mL and 50 μg / mL could significantly inhibit the expression of TNF-α, which may be the source of the post-biotic anti-inflammatory activity of K56.

[0401] (2) Using a high-fat-induced zebrafish model, it was demonstrated that intervention with K56 postbiotic components, malic acid, and sebacic acid alleviated liver damage induced by a high-fat diet, reduced liver pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increased anti-inflammatory factors IL-10 and TGF-β. Simultaneously, malic acid and sebacic acid intervention regulated the expression of intestinal inflammatory factors TNF-α, IL-1β, and IL-10, and significantly upregulated the expression of tight junction proteins ZO-1 and Occludinb. Furthermore, significant changes were observed in the zebrafish gut microbiota under the intervention of malic acid and sebacic acid, with a significant increase in the relative abundance of beneficial bacteria Bosea, Cetacea, and Aeromonas, and a significant decrease in the relative abundance of Vibrio and Shewanella.

[0402] (3) Using a DSS-induced mouse UC model, we demonstrated that post-K56 malic acid and sebacic acid alleviated the basic phenotype of DSS-induced colitis, including the restoration of mouse body weight and basic colon morphology, decreased DAI index and MPO activity, increased colon length, and decreased expression of pro-inflammatory factor mRNA. Based on transcriptomics, we analyzed the changes in transcriptional levels in colonic tissue after malic acid and sebacic acid intervention. KEGG pathway enrichment analysis revealed that differentially expressed genes after malic acid and sebacic acid intervention were highly enriched in the IL-17 signaling pathway. RT-qPCR results showed that malic acid and sebacic acid significantly reduced the expression of inflammation-related S100a8, S100a9, Cxcl2, Cxcl5, IL-11, and Lcn2.

[0403] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of malic acid or a mixture of malic acid and sebacic acid in the preparation of products that improve liver health.

2. The application according to claim 1, characterized in that, The improvements in liver health include: (1) It has an auxiliary protective effect against liver damage; and / or (2) treatment of liver injury; Preferably, when preparing a product that has an auxiliary protective effect against liver injury, the product is a drug, food, or health product; when preparing a product for treating liver injury, the product is a drug. Preferably, the dosage form of the health product is capsules, tablets, powders, granules, pills, or oral liquid; Preferably, the food is selected from beverages, meat, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, electrolyte drinks, drinking water, chewing gum, capsules, tea, and vitamin complexes; Preferably, the dosage form of the drug is selected from: oral liquid, tablet, powder, granule, capsule, enema or gastric enema.

3. The application according to claim 2, characterized in that, The product has at least one use from the group consisting of: (1) Decreased liver weight or a decreasing trend in liver weight; (2) The liver weight index is reduced or the liver weight index shows a decreasing trend; (3) Improve the neatness and / or density of hepatocyte arrangement; (4) The number of nucleoli in hepatocytes is increased or shows a trend of increasing; (5) Cytoplasmic vacuoles shrink or show a tendency to shrink; (6) The hepatic steatosis has decreased or shown a decreasing trend; (7) Reduces liver inflammation; Preferably, the reduction of liver inflammation includes: decreasing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors; Preferably, the pro-inflammatory factor is selected from at least one of TNF-α, IL-6, and IL-1β; Preferably, the anti-inflammatory factor is selected from at least one of TGF-β and IL-10.

4. The application according to any one of claims 1-3, characterized in that, The liver injury is selected from any one of the following: acute liver failure or acute liver injury, subacute liver failure, chronic-on-acute liver failure, chronic liver failure or chronic liver injury, drug-induced liver injury, endotoxin-induced liver injury, liver injury with endotoxemia, and liver failure. Preferably, the chronic liver injury is induced by a high-fat diet; Preferably, the drug is an oral drug or an injectable drug.

5. Application of malic acid in the preparation of products for the prevention or treatment of acute colitis or ulcerative colitis.

6. The application according to claim 5, characterized in that, The product is a medicine; Preferably, the drug has at least one of the following uses: (1) Reduce intestinal barrier damage; (2) Relieves intestinal inflammation; (3) Reduce the relative abundance of intestinal actinomycetes; (4) Increase the relative abundance of the genera *Bosea* and *Cetobacterium* in the gut; (5) Reduce the relative abundance of Vibrio and Shewanella; (6) Increase the abundance of unclassified_f_Kineosporiaceae and norank_f_JG30-KF-CM45; (7) Reduce the abundance of Paraclostridium; (8) Restore the subject's weight; (9) Reduced the subject's disease activity index score; (10) Improves colonic shortening caused by acute colitis or ulcerative colitis; (11) Reduces colonic myeloperoxidase activity; (12) Restore the colonic crypt structure of the subjects; (13) Alleviating DSS-induced acute colitis or ulcerative colitis by inhibiting the IL-17 signaling pathway; Preferably, the reduction of intestinal barrier damage includes: reducing damage to the intestinal mucosa, submucosa, muscularis propria, and serosa; increasing the number of goblet cells; and reducing inflammatory cell infiltration at least one of the following: Preferably, the relief of intestinal inflammation includes: reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors; Preferably, the inhibition of the IL-17 signaling pathway includes inhibiting the mRNA expression of at least one of the genes S100a8, S100a9, Cxcl2, Cxcl5, Lcn2 and IL-11 in the IL-17 signaling pathway.

7. The application of a probiotic-derived product in the preparation of products that improve liver health, characterized in that, The probiotic postbiotic product is prepared from the cell-free supernatant of inactivated probiotics; the probiotics include Lactobacillus paracasei K56, whose preservation number is CGMCC No. 15139 or DSM27447. Preferably, the probiotic postbiotic product further includes: a carrier and / or auxiliary materials; Preferably, the probiotic postbiotic product is in the form of liquid, solid, or semi-solid; Preferably, the inactivated bacteria are the fermentation products of the probiotics; the sterilization conditions are: 70℃-121℃, treatment for 5min-15min.

8. The application according to claim 7, characterized in that, The improvements in liver health include: (1) It has an auxiliary protective effect against liver damage; and / or (2) treatment of liver injury; Preferably, when preparing a product that has an auxiliary protective effect against liver injury, the product is a drug, food, or health product; when preparing a product for treating liver injury, the product is a drug. Preferably, the dosage form of the health product is capsules, tablets, powders, granules, pills, or oral liquid; Preferably, the food is selected from beverages, meat, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, electrolyte drinks, drinking water, chewing gum, capsules, tea, and vitamin complexes; Preferably, the product has at least one use from the group consisting of: (1) Decreased liver weight or a decreasing trend in liver weight; (2) The liver weight index is reduced or the liver weight index shows a decreasing trend; (3) Improve the neatness and / or density of hepatocyte arrangement; (4) The number of nucleoli in hepatocytes is increased or shows a trend of increasing; (5) Cytoplasmic vacuoles shrink or show a tendency to shrink; (6) The hepatic steatosis has decreased or shown a decreasing trend; (7) Reduces liver inflammation.

9. The application according to claim 8, characterized in that, The reduction of liver inflammation includes: decreasing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors; Preferably, the pro-inflammatory factor is selected from at least one of TNF-α, IL-6, and IL-1β; Preferably, the anti-inflammatory factor is selected from at least one of TGF-β and IL-10.

10. The application according to any one of claims 7-9, characterized in that, The liver injury is selected from any one of the following: acute liver failure or acute liver injury, subacute liver failure, chronic-on-acute liver failure, chronic liver failure or chronic liver injury, drug-induced liver injury, endotoxin-induced liver injury, liver injury with endotoxemia, and liver failure. Preferably, the chronic liver injury is induced by a high-fat diet; Preferably, the drug is an oral drug or an injectable drug.

11. Application of sebacic acid in the preparation of products that improve liver health.

12. The application according to claim 11, characterized in that, The improvements in liver health include: (1) It has an auxiliary protective effect against liver damage; and / or (2) treatment of liver injury; Preferably, when preparing a product that has an auxiliary protective effect against liver injury, the product is a drug, food, or health product; when preparing a product for treating liver injury, the product is a drug. Preferably, the dosage form of the health product is capsules, tablets, powders, granules, pills, or oral liquid; Preferably, the food is selected from beverages, meat, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, electrolyte drinks, drinking water, chewing gum, capsules, tea, and vitamin complexes; Preferably, the product has at least one use from the group consisting of: (1) Decreased liver weight or a decreasing trend in liver weight; (2) The liver weight index is reduced or the liver weight index shows a decreasing trend; (3) Improve the neatness and / or density of hepatocyte arrangement; (4) The number of nucleoli in hepatocytes is increased or shows a trend of increasing; (5) Cytoplasmic vacuoles shrink or show a tendency to shrink; (6) The hepatic steatosis has decreased or shown a decreasing trend; (7) Reduces liver inflammation; Preferably, the reduction of liver inflammation includes: decreasing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors; Preferably, the pro-inflammatory factor is selected from at least one of TNF-α, IL-6, and IL-1β; Preferably, the anti-inflammatory factor is selected from at least one of TGF-β and IL-10.

13. The application according to any one of claims 11-12, characterized in that, The liver injury is selected from any one of the following: acute liver failure or acute liver injury, subacute liver failure, chronic-on-acute liver failure, chronic liver failure or chronic liver injury, drug-induced liver injury, endotoxin-induced liver injury, liver injury with endotoxemia, and liver failure. Preferably, the chronic liver injury is induced by a high-fat diet; Preferably, the drug is an oral drug or an injectable drug.

14. The use of sebacic acid or a mixture of sebacic acid and malic acid in the preparation of products for the prevention or treatment of acute colitis or ulcerative colitis; Preferably, the product is a drug; Preferably, the drug has at least one of the following uses: (1) Reduce intestinal barrier damage; (2) Relieves intestinal inflammation; (3) Reduce the relative abundance of intestinal actinomycetes; (4) Increase the relative abundance of the genera *Bosea* and *Cetobacterium* in the gut; (5) Reduce the relative abundance of Vibrio and Shewanella; (6) Increase the abundance of unclassified_f_Kineosporiaceae and norank_f_JG30-KF-CM45; (7) Reduce the abundance of Paraclostridium; (8) Restore the subject's weight; (9) Reduced the subject's disease activity index score; (10) Improves colonic shortening caused by acute colitis or ulcerative colitis; (11) Reduces colonic myeloperoxidase activity; (12) Restore the colonic crypt structure of the subjects; (13) Alleviating DSS-induced acute colitis or ulcerative colitis by inhibiting the IL-17 signaling pathway; Preferably, the reduction of intestinal barrier damage includes: reducing damage to the intestinal mucosa, submucosa, muscularis propria, and serosa; increasing the number of goblet cells; and reducing inflammatory cell infiltration at least one of the following: Preferably, the relief of intestinal inflammation includes: reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors; Preferably, the inhibition of the IL-17 signaling pathway includes inhibiting the mRNA expression of at least one of the genes S100a8, S100a9, Cxcl2, Cxcl5, Lcn2 and IL-11 in the IL-17 signaling pathway.

Images