Application of pediococcus acidilactici ZJUIDS13 in improvement of liver diseases

Abstract

The invention relates to the technical field of microorganisms, in particular to application of pediococcus acidilactici ZJUIDS13 in improvement of liver diseases, including improvement of alcoholic liver diseases and improvement of fatty liver diseases related to metabolic dysfunction. The pediococcus acidilactici ZJUIDS13 has relatively strong capability of protecting liver injury induced by alcohol, and can improve the states of liver injury and lipid metabolism disorder caused by high fat diet.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically to application of Pediococcus acidilactici ZJUIDS13 in improving liver diseases, including improving alcoholic liver disease and improving fatty liver disease associated with metabolic dysfunction. Background Art

[0002] As a probiotic, Pediococcus acidilactici has received extensive attention in scientific research in recent years. This microorganism not only plays an important role in the production of traditional fermented foods such as cheese, kimchi, and sausage, but also has become a research hotspot in the fields of biomedicine and nutrition due to its unique physiological characteristics and health benefits. In terms of maintaining intestinal health, Pediococcus acidilactici inhibits the growth of harmful bacteria and maintains the balance of intestinal microecology by producing metabolites such as organic acids, while enhancing intestinal barrier function, promoting the absorption of nutrients, and improving host immunity. In addition, Pediococcus acidilactici also shows great potential in improving metabolic diseases, participating in immune regulation, and exerting anti-inflammatory effects.

[0003] Alcoholic liver disease (ALD) is a liver disease caused by long-term heavy drinking. It is characterized by fatty degeneration in liver cells and liver metabolic disorders. The initial manifestation of ALD is alcoholic fatty liver (AFL), which then develops into alcoholic steatohepatitis (ASH), liver fibrosis, cirrhosis and hepatocellular carcinoma. Continuous chronic liver damage and inflammation will eventually lead to progressive fibrosis and cirrhosis, which may eventually promote the development of hepatocellular carcinoma. The pathogenesis of ALD includes hepatic steatosis, oxidative stress, acetaldehyde-mediated toxicity, and cytokine- and chemokine-induced inflammation.

[0004] The invention of CN118562674A "A strain of Pediococcus acidilactici MY8 and its application in the preparation of sleep-aiding and whitening food and medicine" informs that the Pediococcus acidilactici MY8 strain can produce and secrete 3-hydroxybutyric acid (3-HB), which prevents and treats chronic syndromes (hypertension, alcoholic fatty liver, enteritis, and intestinal cancer).

[0005] The invention of CN118360201A, "A strain of Pediococcus acidilactici CCFM1364 and its prepared postbiotics for alcohol detoxification and liver protection and application in transforming pueraria lobata flavonoids", increases the activity of alcohol metabolism enzymes (ADH and ALDH), enhances alcohol metabolism; inhibits the upregulation of Cyp2e1 and CYP1A2 in the liver induced by alcohol, exerts alcohol detoxification and liver protection effects,

[0006] The invention of CN118360200A, "Application of a strain of lactic acid bacteria CCFM1365 and its prepared postbiotics for alcohol sobering and liver protection, and conversion of pueraria flavonoids", states that lactic acid bacteria CCFM1365 and its prepared synbiotic preparations, live bacteria and postbiotics have at least one of the following effects: (1) improving liver fatty degeneration and lipid accumulation caused by long-term alcohol exposure; (2) enhancing alcohol metabolism; having anti-oxidative stress and anti-inflammatory response; (3) reducing the expression level of mRNA of genes related to inflammatory mediators, oxidative stress and alcohol metabolism in liver tissue; (4) reducing intestinal permeability and reducing alcohol absorption.

[0007] The invention of CN116891820A "A strain of Pediococcus acidilactici RH2712 capable of alleviating alcoholic liver damage and its application" informs that the strain of Pediococcus acidilactici RH2712 can alleviate alcoholic liver damage.

[0008] Metabolic Dysfunction-Associated Fatty Liver Disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a global public health problem that affects more than a quarter of the adult population. MASLD is characterized by the coexistence of hepatic steatosis and metabolic disorders, has a similar pathogenesis to cardiovascular disease (CVD), and is closely associated with recognized cardiovascular risk factors such as obesity, type 2 diabetes, and atherogenic dyslipidemia. However, there is no safe and effective treatment for MASLD to date.

[0009] The invention of 202211133365.2, "Pediococcus acidilactici ZJUIDS13 with the effect of relieving ulcerative colitis and its application", discloses a Pediococcus acidilactici ZJUIDS13 with the effect of relieving ulcerative colitis, and its preservation number is: CGMCC NO.25529. The patent also discloses the application of ZJUIDS13 in the preparation of products with the effect of relieving ulcerative colitis. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide application of Pediococcus acidilactici ZJUIDS13 in preparing a preparation for improving liver disease.

[0011] In order to solve the above technical problems, the present invention provides an application of Pediococcus acidilactici ZJUIDS13 (CGMCC NO.25529) in preparing a preparation for improving liver disease.

[0012] As an improvement of the application of the present invention: the preparation comprises a drug.

[0013] As a further improvement of the application of the present invention: improving liver disease includes at least any one of the following: improving alcoholic liver disease, improving fatty liver disease associated with metabolic dysfunction.

[0014] As a further improvement of the application of the present invention: comprising Pediococcus acidilactici ZJUIDS13 and inactivated Pediococcus acidilactici ZJUIDS13.

[0015] As a further improvement of the application of the present invention: comprising a suspension prepared by using the bacterial powder of Pediococcus acidilactici ZJUIDS13 and an inactivated ZJUIDS13 bacterial liquid.

[0016] The Pediococcus acidilactici ZJUIDS13 provided by the present invention has the following abilities: strong ability to protect alcohol-induced liver damage, ability to reduce serum and liver triglycerides, ability to improve the oxidative stress state in the body, ability to inhibit inflammation, and ability to inhibit fatty acid synthesis.

[0017] The Pediococcus acidilactici ZJUIDS13 provided by the present invention can improve the liver damage and lipid metabolism disorder caused by alcohol induction, mainly by reducing the levels of ALT and AST in plasma, improving liver lipid metabolism, and exerting an anti-inflammatory effect.

[0018] The present invention provides a derivative of Pediococcus acidilactici ZJUIDS13, which still has a beneficial effect on the health of the host after being inactivated.

[0019] The present invention embodies the following capabilities: strong ability to protect liver damage, ability to improve antioxidant capacity in the body, ability to reduce liver triglycerides, and the effect of regulating blood lipid metabolism, strong ability to protect liver damage, ability to improve antioxidant capacity in the body, ability to reduce liver triglycerides, and the effect of regulating blood lipid metabolism.

[0020] The Pediococcus acidilactici ZJUIDS13 provided by the present invention can improve the liver damage and lipid metabolism disorder caused by a high-fat diet, can effectively reduce the levels of ALT and AST in plasma, improve liver lipid metabolism, regulate blood lipids, and exert the body's antioxidant effect.

[0021] The required dose for humans can be calculated based on the corresponding dose for mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on the liver weight and liver-to-body ratio of mice;

[0024] Figure 2 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on mouse plasma ethanol;

[0025] Figure 3 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on ALT and AST in mouse plasma;

[0026] Figure 4 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on triglycerides in mouse plasma and liver;

[0027] Figure 5 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on fatty acid synthesis genes in mouse liver;

[0028] Figure 6 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on oxidative stress indexes in mouse liver;

[0029] Figure 7 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on the expression of inflammatory factors in mouse liver;

[0030] Figure 8 The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on the liver weight and liver-to-body ratio of mice;

[0031] Fig. 9 The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on mouse plasma ethanol;

[0032] Fig.10 The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on ALT and AST in mouse plasma;

[0033] Fig.11The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on triglycerides in mouse plasma and liver;

[0034] Fig.12 The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on the oxidative stress indexes of mouse liver;

[0035] Fig.13 The present invention shows the effect of the inactivated Pediococcus acidilactici ZJUIDS13 on the expression of inflammatory factors in mouse liver.

[0036] Fig.14 The present invention shows the effects of Pediococcus acidilactici ZJUIDS13 on the body weight, liver weight and liver-to-body ratio of high-fat mice.

[0037] Fig.15 The effect of Pediococcus acidilactici ZJUIDS13 of the present invention on triglyceride (TG) in the liver of high-fat mice;

[0038] Fig.16 The present invention is the effect of Pediococcus acidilactici ZJUIDS13 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in the plasma of high-fat mice;

[0039] Fig.17 The present invention shows the effect of Pediococcus acidilactici ZJUIDS13 on the glucose tolerance level of high-fat mice.

[0040] Fig.18 The present invention shows the effect of Pediococcus acidilactici ZJUIDS13 on the insulin resistance level of high-fat mice.

[0041] Fig.19 The present invention is the effect of Pediococcus acidilactici ZJUIDS13 on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in high-fat mice;

[0042] Fig. 20 The present invention is the effect of Pediococcus acidilactici ZJUIDS13 on malondialdehyde (MDA) and total superoxide dismutase (T-SOD) in the liver of high-fat mice;

[0043] Fig.21 The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on the body weight, liver weight and liver-to-body ratio of high-fat mice;

[0044] Fig. 22 The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on triglycerides (TG) in the liver of high-fat mice;

[0045] Fig.23 The present invention is the effect of the inactivated Pediococcus acidilactici ZJUIDS13 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in the plasma of high-fat mice;

[0046] Fig.24 The present invention discloses the effect of the inactivated Pediococcus acidilactici ZJUIDS13 on the glucose tolerance level of high-fat mice.

[0047] Fig.25 The invention discloses the effect of the inactivated Pediococcus acidilactici ZJUIDS13 on the insulin resistance level in high-fat mice.

[0048] Fig.26 The effect of the inactivated Pediococcus acidilactici ZJUIDS13 of the present invention on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in high-fat mice;

[0049] Fig. 27 The present invention shows the effect of the inactivated Pediococcus acidilactici ZJUIDS13 on malondialdehyde (MDA) and total superoxide dismutase (T-SOD) in the liver of high-fat mice. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0051] The following case uses Pediococcus acidilactici ZJUIDS13 (CGMCCNO.25529).

[0052] Example 1, preparation of Pediococcus acidilactici ZJUIDS13 bacterial powder (i.e., Pediococcus acidilactici ZJUIDS13 freeze-dried bacterial powder):

[0053] It was prepared according to Example 9 of the invention of 202211133365.2 “Lactic acid Pediococcus ZJUIDS13 and its application for relieving ulcerative colitis” and stored at -20°C.

[0054] Example 2: Preparation of inactivated Pediococcus acidilactici ZJUIDS13 bacterial solution

[0055] A single colony of Pediococcus acidilactici ZJUIDS13 was selected and inoculated into 50 mL of MRS liquid culture medium and placed in a 37°C incubator for 18 h. It was activated again in 250 mL of MRS liquid culture medium at a 5% (volume %) inoculation amount and placed in a 37°C incubator for 24 h. Finally, the activated Pediococcus acidilactici ZJUIDS13 was cultured in a 10 L fermenter at a high density anaerobic culture at a 5% inoculation amount and cultured anaerobically at 37°C and pH 6.8 for 18 h. The bacterial liquid density reached 10 9 ~10 10CFU / mL is used as bacterial solution (live bacterial solution).

[0056] The preparation method of the inactivated bacterial solution of Pediococcus acidilactici ZJUIDS13 is as follows: the above-mentioned bacterial solution of ZJUIDS13 is inactivated at 121° C. under high temperature and high pressure for 30 min to obtain the inactivated bacterial solution of ZJUIDS13.

[0057] 1. Alcoholic liver disease

[0058] Experiment 1: Improvement of alcoholic liver disease by Pediococcus acidilactici ZJUIDS13

[0059] 1. Experimental Materials

[0060] Twenty C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center, company license number: SCXK (Shanghai) 2013-0016, and were kept in the SPF animal breeding room of the Experimental Animal Center of Zhejiang Chinese Medical University.

[0061] Reagents: ALT kit (catalog number: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (catalog number: C010-2 Nanjing Jiancheng Bioengineering Institute), TG kit (catalog number: A110-1-1 Nanjing Jiancheng Bioengineering Institute), TC kit (catalog number: A111-1-1 Nanjing Jiancheng Bioengineering Institute), FFA kit (catalog number: A042-2-1 Nanjing Jiancheng Bioengineering Institute), HDL-C kit (catalog number: A112-1-1 Nanjing Jiancheng Bioengineering Institute), LDL-C kit (catalog number: A113-1-1 Nanjing Jiancheng Bioengineering Institute), MDA kit (catalog number: S0131S Beyotime Biotechnology Co., Ltd.), T-SOD (catalog number: S0101S Beyotime Biotechnology Co., Ltd.), GSH-px kit (catalog number: S0057S Beyotime Biotechnology Co., Ltd.), CAT kit (catalog number: S0051 Beyotime Biotechnology Co., Ltd.).

[0062] Lieber-DeCarli alcohol liquid model feed (product code TP4030B, Nantong Trophy Feed Technology Co., Ltd.); Lieber-DeCarli control liquid feed (product code TP4030BC, Nantong Trophy Feed Technology Co., Ltd.).

[0063] Before intragastric administration, the freeze-dried powder of Pediococcus acidilactici ZJUIDS13 (hereinafter referred to as ZJUIDS13 freeze-dried powder) was prepared into a ZJUIDS13 suspension with PBS buffer (0.01 M, pH = 7.2). The number of viable cells in the ZJUIDS13 suspension for intragastric administration was about 5 × 10 9CFU / mL. The intragastric volume of each mouse was 200 μL. The number of viable bacteria per gram of freeze-dried bacterial powder was counted and corrected on MRS agar plates every week during the experimental period.

[0064] 2. Animal husbandry

[0065] Breeding environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good sanitary environment in the cage.

[0066] After one week of adaptive feeding, C57BL / 6 mice were randomly divided into four groups, namely, pair-fed group (PF group), alcohol-fed group (AF group), pair-fed group + Pediococcus acidilactici ZJUIDS13 (PF+ZJUIDS13 group), and alcohol-fed group + Pediococcus acidilactici ZJUIDS13 (AF+ZJUIDS13 group). The alcoholic liver disease mouse model was established by feeding with Lieber-DeCarli liquid alcohol feed, which was purchased from Nantong Trophy Feed Technology Co., Ltd. Each group of mice was adaptively fed with non-alcoholic Lieber-DeCarli control liquid feed for the first three days, and the daily liquid feed amount for each mouse was 15g. After the adaptive feeding, the AF group and the AF+ZJUIDS13 group were fed with alcohol-containing Lieber-DeCarli liquid feed for 4 weeks, and the PF group and the PF+ZJUIDS13 group continued to be fed with Lieber-DeCarli control liquid feed for 4 weeks. During this period, the amount of control liquid feed for the PF group mice was adjusted according to the daily alcohol liquid feed intake of the AF group mice. From the first day of feeding liquid feed, the PF and AF groups were gavaged with 200 μL PBS every day, and the PF+ZJUIDS13 group and the AF+ZJUIDS13 group were gavaged with 200 μL ZJUIDS13 suspension.

[0067] The feed consumed by the mice was changed every day and the food intake was recorded. The weight of the mice was recorded every week. After feeding, the mice were anesthetized with 1% pentobarbital intraperitoneal injection, and blood was taken from the inferior vena cava to measure biochemical indicators such as ALT, AST, and TG. The liver tissue of the mice was weighed and tested for related indicators. All experiments complied with the Animal Ethics Code and the relevant regulations of the Laboratory Animal Ethics Committee, and were specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood was taken from the abdominal aorta under anesthesia, and the bodies were uniformly handled harmlessly.

[0068] 3. Index determination

[0069] According to the instructions for use of the corresponding kits, the components used are provided by the corresponding kits. The details are as follows:

[0070] 3.1 Plasma ALT detection

[0071] Mouse plasma samples were directly sampled for determination. Alanine aminotransferase matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the determination wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 505 nm, and the standard curve was checked to obtain the corresponding ALT / GPT activity unit.

[0072] 3.2 Plasma AST detection

[0073] Mouse plasma samples were directly sampled for determination. The matrix solution of aspartate aminotransferase was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the determination well, mixed, and 20 μL matrix solution was added to the control well, and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination well and the control well, and 5 μL sample to be tested was added to the control well, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding AST / GOT activity unit.

[0074] 3.3 Plasma TG detection

[0075] Mouse plasma samples were directly sampled for determination. 2.5 μL of the sample to be tested and 250 μL of working solution were added to the sample well, 2.5 μL of distilled water and 250 μL of working solution were added to the blank well, 2.5 μL of calibrator and 250 μL of working solution were added to the standard well, the well plate was shaken to mix, incubated at 37°C for 10 minutes, and the OD value of each well was measured by an enzyme-labeled instrument at a wavelength of 500 nm. The standard curve was checked to obtain the corresponding triglyceride content.

[0076] 3.4 Plasma FFA detection

[0077] Mouse plasma samples were directly sampled and measured, 4 μL of the sample to be tested and 200 μL of reagent 1 solution were added to the sample well, 4 μL of distilled water and 200 μL of reagent 1 solution were added to the blank well, 4 μL of calibrator and 200 μL of reagent 1 solution were added to the standard well, the plate was shaken to mix, incubated at 37°C for 5 minutes, the OD value of each well was measured by a microplate reader at a main wavelength of 546 nm and an auxiliary wavelength of 600 nm, A1, then 50 μL of reagent 2 solution was added to each well, the plate was shaken to mix, incubated at 37°C for 5 minutes, the main wavelength was 546 nm, and the auxiliary wavelength was 600 nm, the OD value of each well was measured by a microplate reader, A2, △A=A2-A1 was calculated, and the corresponding free fatty acid content was obtained.

[0078] 3.5 Liver TG detection

[0079] Weigh 20 mg of liver accurately and add lysate at a ratio of 1 mg liver to 20 μL lysate. Use an automatic sample rapid grinder to break up the tissue, then let it stand for 10 minutes, take an appropriate amount of supernatant and transfer it to a 1.5 mL centrifuge tube, and perform the following steps. The remaining lysate can be used for protein quantification by the BCA method. Heat the supernatant in a 70°C metal bath for 10 minutes. Centrifuge at room temperature for 5 minutes at 2000 rpm, and take the supernatant for TG determination. Take 10 μL of supernatant in a 96-well plate, and dilute the 4 mM glycerol standard to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μmol / L, and take 10 μL of each dilution in a 96-well plate, and prepare the working solution according to the instructions of the Beijing Prilai Liquid TG Kit. Add 190 μL of working solution to the sample, incubate at 37°C for 15 minutes, and measure the OD value at a working wavelength of 550 nm.

[0080] 3.6 Liver MDA detection

[0081] Accurately weigh the liver tissue, add 9 times the volume of physiological saline according to the ratio of weight (g): volume (mL) = 1:9, cut the tissue into pieces, prepare homogenate in an ice water bath, centrifuge at 3000 rpm for 10 min, and take the supernatant, i.e. 10% homogenate supernatant for testing. Add 0.1 mL of anhydrous ethanol to the blank tube, add 0.1 mL of 10 nmol / mL standard to the standard tube, add 0.1 mL of test sample to the assay tube and control tube, add 0.1 mL of reagent 1 to each of the four tubes, mix well, add 3 mL of reagent 2 application solution to the four tubes, add 1 mL of reagent 3 application solution to the blank tube, standard tube, and assay tube, and add 1 mL of 50% glacial acetic acid to the control tube. Mix well, incubate at 95℃ for 40 min, take out and cool, then centrifuge at 3500-4000 r / min for 10 min to take the supernatant, and measure the OD value at 532 nm. Calculate the MDA content between each group according to the calculation formula.

[0082] 3.7 Liver T-SOD detection

[0083] Take 10% liver homogenate supernatant for testing. Add 1mL of reagent 1 application solution to two tubes, add 0.05mL of sample to the test tube, add 0.05mL of distilled water to the control tube, add 0.1mL of reagent 2, reagent 3, and reagent 4 application solution to the two tubes respectively, mix thoroughly with a vortex mixer, incubate at 37℃ for 40min, and add 2mL of color developer to the two tubes respectively. Mix well, leave at room temperature for 10min, measure the OD value at a wavelength of 550nm, and calculate the T-SOD content of each group according to the calculation formula.

[0084] 3.8 Liver GSH-px detection

[0085] 10% liver homogenate supernatant was taken for testing. 183 μL glutathione peroxidase detection buffer and 5 μL 10 mM GSH solution were added to the sample wells and blank control wells in turn, and then 10 μL sample was added to the sample wells, and 12 μL 15 mM peroxide reagent solution was added to each well, and the plate was shaken to mix, incubated at room temperature for 10 minutes, and 6.6 μL DTNB solution was added to each well, incubated at 25 ° C for 10 minutes, and the OD value of each well was measured by using an enzyme reader at a wavelength of 412 nm, and the GSH-px content of each group was calculated according to the calculation formula.

[0086] 3.9 Liver CAT test

[0087] Take 10% liver homogenate supernatant for testing. Take 20μL of supernatant from the sample tube to a 1.5ml plastic centrifuge tube, do not add sample to the blank tube, and add catalase detection buffer to each tube to a volume of 40μL. Then add 10μL of 250mM hydrogen peroxide solution, mix quickly with a pipette, and react at 25℃ for 5 minutes. Add 450μL of catalase reaction termination solution to each tube, invert or Vortex to terminate the reaction. Add 40μL of catalase detection buffer to a clean plastic centrifuge tube, and then add 10μL of the above reaction system that has been terminated and mixed, and mix. Take 10 microliters from the 50μL system in the previous step and add it to one well of a 96-well plate. Add 200μL of color working solution, incubate at 25℃ for 15 minutes, measure the OD value of each well with an enzyme reader at a wavelength of 520nm, and calculate the CAT content of each group according to the calculation formula.

[0088] 3.10 Real-time PCR

[0089] (1) Take out the tissue from the -80°C freezer and place it in an ice box. Use an electronic balance to weigh about 0.02 g of tissue into an EP tube and precool the centrifuge at 4°C.

[0090] (2) Add 1 mL of Trizol and 3 steel beads to the EP tube containing the tissue block, grind with a grinder, remove the liquid, pour out the steel beads, and react at room temperature for 10 min;

[0091] (3) Add 200 μL of chloroform to the EP tube, shake vigorously for 30 seconds, and place on ice for 5-10 minutes;

[0092] (4) After standing, place the centrifuge tube in a 4°C centrifuge at 12,000 rpm for 15 min;

[0093] (5) Pipette the aqueous phase (supernatant) of the centrifuged sample into a new 1.5 mL centrifuge tube;

[0094] (6) Add an equal volume of isopropanol solution to the centrifuged solution, gently invert to mix, and let stand at -20°C for 20 min;

[0095] (7) Take out the sample from -20°C and centrifuge it at 12000 rpm and 4°C for 15 min;

[0096] (8) After centrifugation, discard the supernatant to obtain a white (or colorless and transparent) precipitate. Add 100-300 μL of pre-cooled 75% ethanol prepared with DEPC water along the inner wall of the centrifuge tube and wash 2-3 times.

[0097] (9) After discarding the liquid, air-dry at room temperature for about 15 min, add 20-50 μL of pre-cooled DEPC water to the centrifuge tube to dissolve the air-dried precipitate (RNA) at the bottom, and store in a -20°C refrigerator for later use;

[0098] (10) Use an ultra-micro UV spectrophotometer to measure the RNA concentration, record the results and calculate the sample loading amount for each group;

[0099] (11) Add the corresponding reaction solution in the reverse transcription kit to premix, add the sample for reverse transcription, shake and centrifuge, place in the PCR instrument, and set the corresponding PCR reaction conditions according to the reverse transcription kit. Store the cDNA sample reverse transcribed by the reverse transcription instrument in a -20°C refrigerator for later use;

[0100] (12) Use a 0.2 mL fluorescent quantitative PCR eight-tube concatenation, add the reactants, use cDNA as a template, and use a fluorescent quantitative PCR amplification instrument to amplify the experimental target gene and perform fluorescent quantitative PCR determination;

[0101] (13) The mixed reactants were shaken and mixed, and after centrifugation, the eight-tube strip was placed in a qRT PCR reactor. The PCR reaction program was set as follows: pre-denaturation: 94°C, 5 min; denaturation: 94°C, 30 s, 60°C, 30 s, 72°C, 30 s, 40 cycles; extension: 72°C, 5 min, and storage at 4°C. The hot cover temperature of the PCR instrument was set to 105°C;

[0102] (14) The expression changes of the target genes were calculated using the 2–ΔΔCT method.

[0103] 3.11 Detection of plasma ethanol content

[0104] The sample to be tested (mouse plasma) was diluted with ultrapure water (1:1). Shake gently. If the sample contains more impurities, centrifuge gently (4°C, 3000×g, 5 minutes) and take the supernatant. After mixing the supernatant with chromatography-grade tert-butyl alcohol (1:5), filter it with a 0.45μm disposable filter and inject it into a chromatographic bottle. The gas chromatograph includes an automatic sampler AOC-20S, a flame ionization detector (FID), a GC-2010 and an Rtx-BAC1 chromatographic column (specifications are 30m×0.32mm×1.8μm). Injection mode: split injection, split ratio 20:1, carrier gas control mode: constant linear velocity, carrier gas is nitrogen (2ml / min), injection volume 0.2μL. The column temperature program can be adjusted as follows: the initial temperature is 40°C for 2 minutes, the temperature is increased to 240°C at a rate of 10°C / min, and it is maintained for 1 minute, and the total time is 23min.

[0105] 4. Experimental Results

[0106] 4.1. The liver weights of mice in the control feed feeding group (PF group), alcohol feed feeding group (AF group), control feed feeding group + Pediococcus acidilactici ZJUIDS13 (PF+ZJUIDS13 group), and alcohol feed feeding group + Pediococcus acidilactici ZJUIDS13 (AF+ZJUIDS13 group) were as follows: Figure 1 As shown in the left figure, the liver index (calculated based on the liver weight and body weight of the mouse) is Figure 1 As described in the figure on the right.

[0107] Figure 1 The results showed that Pediococcus acidilactici ZJUIDS13 could reduce the increase of liver weight and liver index caused by alcohol. Alcohol feeding caused the liver weight and liver index of mice to be significantly higher than that of the normal control group, while ZJUIDS13 intervention could significantly reduce the liver weight and liver index of mice.

[0108] 4.2. The results of the above plasma ethanol content experiment are as follows Figure 2 shown.

[0109] Figure 2 The results showed that Pediococcus acidilactici ZJUIDS13 could accelerate the metabolism of ethanol. Compared with the PF group, the ethanol content in the plasma of mice in the AF group was significantly increased. After intervention with ZJUIDS13, the ethanol content in the plasma of mice was significantly reduced, accelerating the metabolism of ethanol.

[0110] 4.3. The results of the above plasma AST and ALT detection experiments are as follows Figure 3 shown.

[0111] Figure 3 The results showed that Pediococcus acidilactici ZJUIDS13 could significantly reduce the increase of ALT and AST caused by alcohol feeding. Alanine aminotransferase and aspartate aminotransferase are recommended by the World Health Organization as the most sensitive detection indicators of liver function damage. Compared with the PF group, the plasma ALT and AST levels of mice in the AF group were significantly increased, while the ALT and AST levels of the AF+ZJUIDS13 group were significantly lower than those in the AF group, indicating that ZJUIDS13 intervention can significantly improve liver damage caused by alcohol feeding.

[0112] 4.4. The results of the above plasma TG, FFA and liver TG detection experiments are as follows Figure 4 shown.

[0113] Figure 4 The results showed that alcohol feeding caused lipid metabolism disorders in mice. The plasma TG, FFA and liver TG of mice in the AF group were significantly increased compared with those in the PF group, while the plasma triglyceride and free fatty acid content and liver triglyceride content of the AF+Pediococcus acidilactici ZJUIDS13 group were significantly reduced compared with the AF group. Therefore, it was judged that Pediococcus acidilactici ZJUIDS13 could effectively reduce the content of plasma and liver triglyceride and plasma free fatty acid content in mice.

[0114] 4.5. The results of the above-mentioned fluorescence quantitative PCR experiment on liver lipid metabolism-related factors are as follows Figure 5 shown.

[0115] Figure 5 The results showed that Pediococcus acidilactici ZJUIDS13 could reduce fatty acid synthesis in the liver and improve lipid metabolism. SREBP1c is a member of the SREBPs family and a transcriptional regulator that binds to the endoplasmic reticulum. It regulates the expression of important genes for lipid synthesis and uptake. FASN is a core enzyme in lipid metabolism, which is mainly responsible for catalyzing the biosynthesis of de novo fatty acids (FA). This is a process necessary for cell growth and survival. Compared with the PF group, the expression levels of Srebp1c and Fasn mRNA in the liver of mice in the AF group were significantly increased, indicating that alcohol feeding increased fatty acid synthesis in the liver of mice, and the intervention of Pediococcus acidilactici ZJUIDS13 could significantly reduce the expression levels of Srebp1c and Fasn mRNA in the liver of mice.

[0116] 4.6. The results of the above liver T-SOD, MDA, CAT, and GSH-px detection experiments are as follows Figure 6 shown.

[0117] Figure 6 The results showed that Pediococcus acidilactici ZJUIDS13 has strong antioxidant capacity. Free radicals are unstable molecules produced by the human body during metabolism. They are highly oxidizing and can damage biological macromolecules such as cell membranes, proteins and DNA, leading to cell damage, aging and even death. The antioxidant system can neutralize these free radicals and reduce their damage to cells, thereby protecting the normal structure and function of cells. Alcohol feeding significantly increased the content of lipid peroxide MDA in the liver of mice and reduced the content of antioxidant enzymes GSH-px, T-SOD and CAT. After intervention, Pediococcus acidilactici ZJUIDS13 was able to reverse the oxidative stress state caused by alcohol and exert antioxidant capacity.

[0118] 4.7. The results of the above-mentioned fluorescence quantitative PCR experiment on liver inflammation-related factors are as follows Figure 7 shown.

[0119] Figure 7 The results showed that Pediococcus acidilactici ZJUIDS13 had anti-inflammatory effects. Compared with the PF group, the levels of IL-1β, IL-6, and TNF-α mRNA in the liver of alcohol-fed mice were significantly increased, while the levels of IL-1β, IL-6, and TNF-α mRNA in the liver of AF+ZJUIDS13 group mice were significantly decreased compared with those of AF group mice.

[0120] Experiment 2: Improvement of alcoholic liver disease by inactivated Pediococcus acidilactici ZJUIDS13

[0121] The “5×10 9 CFU / mL of ZJUIDS13 suspension" was changed to "5×10 9 CFU / mL of inactivated ZJUIDS13 bacterial liquid", thus forming the control feed feeding group + inactivated Pediococcus acidilactici ZJUIDS13 (PF+ZJUIDS13(heat-killed) group) and the alcohol feed feeding group + inactivated Pediococcus acidilactici ZJUIDS13 (AF+ZJUIDS13(heat-killed) group).

[0122] That is, the PF and AF groups were intragastrically administered with 200 μL PBS every day, and the PF+ZJUIDS13 (heat-killed) group and the AF+ZJUIDS13 (heat-killed) group were intragastrically administered with 200 μL inactivated Pediococcus acidilactici ZJUIDS13 bacterial solution.

[0123] The rest is the same as Experiment 1.

[0124] The experimental results are as follows:

[0125] Figure 8 The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 could reduce the increase of liver weight and liver index caused by alcohol. Compared with the PF group, alcohol feeding caused a significant increase in the liver weight and liver index of mice in the AF group, while the liver weight and liver index of mice in the AF+ZJUIDS13 (heat-killed) group were significantly reduced compared with the AF group.

[0126] Fig. 9 The results showed that inactivated Pediococcus acidilactici ZJUIDS13 could accelerate the metabolism of ethanol. Compared with the PF group, the ethanol content in the plasma of mice in the AF group was significantly increased, and the inactivated ZJUIDS13 intervention could significantly reduce the ethanol content in the plasma of mice.

[0127] Fig.10 The results showed that the inactivated lactic acid bacteria ZJUIDS13 can improve liver damage caused by alcohol feeding. The plasma ALT and AST levels of mice in the AF group were significantly higher than those in the PF group, while the ALT and AST levels in the AF+ZJUIDS13 group were significantly lower than those in the AF group, indicating that ZJUIDS13 intervention can significantly improve liver damage caused by alcohol feeding.

[0128] Fig.11 The results showed that alcohol feeding caused lipid metabolism disorders in mice. Compared with the PF group, the plasma TG, FFA and liver TG of mice in the AF group were significantly increased, while the plasma triglyceride and free fatty acid content and liver triglyceride content of mice in the AF+ZJUIDS13 (heat-killed) group were significantly reduced compared with the AF group. Therefore, it was judged that the inactivated lactic acid bacteria ZJUIDS13 can effectively reduce the content of plasma and liver triglyceride and plasma free fatty acid content in mice.

[0129] Fig.12 The results showed that inactivated Pediococcus acidilactici ZJUIDS13 could improve the oxidative stress state caused by alcohol. Compared with the PF group, alcohol feeding significantly increased the content of lipid peroxide MDA in the liver of mice and reduced the content of antioxidant enzymes GSH-px, T-SOD and CAT, while inactivated Pediococcus acidilactici ZJUIDS13 intervention could reduce the content of MDA in the liver of mice and increase the content of antioxidant enzymes GSH-px, T-SOD and CAT, exerting antioxidant capacity.

[0130] Fig.13The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 had anti-inflammatory effects. Compared with the PF group, the IL-1β, IL-6 and TNF-α mRNA levels in the liver of the AF group mice were significantly increased, while the IL-1β, IL-6 and TNF-α mRNA levels in the liver of the AF+ZJUIDS13 (heat-killed) group mice were significantly reduced compared with the AF group mice.

[0131] 2. Non-alcoholic liver disease

[0132] Experiment 3: Pediococcus acidilactici ZJUIDS13 improves non-alcoholic liver disease:

[0133] 1. Experimental Materials

[0134] Experimental animals: 24 C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center, company license number: SCXK (Shanghai) 2013-0016, and were raised in the Experimental Animal Center of Zhejiang Chinese Medical University in an SPF environment.

[0135] Reagents: ALT kit (catalog number: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (catalog number: C010-2 Nanjing Jiancheng Bioengineering Institute), TG kit (catalog number: A110-1-1 Nanjing Jiancheng Bioengineering Institute), HDL-C kit (catalog number: A112-1-1 Nanjing Jiancheng Bioengineering Institute), LDL-C kit (catalog number: A113-1-1 Nanjing Jiancheng Bioengineering Institute), MDA kit (catalog number: S0057S Beyotime Biotechnology Co., Ltd.), T-SOD (catalog number: S0057S Beyotime Biotechnology Co., Ltd.).

[0136] The freeze-dried bacterial powder of Pediococcus acidilactici ZJUIDS13 (hereinafter referred to as ZJUIDS13 freeze-dried bacterial powder) was dissolved in physiological saline to obtain 5×10 9 CFU / ml of bacterial solution.

[0137] 2. Methods:

[0138] 2.1 Animal husbandry

[0139] After one week of adaptation feeding in an SPF-level animal laboratory, C57BL / 6 mice at age of 8 weeks were randomly divided into three groups, with 8 mice in each group, namely, a normal feed group (NFD group), a 45% high-fat feed group (HFD group), and a 45% high-fat feed + Pediococcus acidilactici ZJUIDS13 group (HFD + Pediococcus acidilactici ZJUIDS13 group).

[0140] The NFD group was fed with a control diet for 12 weeks, and the HFD group and HFD+Pediococcus acidilactici ZJUIDS13 group were fed with a 45% high-fat diet for 12 weeks. During this period, the HFD+Pediococcus acidilactici ZJUIDS13 group was gavaged with 0.2 mL of Pediococcus acidilactici ZJUIDS13 bacterial solution every other day (10 9 CFU / mouse, the solvent was normal saline).

[0141] The feed consumed by the mice was replaced with new feed every two days, and the weight of the mice was recorded every week to detect changes. After feeding, the mice were anesthetized with 1% pentobarbital intraperitoneal injection, blood was drawn from the inferior vena cava to measure biochemical indicators such as ALT and AST, and the liver tissue of the mice was taken for the measurement of relevant indicators.

[0142] 3. Index determination

[0143] 3.1 Plasma ALT detection: The detection method is the same as the above experiment 1;

[0144] 3.2 Plasma AST detection: The detection method is the same as the above experiment 1;

[0145] 3.3 Glucose tolerance test

[0146] After fasting for 12 h, mice were intraperitoneally injected with 2.5 g / kg of glucose solution according to their body weight. Blood samples were collected at 0, 30, 60, and 120 min after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0147] 3.4 Insulin resistance detection

[0148] After 4 h of fasting, mice were intraperitoneally injected with 0.75 U / kg of insulin according to their body weight. Blood samples were collected at 0, 30, 60, and 120 min after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0149] 3.5 Detection of liver TG: The detection method is the same as that of experiment 1 above;

[0150] 3.6 Malondialdehyde (MDA) detection: The detection method is the same as the above experiment 1;

[0151] 3.7 Detection of superoxide dismutase (T-SOD): The detection method is the same as that of experiment 1 above.

[0152] 4. Experimental results:

[0153] Fig.14The results showed that Pediococcus acidilactici ZJUIDS13 can significantly reduce the weight gain caused by a high-fat diet. A 12-week high-fat diet caused the mice to weigh significantly more than the normal group, while ZJUIDS13 intervention could significantly reduce the weight gain of mice caused by a high-fat diet. The liver weight and liver-to-body ratio of mice were also significantly lower than those of the high-fat diet group under ZJUIDS13 intervention.

[0154] Fig.15 The results showed that lactic acid bacteria ZJUIDS13 can significantly reduce the increase of liver TG caused by a high-fat diet. The triglyceride content in the liver of mice in the 45% high-fat diet group (HFD) was significantly increased compared with the control group (NFD), while the triglyceride content in the liver of the HFD+ lactic acid bacteria ZJUIDS13 group was significantly reduced compared with the HFD group. Therefore, it is judged that lactic acid bacteria ZJUIDS13 can effectively reduce the triglyceride content in the liver of mice.

[0155] Fig.16 The results showed that Pediococcus acidilactici ZJUIDS13 can significantly reduce the increase of ALT and AST caused by a high-fat diet. ALT, or alanine aminotransferase, is mainly present in the cytoplasm of liver cells. The intracellular concentration is 1000-3000 times higher than that in serum. As long as 1% of liver cells are destroyed, the serum enzyme can be doubled. Therefore, alanine aminotransferase is recommended by the World Health Organization as the most sensitive detection indicator of liver function damage. A decrease in ALT means a decrease in liver damage. AST, or aspartate aminotransferase, is also called aspartate aminotransferase. AST is mainly distributed in the mitochondria of liver cells and is also one of the sensitive indicators of liver cell damage.

[0156] Fig.17 , Fig.18 The results showed that Pediococcus acidigensis ZJUIDS13 could significantly improve insulin resistance and glucose tolerance caused by a high-fat diet.

[0157] Fig.19 The results showed that Pediococcus acidilactici ZJUIDS13 could significantly reduce the increase of low-density lipoprotein (LDL) caused by a high-fat diet and increase the content of high-density lipoprotein (HDL) in plasma, thereby improving blood lipid metabolism.

[0158] Fig. 20The results showed that Pediococcus acidilactici ZJUIDS13 has strong antioxidant capacity. Under normal circumstances, the antioxidant enzymes in the natural antioxidant defense system of the organism can work synergistically with antioxidants in diet or medicine to remove peroxides. The most important antioxidant enzymes include superoxide dismutase (SOD). SOD is responsible for dismutating superoxide anions into hydrogen peroxide, thereby preventing the production of highly toxic hydroxyl free radicals. On the other hand, the level of lipid peroxidation product MDA is detected to indirectly determine the severity of free radical attack on cells.

[0159] Experiment 4: Improvement of non-alcoholic liver disease by inactivated Pediococcus acidilactici ZJUIDS13

[0160] The "Pediococcus acidilactici ZJUIDS13 intervention" in Experiment 3 was changed to inactivated Pediococcus acidilactici ZJUIDS13 intervention. The rest was the same as Experiment 3.

[0161] The experimental results are as follows:

[0162] Fig.21 The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 could significantly reduce the weight gain caused by a high-fat diet. A 12-week high-fat diet caused the mice to weigh significantly more than the normal group, while the inactivated ZJUIDS13 intervention could significantly reduce the weight gain of mice caused by the high-fat diet. The liver weight and liver-to-body ratio of mice were also significantly lower than those of the high-fat diet group under the intervention of inactivated ZJUIDS13.

[0163] Fig. 22 The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 could significantly reduce the increase of liver TG caused by a high-fat diet. The triglyceride content in the liver of mice in the 45% high-fat diet group (HFD) was significantly increased compared with that in the control group (NFD), while the triglyceride content in the liver of the HFD+ZJUIDS13 (heat-killed) group was significantly decreased compared with that in the HFD group.

[0164] Fig.23 The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 could significantly reduce the increase of ALT and AST caused by a high-fat diet.

[0165] Fig.24 , Fig.25 The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 could significantly improve insulin resistance and glucose tolerance caused by a high-fat diet.

[0166] Fig.26 The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 could significantly reduce the increase of low-density lipoprotein (LDL) caused by a high-fat diet, and increase the content of high-density lipoprotein (HDL) in plasma, which could play a role in regulating blood lipids.

[0167] Fig. 27 The results showed that the inactivated Pediococcus acidilactici ZJUIDS13 had strong antioxidant capacity and could effectively remove free radicals and peroxides from the body.

[0168] The present invention also conducted the following comparative test: Pediococcus acidilactici MY8, Pediococcus acidilactici CCFM1364, Pediococcus acidilactici CCFM1365, and Pediococcus acidilactici RH2712 were tested according to the above experiment, and the experimental results were compared with those of the present invention as follows:

[0169] Pediococcus acidilactici MY8 was shown to effectively inhibit the activity of α-glucosidase and reduce blood sugar; Pediococcus acidilactici CCFM1364 and Pediococcus acidilactici CCFM1365 were shown to improve the oxidative stress state of mouse liver and reduce the expression of liver inflammatory factors; Pediococcus acidilactici RH2712 was shown to have a strong ability to degrade ethanol. However, none of them has the function of improving fatty liver disease associated with metabolic dysfunction.

[0170] Only the ZJUIDS13 of the present invention can reduce the ethanol content in the plasma of mice with alcoholic liver disease, and ZJUIDS13 can also reduce liver lipid synthesis and improve the degree of liver fatty degeneration. In other words, only the ZJUIDS13 of the present invention has the function of improving alcoholic liver disease and improving fatty liver disease associated with metabolic dysfunction at the same time.

[0171] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of Pediococcus acidilactici ZJUIDS13 in the preparation of preparations for improving liver diseases.

2. The use according to claim 1, characterized in that: The formulation includes a medicament.

3. The use according to claim 1 or 2, characterized in that Improvement of liver disease includes at least one of the following: Improve alcoholic liver disease and fatty liver disease associated with metabolic dysfunction.

4. The use according to claim 3, characterized in that: Including Pediococcus acidilactici ZJUIDS13 and inactivated Pediococcus acidilactici ZJUIDS13.

5. The use according to claim 3, characterized in that: It includes a suspension prepared by using the bacterial powder of Pediococcus acidilactici ZJUIDS13 and an inactivated ZJUIDS13 bacterial liquid.

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