A kind of lactobacillus amylovora and its application in improving cholestatic liver disease

Abstract

The present invention discloses a lactobacillus amylovorus, whose name is QC1H, which is isolated from nature and has a deposit number of CCTCC NO: M2023427. The fermentation broth of the lactobacillus amylovorus or the extracellular vesicles prepared therefrom can be used to improve cholestatic liver disease, specifically: improve liver function damage, inflammatory lesions and liver fibrosis caused by cholestatic liver disease, can significantly improve DDC-induced cholestatic liver damage and liver fibrosis in mice, reduce the levels of liver enzymes such as ALT, AST, ALP in serum, reduce the level of liver bile acid, and alleviate the degree of liver fibrosis. The preparation prepared by the lactobacillus amylovorus is a high-concentration, efficient and safe biological preparation, which can be used for the treatment and protection of liver disease.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus amylovorus and application thereof in improving cholestatic liver disease. Background Art

[0002] Cholestatic liver disease is a hepatobiliary disease caused by various reasons, which is caused by the disorder of bile production, secretion and excretion. It cannot be actively discharged into the intestinal cavity through the bile ducts, accumulates in the liver, and refluxes into the blood, causing a series of organic damage, metabolic disorders and functional disorders. The causes of cholestatic liver disease are relatively complex, and the main causes include genetics, immunity, degeneration, infection, stones and tumors. If not treated promptly and effectively, it may even progress to liver fibrosis, cirrhosis, liver failure and liver tumors. Epidemiological evidence shows that the incidence of cholestatic hepatitis in my country accounts for more than 10% of patients with chronic liver disease. At present, the main clinical therapeutic drugs are ursodeoxycholic acid (UDCA), S-adenosylmethionine, cholestyramide, obeticholic acid and fibrates, among which UDCA is the first-line drug recommended in clinical practice, and its main purpose is to improve clinical symptoms and liver damage caused by cholestasis. However, the clinical efficacy is still unsatisfactory. About 30% of patients are ineffective against UDCA. In addition, UDCA has a slow onset of effect and is relatively expensive. Therefore, more effective drugs are urgently needed clinically.

[0003] Bacteria can actively release nano-sized membrane vesicles into the extracellular environment. These spherical structures of 20-400 nm are called extracellular vesicles (EVs). EVs can carry a variety of biological genetic information, such as nucleic acids, enzymes, effector proteins and virulence factors. Their structure enables EVs to play an important role in bacterial competition, survival, material exchange, host immune escape, regulation, infection and invasion. Based on the many health-promoting effects of probiotics, the beneficial effects of probiotic-derived extracellular vesicles EVs, such as anti-inflammatory and anti-tumor, have been clearly confirmed in several studies. Recently, the use of EVs as therapeutic agents has become a promising research area. In addition, the clearance and processing of nanomaterials in the body are very different from those of small molecules. In particular, systemically administered small molecules usually undergo rapid renal clearance, while nanoparticles accumulate preferentially in the liver. Biological nanoparticles show similar advantages to synthetic nanoparticles, including multifunctionality and reduced renal clearance. In addition, due to optimization through evolutionary selection, biologically derived materials generally show higher biocompatibility and physiological activity. Therefore, bionanoparticles may have superior properties as therapeutic agents compared to artificial particles. In addition, bionanoparticles can be further modified to enhance certain properties, such as content or membrane characteristics.

[0004] With the discovery of starch-eating lactic acid bacteria (Lactobacillus amylovorus, La) in dairy farm waste and corn fermentation broth, scholars around the world have never given up on its research and use. A lot of research has been done in the fields of industry, food industry, agriculture and medicine at home and abroad on improving the utilization efficiency of starch in food and extending the shelf life of food. Its biodegradation and synthesis functions, as well as its antibacterial and antiviral abilities, all come from an enzyme or an active ingredient secreted by Lactobacillus amylovorus. Foods fermented by Lactobacillus amylovorus can lower cholesterol in the body, enhance the activity of digestive enzymes, stimulate beneficial microorganisms to hydrolyze sugars, and prevent and treat diarrhea. However, the current research on starch-eating lactic acid bacteria, its fermentation broth and extracellular vesicles (EVs) is very limited, and there are even fewer reports on its use in the treatment of liver disease. Summary of the invention

[0005] In order to overcome at least one problem existing in the prior art, the present invention isolates a strain of Lactobacillus amylovora from nature, and prepares a Lactobacillus amylovora fermentation broth or prepares Lactobacillus amylovora extracellular vesicles through an appropriate extraction method, which can be used in the treatment and prevention of liver disease, and the production process is simple and efficient, and does not pollute the environment.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide a starch-eating lactobacillus, whose name is QC1H, which is classified as Lactobacillus amylovorus, whose preservation number is CCTCC NO: M2023427, the preservation date is March 28, 2023, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0008] Furthermore, the amylovorus lactobacillus is screened and isolated from pig manure, and the homology of its 16sRNA sequence with the base sequence of the existing amylovorus lactobacillus (Lactobacillus amylovorus) in NCBI reaches 100%.

[0009] The second aspect of the present invention is to provide a microbial agent prepared by any of the above-mentioned Lactobacillus amylovora, which contains the Lactobacillus amylovora or is prepared by fermenting the Lactobacillus amylovora.

[0010] Furthermore, the microbial agent comprises Lactobacillus amylovora extracellular vesicles (LaEVs) extracted from the supernatant of the fermentation broth of the Lactobacillus amylovora.

[0011] Furthermore, the particle size of the extracellular vesicles of Lactobacillus amylovora is between 131 and 369 nm.

[0012] Furthermore, the extracellular vesicles of Lactobacillus amylovora exhibit a typical bubble structure containing a phospholipid bilayer and are spherical particles, among which the vesicles with a particle size of 131 nm are the most numerous, accounting for about 56.7%.

[0013] Furthermore, the protein concentration of the extracellular vesicles of Lactobacillus amylovora is 3.94 μg / μL.

[0014] Furthermore, the step of preparing the fermentation liquid of the Lactobacillus amylovora comprises: inoculating the Lactobacillus amylovora for culturing to obtain the fermentation liquid.

[0015] Furthermore, the method for preparing the extracellular vesicles of Lactobacillus amylovora comprises the steps of:

[0016] 1) inoculating the Lactobacillus amylovora for culturing to obtain a fermentation solution;

[0017] 2) centrifuging the fermentation broth obtained in step 1) and collecting the supernatant;

[0018] 3) filtering, ultrafiltering, and centrifuging the supernatant obtained in step 2) to collect the precipitate;

[0019] 4) resuspending and filtering the precipitate obtained in step 3) to obtain the extracellular vesicles of Lactobacillus amylovora.

[0020] Specifically, the method for preparing extracellular vesicles of Lactobacillus amylovora comprises the steps of:

[0021] A) activating the frozen Lactobacillus amylovora strain, inoculating it into MRS broth at an inoculum amount of 2 wt%, and culturing it at 37° C. for 18 h to obtain a fermentation broth;

[0022] B) Centrifuge the fermented bacterial broth at 5000 g and 4°C for 15 minutes, remove the precipitate, and collect the supernatant;

[0023] C) The supernatant was filtered through a 0.22 μm filter, and after filtration, the supernatant was centrifuged at 5000 g and 4° C. for 30 minutes using an ultrafiltration tube. The obtained ultrafiltrate was ultracentrifuged at 120,000 g and 4° C. for 2 hours, and the precipitate was collected;

[0024] D) The precipitate was resuspended in PBS and filtered using a 0.22 μm filter to obtain the extracellular vesicles of Lactobacillus amylovora.

[0025] The third aspect of the present invention is to provide a use of any of the above-mentioned Lactobacillus amylovora or any of the above-mentioned microbial agents in the preparation of a preparation for preventing or treating cholestatic liver disease.

[0026] Furthermore, the preparation uses Lactobacillus amylovora fermentation broth or Lactobacillus amylovora extracellular vesicles as main active ingredients.

[0027] Furthermore, the preparation also includes pharmaceutically acceptable auxiliary ingredients.

[0028] Furthermore, the effective dosage of the extracellular vesicles of Lactobacillus amylovora in the preparation is 1.2 μg / Kg.

[0029] Furthermore, the cholestatic liver disease is cholestatic liver disease induced by DDC.

[0030] Furthermore, the preparation for preventing or treating cholestatic liver disease includes a preparation for improving liver function damage, inflammatory lesions and / or liver fibrosis caused by cholestatic liver disease.

[0031] Further, the improvement of liver injury inflammatory lesions and / or liver fibrosis caused by cholestatic liver disease includes at least one of the following functions: reducing liver inflammatory cell infiltration and focal necrosis area, reducing serum liver enzyme levels, reducing liver bile acid levels, reducing the expression of liver inflammatory cytokines, reducing liver fibrosis markers and / or related gene expression. The above-mentioned reduction of serum liver enzyme levels includes reducing serum ALT, AST, ALP and other liver enzyme levels, reducing the expression of liver inflammatory cytokines includes reducing the mRNA expression of liver inflammatory cytokines IL-6, IL-1β, TNF-α, and reducing the expression of liver fibrosis markers and / or related genes includes the mRNA expression of liver fibrosis α-SMA and Vimentin.

[0032] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0033] According to the study of synthetic nanoparticles, it has been proved that intravenously injected EVs are mainly accumulated in the liver, making it particularly suitable for the treatment of liver diseases, and studies have shown that liver damage will further increase the absorption of EVs in this organ, thereby improving the organic matter delivery rate. The present invention isolates a strain of Lactobacillus amylovora QC1H, and the fermentation broth prepared therefrom or the extracellular vesicles extracted from the fermentation broth have good therapeutic effects on cholestatic liver injury, inflammatory lesions, and liver fibrosis, and the total protein content of the extracellular vesicles is 3.94μg / μL. The production process of the above-mentioned extracellular vesicles is simple and efficient, has no pollution to the environment, and has the advantages of high concentration, high efficiency, high cost performance, safety, and low cost, and has good application prospects in the treatment and protection of liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0035] Figure 1Schematic diagram of Lactobacillus amylovora extracellular vesicles (LaEVs) under high-resolution transmission electron microscopy in one embodiment of the present invention;

[0036] Figure 2 This is a particle size diagram of Lactobacillus amylovora extracellular vesicles (LaEVs) measured by a particle size analyzer in one embodiment of the present invention;

[0037] Figure 3 The results of the effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on ALT, AST, ALP and liver total bile acid levels in mice with DDC-induced cholestatic liver injury and fibrosis are shown in one embodiment of the present invention; specifically: serum ALT activity, serum AST activity, serum ALP activity, liver TBA level;

[0038] Figure 4 This is a result diagram of the effect of extracellular vesicles of Lactobacillus amylovora (LaEVs) on the pathological changes of liver tissues in mice with DDC-induced cholestatic liver injury and fibrosis in one embodiment of the present invention; wherein, H&E and Sirius red staining (100X);

[0039] Figure 5 This is a result diagram showing the effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on the gene expression levels of IL-1β, IL-6, and TNF-α in the liver of mice with DDC-induced cholestatic liver injury and fibrosis in one embodiment of the present invention;

[0040] Figure 6 This is a graph showing the effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on the gene expression levels of α-SMA and Vimentin in the liver of mice with DDC-induced cholestatic liver injury and fibrosis in one embodiment of the present invention.

[0041] All test results in the above figures are expressed as means±SEM (n=5), and different letters between data groups indicate significant differences between them (P<0.05). DETAILED DESCRIPTION

[0042] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0043] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0044] In the following examples, the composition of MRS broth: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 5.0 g, dipotassium hydrogen phosphate 2.0 g, glucose 20.0 g, Tween 80 1 mL, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2, manganese sulfate 0.04, distilled water 1000 mL, 118 ° C high pressure sterilization for 15 minutes before use.

[0045] Example 1 - Isolation, screening and identification of starch-eating lactic acid bacteria (Lactobacillus amylovorus)

[0046] In this embodiment, the following method is used to obtain starch-eating lactic acid bacteria:

[0047] (1) Isolation and screening of starch-eating lactic acid bacteria

[0048] Weigh 0.1 g of feces from piglets, sows, etc. and add them to 0.9 mL of sterile saline. Mix by pipetting and dilute with saline by 10 times. -5 -10 -7times, take 100μL of each dilution multiple and spread it on MRS agar plate, invert it in a constant temperature incubator at 37℃ and culture it for 48h; then use a sterile inoculation loop to pick colonies with lactic acid bacteria morphology, inoculate them in MRS broth, and culture them at 37℃ for 48h; then take the liquid bacterial liquid to streak the MRS agar plate, invert it in a constant temperature incubator at 37℃ and culture it for 48h; pick a single colony and inoculate it in MRS broth again, repeat the above steps 3 times to obtain the purified primary screening bacteria. After further screening by Gram staining, it was identified as amylovorax lactobacillus.

[0049] Main biological characteristics: Under the microscope, it is blue-purple, long rod-shaped, mostly single, not chain-shaped; after culturing at 37°C on an MRS plate for 24 hours, it appears as yellow colonies with a smooth surface, slightly raised, powdery and opaque, uniform texture, neat edges, and a diameter of about 2-3 mm.

[0050] (2) Identification of starch-eating lactic acid bacteria

[0051] The selected strains were activated by MRS agar plates and MRS broth culture, and then sent to Sangon Biotech Co., Ltd. for strain sequencing. The sequencing results were compared with the GenBank database of the National Center for Biotechnology Information (NCBI) of the United States, and strains with more than 99% homology were found for comparison to determine the type of lactic acid bacteria to be measured. The sequence (5'-3') measured by the strain is shown below, and the homology with the base sequence of Lactobacillus amylovorus currently available on NCBI reached 100%.

[0052]

[0053]

[0054] The amylovorus lactobacillus identified above was named QC1H, has been preserved, and its classification name is Lactobacillus amylovorus. Its preservation number is CCTCC NO: M2023427. The preservation date is March 28, 2023. The preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0055] Example 2 - Preparation and characterization of extracellular vesicles (LaEVs) of lactic acid bacteria

[0056] This example uses a preferred method to prepare extracellular vesicles of lactic acid bacteria and characterize and analyze them, including the following steps:

[0057] (1) Preparation of extracellular vesicles of lactic acid bacteria

[0058] In a UV-sterilized clean bench, resuscitate the frozen Lactobacillus amylovora strain, pick a single colony in MRS broth, place it in a constant temperature incubator at 37°C for 6 hours, inoculate it in 2L of MRS broth at a 2% inoculum, place it in a constant temperature incubator at 37°C, and culture it for 18 hours. The obtained bacterial solution was divided into 50mL EP tubes and centrifuged at 5000g and 4°C for 15min. The precipitate is the bacterial body, discard the precipitate, filter the supernatant with a 0.22μm filter, and then use a high-grade centrifuge tube (ultrafiltration tube is The supernatant was discarded and the precipitate was resuspended in a small amount of sterile PBS buffer. The suspensions in multiple tubes were mixed and filtered and sterilized again using a 0.22 μm filter to obtain sterile, high-concentration target extracellular vesicles.

[0059] (2) Characterization and analysis of extracellular vesicles of lactic acid bacteria

[0060] After diluting the extracted extracellular vesicles, the total protein concentration was measured using the BCA protein quantification kit of Novozyme. The protein concentration standard curve can be obtained by the absorbance of the gradient concentration protein standard at 562nm. After prior fitting, the standard equation y = 2138.8x-430.11, R2 = 0.99 can be obtained. When the protein content is 0-1000μg, the linear relationship of the standard curve is good, and the measured value is accurate and reliable. Substituting the measured OD value into the standard equation and the following formula, the protein concentration can be obtained.

[0061] The obtained extracellular vesicles were observed for their morphology using a high-resolution transmission electron microscope. 15 μL of EVs solution was dropped onto a copper mesh, allowed to stand for 2 minutes for adsorption, and excess solution on the copper mesh was removed with filter paper. 15 μL of 2% phosphotungstic acid stain was used for negative staining for 2-3 minutes, excess stain was removed with filter paper, dried for 20-30 minutes, and then examined by transmission electron microscopy.

[0062] Use a particle size analyzer to detect its molecular size. Load the sample with a sample needle and place the sample in the sample slot of the Zeta View S / N 22-779 nanoparticle tracking analyzer. Use Zeta View software to analyze the acquired data to obtain the sample particle size distribution diagram. The instrument detection parameters are set as follows: Laser wavelength is 520nm, Filter Wavelength is Scatter, and other parameters use default parameters. Among them, the EVs particle concentration calculation formula is:

[0063] EVs particle concentration = detected concentration × sample dilution factor

[0064] After testing, the protein concentration of the isolated Lactobacillus amylovora EVs sample was 3.94 μg / μl when the fermentation volume was 2 L and the ultracentrifugation speed was 120,000 g. The morphology of the obtained extracellular vesicles was observed using a high-resolution transmission electron microscope ( Figure 1 ), and the molecular size was measured using a particle size analyzer ( Figure 2 ). Characterization and analysis showed that the prepared solution contained extracellular vesicles of Lactobacillus amylovora. Figure 1 These vesicles present a typical vesicle structure containing a phospholipid bilayer, are spherical particles, and have a diameter of about 131nm, which is consistent with the results of the nanoparticle size analyzer (Zeta View S / N 22-779). Figure 2 As shown in the figure, it can be seen that the LaEVs particles are evenly distributed and the peaks are relatively neat and uniform, which indicates that the concentration of LaEVs prepared in this experiment is high and contains fewer impurities, which also ensures the reliability of subsequent experiments. Finally, the particle size distribution and concentration of LaEVs were obtained by NTA software analysis. The results showed that the particle size of LaEVs was between 131-369nm, and the vesicles with a particle size of 131nm were the largest, accounting for about 56.7%, and the particle size concentration was 4.8×10 9 This is consistent with the results observed by electron microscopy and the particle size distribution range of bacterial EVs reported in other literature.

[0065] Example 3 - Effect of Lactobacillus amylovora extracellular vesicles (LaEVs) on the alleviation of cholestatic liver injury

[0066] This example verifies the effect of the extracellular vesicles of lactic acid bacteria starch prepared in Example 2 in alleviating cholestatic liver injury, which includes the steps of:

[0067] (1) Materials and methods

[0068] 1.1- Chemical reagents;

[0069] Main reagents: alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bile acid detection kit (Nanjing Jiancheng Bioengineering Institute), RNAtrizol (Nanjing Novozyme Biotechnology Co., Ltd.), RT-PCR kit (Eric Biotech).

[0070] 1.2-Animal testing methods;

[0071] Eighteen 7-week-old specific-pathogen-free (SPF) male C57BL / 6 mice purchased from Jicui Yaokang were used in this experiment (weight 22.27±0.40g). The mice were randomly divided into 3 groups, 6 in each group: control group; DDC group; DDC+LaEVs group. The mice were kept in an SPF environment (12h light and 12h dark alternately, free access to food and water, constant temperature and humidity suitable for automatic control) in 1 cage per 6 mice. The experiment began after 1 week of adaptive feeding. The control group was fed with normal mouse diet for 14 consecutive days. On the first day, DDC purified diet and normal mouse diet were given to the DDC group and DDC+LaEVs group at a ratio of 1:2; on the second day, DDC purified diet and normal mouse diet were given to the DDC group and DDC+LaEVs group at a ratio of 2:1; on the 3rd to 14th day, the DDC group and DDC+LaEVs group were only given DDC purified diet. The mice in the DDC+LaEVs group were gavaged with 1.2 μg / kg extracellular vesicles of Lactobacillus amylovora for 14 consecutive days at the same time every day. The experiment lasted for 2 weeks, and the mice had free access to food and water throughout the experiment.

[0072] 1.3-Sample collection

[0073] After the experiment, the mice were fasted for 12 h before euthanasia, and their blood was collected by enucleation of the eyeballs, centrifuged at 3500 rpm × 25 min, and the serum was separated and stored at -80 ° C. After the death of the mice, the abdominal cavity was opened and liver tissue samples were collected. One part of the tissue was fixed with 4% paraformaldehyde solution for subsequent tissue morphology analysis, and the other part was quickly frozen with liquid nitrogen and stored at -80 ° C for subsequent molecular biology analysis.

[0074] 1.4- Serum biochemical indexes and liver total bile acid determination

[0075] Alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bile acid in the liver were detected using kits.

[0076] 1.5-H&E and Sirius Red Staining of Liver Tissue

[0077] The liver tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. A piece of tissue (1.5 cm × 1 cm × 0.5 cm) was cut and placed in an embedding box. After dehydration, wax immersion, embedding, freezing and other operations, the liver tissue was cut into 3 μm thick slices using a tissue slicer. The prepared tissue slices were stained with HE and Sirius red, and then dehydrated, transparentized, and sealed for observation under a microscope. The slices were observed and photographed using an upright optical microscope.

[0078] 1.6-Real-Time PCR detection of relative expression levels of IL-1β, IL-6, TNF-α, α-SMA, and Vimentin in liver tissue

[0079] According to the sequences of GAPDH, IL-1β, IL-6, TNF-α, α-SMA, and Vimentin of C67BL / 6 mice in GenBank, the primers were designed using Premier 5.0 in accordance with the principles of quantitative PCR primer design. Each primer was synthesized by Sangon Biotech Co., Ltd. The specific sequences are shown in Table 1. The total RNA was extracted using the TRIZOL kit according to the instructions, and the concentration of the extracted RNA was finally detected by the Nanodrop 2000 (Thermoscientific, USA) analyzer. The real-time PCR reaction system and PCR amplification conditions were referred to the instructions of the PrimeScript RT-PCR Kit of Myrica. Real-time PCR amplification was performed on the 7300 Real-time PCR System produced by Applied Biosystems (ABI).

[0080] Table 1 Primer sequences

[0081]

[0082] (2) Results

[0083] 2.1-Effects of extracellular vesicles of Lactobacillus amylovora on serum ALP, AST, ALT, and liver TBA;

[0084] Changes in serum liver enzyme indicators such as Figure 3 As shown (C is serum ALT activity, D is serum AST activity, E is serum ALP activity, F is liver TBA level): compared with the control group, serum ALP, AST, and ALT in the DDC group increased significantly (P < 0.05), and compared with the DDC group, ALP, AST, and ALT in the DDC+LaEVs group decreased significantly (P < 0.05). Under the influence of LaEVs, the level of total bile acid in the liver decreased significantly (P < 0.05).

[0085] 2.2-Effects of extracellular vesicles of Lactobacillus amylovora on pathological changes in liver tissues;

[0086] H&E staining showed that ( Figure 4, H&E, Sirius red staining (100×): In the control group, liver sections showed hepatic lobules composed of normal central veins and cell structures. In the DDC group, obvious punctate hemorrhage necrosis was observed in the liver, with a large number of inflammatory granulocytes and lymphocytes infiltrating the necrosis. Most of the hepatic lobules had disordered structures, the morphology of hepatocyte cords disappeared, the cells in the hepatic lobules were swollen, the cell cytoplasm was lightly stained and loose, and showed balloon-like changes. LaEVs can improve DDC-induced liver lesions.

[0087] Sirius red staining was used to detect collagen deposition in the liver ( Figure 4 ), in the DDC group, there was a large amount of collagen deposition in the portal vein and portal duct areas. LaEVs could significantly reduce the collagen deposition induced by DDC.

[0088] 2.3-Effect of extracellular vesicles of Lactobacillus amylovora on the gene expression levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in the liver;

[0089] like Figure 5 As shown, compared with the control group, the gene expression levels of IL-6, IL-1β, and TNF-α in the DDC group were significantly increased (P<0.05). Compared with the DDC group, the gene expression levels of IL-6, IL-1β, and TNF-α in the LaEVs group were significantly decreased (P<0.05).

[0090] 2.4 Effects of extracellular vesicles of Lactobacillus amylovora on the expression levels of α-SMA and Vimentin genes in the liver

[0091] like Figure 6 As shown, compared with the control group, the gene expression levels of α-SMA and Vimentin in the DDC group were significantly increased (P<0.05). Compared with the DDC group, the gene expression levels of α-SMA and Vimentin in the LaEVs group were significantly decreased (P<0.05).

[0092] The fermentation broth of Lactobacillus amylovora described in the present invention also contains Lactobacillus amylovora extracellular vesicles, which can also improve symptoms such as cholestatic liver injury, but its improvement effect is inferior to that of high-purity and high-concentration Lactobacillus amylovora extracellular vesicles.

[0093] It can be seen from the above embodiments that the amylovorax isolated by the present invention can be used to prepare extracellular vesicles using a safe, low-cost and efficient production method. The extracellular vesicles can improve cholestatic liver damage and liver fibrosis, reduce liver inflammatory cell infiltration and focal necrosis area, and reduce serum ALT, AST, ALP and other liver enzyme levels; reduce liver oxidative stress levels, reduce liver bile acid levels, and improve liver fibrosis.

[0094] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method of producing a lactobacillus starch Lactobacillus amylovorus ) prepared by the microbial agent, characterized in that The microbial agent comprises extracellular vesicles of Lactobacillus amylovora extracted from the supernatant of the fermentation broth of the Lactobacillus amylovora; wherein the name of the Lactobacillus amylovora is QC1H, and its preservation number is CCTCC NO: M 2023427.

2. The microbial agent according to claim 1, characterized in that The particle size of the extracellular vesicles of Lactobacillus amylovora is between 131 and 369 nm.

3. Use of the microbial agent according to any one of claims 1 to 2 in the preparation of a preparation for treating cholestatic liver disease.

4. The use according to claim 3, characterized in that: The preparation uses extracellular vesicles of Lactobacillus amylovora as a main active ingredient.

5. The use according to claim 4, characterized in that The effective dosage of the extracellular vesicles of Lactobacillus amylovora in the preparation is 1.2 μg / Kg.

6. The use according to claim 5, characterized in that: The preparation for treating cholestatic liver disease includes a preparation for improving liver function damage, inflammatory lesions and / or liver fibrosis caused by cholestatic liver disease.

7. The use according to claim 6, characterized in that: The improvement of liver function damage, inflammatory lesions and / or liver fibrosis caused by cholestatic liver disease includes: reducing liver inflammatory cell infiltration and focal necrosis area, reducing serum liver enzyme levels, reducing liver bile acid levels, reducing the expression of liver inflammatory cytokines, and reducing the expression of liver fibrosis markers and / or related genes.

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