Probiotics and probiotic agent for relieving acute alcoholic liver injury and application of probiotics and probiotic agent

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to probiotics and a probiotic agent for relieving acute alcoholic liver injury and application of the probiotics and the probiotic agent. The bacterial strain for relieving the acute alcoholic liver injury is clostridium butyricum JJ100, the bacterial strain is independently used or is compounded with lactobacillus rhamnosus LR to obtain a probiotic agent, the acute alcoholic liver injury can be remarkably relieved, and the effect of the JJ100 and LR composite bacterial agent with the same total bacterial count for relieving the acute alcoholic liver injury is generally superior to that of the JJ100 which is independently used. The traditional Chinese medicine composition has the effects that the levels of LPS, ALT, AST, TC and TG in serum of a mouse with alcoholic liver injury are reduced, the liver injury is effectively relieved, the increase of the blood lipid level of the mouse caused by acute alcohol is inhibited, and the effects of reducing lipid and protecting the liver are achieved; the activity of SOD and CAT in the liver is improved, and oxidative stress in a mouse body is reduced; the activity of TNF-alpha, INF-gamma, IL-4, IL-6 and MPO in the liver of a mouse is reduced, so that the alcoholic liver inflammation is inhibited, and the application prospect in prevention or treatment of acute alcoholic liver injury is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and in particular relates to a probiotic for alleviating acute alcoholic liver injury, a probiotic agent and an application thereof. Background Art

[0002] Alcohol consumption is a global, long-term, and major health problem. Excessive drinkers are highly susceptible to alcoholic liver disease (ALD), and the pathogenesis is complex. Drinking a large amount of alcohol at one time can cause acute ethanol poisoning, which is seriously harmful to the health of the body. Alcohol induces quantitative and qualitative changes in the intestinal microbiota and increases intestinal permeability, which leads to the transfer of live bacteria and microbial products to the liver, inducing and promoting inflammation and promoting hepatocyte death and fibrosis, playing a central role in the cause of ALD (Louvet A, Mathurin P. Alcoholic liver disease: Mechanisms of injury and targeted treatment. Nat Rev Gastroenterol Hepatol. 2015; 12(4): 231–242; Bajaj JS. Alcohol, liver disease and the gutmicrobiota. Nat Rev Gastroenterol Hepatol. 2019; 16(4): 235–246).

[0003] The pathogenesis of alcohol-induced liver damage is mainly divided into fat metabolism disorders, abnormal inflammation, and liver oxidative stress. The beneficial effects of probiotics on the host in a variety of metabolic diseases have been widely confirmed. For example, Lactobacillus probiotics can alleviate alcohol-induced intestinal barrier damage and liver damage. Studies have shown that Lactobacillus rhamnosus (formerly known as Lactobacillus rhamnosus) can reduce intestinal G -Lactobacillus rhamnosus can improve the accumulation of enteric endotoxin LPS and liver fat accumulation, liver lipid peroxidation and cellular inflammatory response (NanjiAA, et al., Lactobacillus Feeding Reduces Endotoxemia and Severity of Experimental Alcoholic Liver (Disease). Proc Soc Exp Biol Med. 1994; 205(3): 243-247; Wu Yanfeng. Study on the effect of Lactobacillus rhamnosus on the alleviation of chronic alcoholic liver damage by adjusting normal intestinal flora. Jilin Agricultural University; 2016; Yeung CY, et al. In vitro prevention of salmonella lipopolysaccharide-induced damages in epithelial barrier function by various lactobacillus strains. Gastroenterol Res Pract. 2013: 1–7), promote the expression of tight junction proteins ZO-1, Claudin-1 and Occludin, improve intestinal barrier function and other mechanisms to improve the liver damage of mice caused by chronic alcohol exposure (Wang, Y. et al., al.,Lactobacillus rhamnosus GG treatment potentiatesintestinal hypoxia-inducible factor,promotes intestinal integrity andameliorates alcohol-induced liver injury.Am.J.Pathol.2011,179,2866–2875; Zhao,H.et al.Inhibition of miR122a by Lactobacillus rhamnosus GG culturesupernatant increases intestinal occludin expression and protects mice from alcoholic liver disease. Toxicol. Lett. 2015, 234, 194–200).Zhu Shiya et al. found that Lactobacillus casei (now renamed Lactobacillus casei) RS8-5 and Lactobacillus helveticus D-7-S146 can inhibit the downregulation of intestinal tight junction protein expression levels and endotoxemia in mice caused by chronic alcohol exposure, restore the balance of intestinal flora, and reduce liver damage in mice (Zhu Shiya. Screening and efficacy evaluation of lactobacilli with the function of alleviating chronic alcoholic liver damage. Jiangnan University, Master's thesis, 2020); Lactobacillus plantarum (formerly known as Lactobacillus plantarum) LP45 and Lactobacillus rhamnosus L519 can improve chronic alcohol-induced liver damage in mice to a certain extent (Zhu Shiya et al. Comparison of the effects of different lactobacilli in alleviating chronic alcoholic liver damage. Food and Fermentation Industries, 2019, 45(22): 20-26). Combination of Lactobacillus plantarum KLDS1.0344 and Lactobacillus acidophilus KLDS1.0901 (Li, H. et al. Lactobacillus plantarum KLDS1.0344 and Lactobacillus acidophilus KLDS1.0901 Mixture Prevents Chronic Alcoholic Liver Injury in Mice by Protecting the Intestinal Barrier and Regulating Gut Microbiota and Liver-Related Pathways. J. Agric. Food Chem. 2021, 69, 183–197.), Bifidobacterium breve ATCC15700 (Tian, ​​X. et al. Bifidobacterium breve ATCC15700 pretreatment prevents alcoholic liver disease through modulating gut microbiota in mice exposed to chronic alcohol intake.J.Funct.Foods,2020,72,104045) and other probiotics can regulate the intestinal microbiota, inhibit the occurrence of alcohol-induced ALD or reduce alcohol damage to the liver through the gut-liver axis.Lactobacillus rhamnosus WKA55 (Chinese patent CN114574406A), LR-Y12 (Chinese patent CN118421538A) and CCFM1107 (CN102618456A) have been used to alleviate alcoholic liver damage, but their protective effects are relatively limited. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a probiotic, a probiotic agent and applications thereof for alleviating acute alcoholic liver injury.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a probiotic for alleviating acute alcoholic liver injury. The probiotic for alleviating acute alcoholic liver injury is Clostridium butyricum JJ-100, which was deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms on January 3, 2025, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a preservation number of CGMCC No. 33288.

[0007] The present invention also provides a probiotic for alleviating acute alcoholic liver damage, wherein the strain in the probiotic is the above-mentioned JJ-100 strain.

[0008] Furthermore, in the probiotic agent, the total number of viable bacteria is not less than 2×10 9 CFU / g or 2×10 9 CFU / ml.

[0009] Furthermore, the probiotics also contain Lactobacillus rhamnosus LR with a preservation number of CGMCC No.33287.

[0010] Among them, Lacticaseibacillus rhamnosus LR, the depository is the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is January 3, 2025, the deposit number is CGMCC No.33287, and the address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0011] The ratio of the viable count of the JJ-100 strain to the LR strain is 1:5 to 5:1;

[0012] The total number of viable bacteria of the JJ-100 strain and the LR strain is not less than 2×10 9 CFU / g or 2×10 9 CFU / ml.

[0013] Furthermore, the above-mentioned probiotics also include anhydrous glucose and skim milk powder.

[0014] Wherein, the probiotic agent can be prepared illustratively by the following method:

[0015] The butyric clostridium JJ-100 or the rhamnosus lactobacillus LR are inoculated into a culture medium for cultivation to obtain a fermentation liquid; the fermentation liquid is centrifuged to obtain bacterial cells; the bacterial cells are evenly mixed with skim milk powder, and then freeze-dried to obtain a freeze-dried bacterial powder of the butyric clostridium JJ-100 or a freeze-dried bacterial powder of the rhamnosus lactobacillus LR;

[0016] By mixing, by weight, 1 part of freeze-dried Clostridium butyricum JJ-100 powder, 1-3 parts of anhydrous glucose and 4-7 parts of skim milk powder, a probiotic agent containing Clostridium butyricum JJ-100 as the strain (Clostridium butyricum JJ-100 composition) can be prepared;

[0017] By weight, 1-3 parts of rhamnosus Lactobacillus LR freeze-dried powder, 10-30 parts of anhydrous glucose and 40-70 parts of skim milk powder are mixed, and then the composition is compounded with the above-mentioned Clostridium butyricum JJ-100 composition according to the ratio of viable bacteria count of 1:5 to 5:1, so as to prepare a probiotic agent compounded with Clostridium butyricum JJ-100 and rhamnosus Lactobacillus LR. Clostridium butyricum JJ-100 and rhamnosus Lactobacillus LR in the probiotic agent have a synergistic effect.

[0018] Furthermore, the dosage form of the probiotics includes probiotics obtained by freeze drying, spray drying, embedding, microencapsulation and other methods and their products.

[0019] The present invention also provides the use of the probiotic agent in preparing a medicine for improving or treating acute alcoholic liver injury.

[0020] Furthermore, the drug also contains excipients; the excipients include any one of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0021] The beneficial effects of the present invention are:

[0022] The invention provides a Clostridium butyricum JJ100 strain capable of relieving acute alcoholic liver injury. The probiotic agent obtained by using the strain alone or in combination with Lactobacillus rhamnosus LR can significantly reduce the levels of lipopolysaccharide (LPS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TC), and total cholesterol (TG) in the serum of mice with alcoholic liver injury, effectively relieve liver injury and inhibit the increase of blood lipid levels in mice caused by acute alcohol, thereby playing the role of lowering lipids and protecting the liver; improve the activities of SOD and CAT in the liver, and reduce oxidative stress in mice; reduce the activities of TNF-α, INF-γ, IL-4, IL-6, and MPO in the liver of mice, thereby inhibiting alcoholic liver inflammation, and has broad application prospects in preventing or treating acute alcoholic liver injury.

[0023] The Clostridium butyricum JJ-100 and Lactobacillus rhamnosus LR of the present invention both have strong digestive tract tolerance and high survival rate in the gastrointestinal tract, thereby ensuring the beneficial activity of the probiotic. The two are compounded with a specific ratio of viable bacteria to obtain a probiotic, which can cooperate with each other, promote each other, and synergize in the effect of relieving liver damage caused by acute alcohol. When the total bacterial count is the same, compared with a single JJ-100 strain or a single LR strain, the probiotic containing both the JJ-100 strain and the LR strain can significantly reduce inflammation, improve antioxidant capacity, reduce lipids and protect the liver, and maintain intestinal barrier function, thereby relieving alcoholic liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the test results of the DPPH free radical scavenging ability, reducing activity and lipid peroxidation inhibition ability of different strains during the screening process of Clostridium butyricum JJ-100.

[0025] Figure 2 These are the test results of the DPPH free radical scavenging, reducing activity and lipid peroxidation inhibiting abilities of different strains during the screening process of Lactobacillus rhamnosus LR.

[0026] Figure 3 The statistical results of serum alanine aminotransferase (ALT) (Figure A), aspartate aminotransferase (AST) (Figure B) and lipopolysaccharide (LPS) (Figure C) levels of mice in each group; Control is the control group, Ethanol is the alcohol model group, CB is the JJ100 bacterial agent group, LR is the LR bacterial agent group, and CL is the JJ100 and LR combined bacterial agent group.

[0027] Figure 4The figures are the statistical results of the levels of triglyceride (TG) (Figure A) and total cholesterol (TC) (Figure B) in the serum of each group of mice; Control is the control group, Ethanol is the alcohol model group, CB is the JJ100 bacterial agent group, LR is the LR bacterial agent group, and CL is the JJ100 and LR combined bacterial agent group.

[0028] Figure 5 The figures are the statistical results of catalase (CAT) (Figure A) and superoxide dismutase (SOD) (Figure B) levels in the livers of mice in each group; Control is the control group, Ethanol is the alcohol model group, CB is the JJ100 bacterial agent group, LR is the LR bacterial agent group, and CL is the JJ100 and LR combined bacterial agent group.

[0029] Figure 6 The figures are the statistical results of the activities of tumor necrosis factor (TNF-α) (Figure A), interleukin-4 (IL-4) (Figure B), interleukin-6 (IL-6) (Figure C) and myeloperoxidase (MPO) (Figure D) in the liver of each group of mice; Control is the control group, Ethanol is the alcohol model group, CB is the JJ100 bacterial agent group, LR is the LR bacterial agent group, and CL is the JJ100 and LR combined bacterial agent group.

[0030] Figure 7 The figure is a statistical result of the intestinal barrier function level of each group of mice; Control is the control group, Ethanol is the alcohol model group, CB is the JJ100 bacterial agent group, LR is the LR bacterial agent group, and CL is the JJ100 and LR combined bacterial agent group.

[0031] Figure 8 HE staining sections of the liver of mice in each group; Control is the control group, Ethanol is the alcohol model group, CB is the JJ100 bacterial agent group, LR is the LR bacterial agent group, and CL is the JJ100 and LR combined bacterial agent group. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0033] Example 1 Isolation, Screening and Identification of Clostridium butyricum JJ-100

[0034] 1. Isolation of strains

[0035] The collected 3g fecal samples of healthy young men who drank alcohol for a long time were diluted to 10 -3 , 10 -4 , 10 -5, 10 -6 , 10 -7 There are 5 gradients in total. Use a pipette to pipette 100 μl onto TSN solid medium (tryptone 15.0 g, yeast extract 5.0 g, soy peptone 5.0 g, ammonium ferric citrate 1.0 g, sodium metabisulfite 1.0 g, agar 20.0 g, distilled water 1 000 mL, pH 7.6 ± 0.2, autoclaved at 121 ° C for 20 min) and spread evenly. Set up 3 parallels for each group.

[0036] The culture medium was transferred to an anaerobic incubator and cultured anaerobically at 37°C for 24 hours. When colonies were formed, a single colony was picked for streaking on a plate and examined under a microscope to observe the morphological characteristics of the colony.

[0037] 2. Screening of strains

[0038] After multiple single colonies were subcultured in RCM medium (10.0 g peptone, 10.0 g beef extract powder, 5.0 g glucose, 3.0 g yeast powder, 1.0 g soluble starch, 5.0 g sodium chloride, 3.0 g sodium acetate, 0.5 g L-cysteine ​​hydrochloride, 1 000 mL distilled water, pH 6.8±0.1, autoclaved at 121°C for 15 min), bacterial suspensions were prepared. The DPPH free radical scavenging ability, reducing activity and lipid peroxidation inhibition ability of the bacterial suspensions of each strain were measured. The antioxidant activity of each strain was comprehensively evaluated by analyzing various indicators. Finally, it was found that strain JJ-100 had the highest antioxidant activity ( Figure 1 ).

[0039] The specific methods for determining the ability of each strain to scavenge DPPH free radicals, reduce activity, and inhibit lipid peroxidation are as follows:

[0040] (1) Determination of DPPH free radical scavenging ability:

[0041] 0.8 mL of sample (the number of viable bacteria was controlled to 10 9 CUF / mL concentration) was added to 1mL of 0.2mmol / L freshly prepared DPPH-anhydrous ethanol solution, mixed, reacted at room temperature in the dark for 30min, centrifuged at 6000rpm for 10min, and the supernatant was taken to measure the absorbance at a wavelength of 517nm, which was recorded as A 样品 An equal volume of saline was used to replace the sample solution as the control group, and an equal volume of a mixture of saline and anhydrous ethanol was used as a blank to adjust the zero.

[0042] Among them, A 样品 is the absorbance value of the sample group; A 样品 is the absorbance value of the control group (normal saline instead of sample);

[0043] DPPH free radical scavenging rate = (A 对照 -A 样品 ) / A 对照 ×100%.

[0044] (2) Determination of reducing activity:

[0045] 0.8 mL of sample (the number of viable bacteria was controlled to 10 9 CUF / mL concentration) 1% potassium ferricyanide 0.5ml, 0.2mol / L PBS buffer (pH=6.6) 0.5ml, mixed and placed in a 50℃ water bath for 20min, the control group sample was distilled water. After cooling, add 0.5ml 10% trichloroacetic acid, centrifuge at 3000rpm for 5min, take 1ml of the supernatant, add 1ml 0.1% FeCl3, and measure the OD value at 700nm after 10min.

[0046] Among them, A 样品 is the absorbance value of the sample group; A 样品 is the absorbance value of the control group (distilled water replaces the sample);

[0047] Reduction activity = (A 样品 -A 对照 ) / A 对照 ×100%.

[0048] (3) Determination of lipid peroxidation inhibition ability:

[0049] Add 1 ml of linoleic acid emulsion to 0.5 ml of 0.02 mol / L PBS buffer, add 0.01% FeSO4 and 0.2 ml of ascorbic acid, and then add 0.8 ml of sample (the number of viable bacteria is controlled to be 10 9 CUF / mL concentration), react in a water bath at 37℃ for 12h, take 2ml, add 0.2ml 4% trichloroacetic acid, 2ml 0.8% thiobarbituric acid, 0.2ml 0.4% BHT, react at 100℃ for 30min, cool, extract with 2ml chloroform, centrifuge and collect supernatant. OD was measured at 532nm, and PBS was used as blank control.

[0050] Among them, A 样品 is the absorbance value of the sample group; A 对照 is the absorbance value of the control group (PBS replaces the sample);

[0051] Anti-lipid peroxidation rate = (1-A 样品 / A 对照 )×100%.

[0052] Physiological and biochemical experiments were conducted on the purified strain JJ-100. The results showed that the colonies of the strain were small and smooth, round in shape, moist and shiny on the surface, and opaque milky white in color.

[0053] 3. Identification of strains

[0054] The strain was cultured in a 37°C incubator until mid-logarithmic phase, and sufficient bacteria were collected after washing with PBS. DNA was extracted using a bacterial DNA extraction kit and the quality of the extracted DNA was tested. The 16S rRNA gene was amplified by PCR using primers 27f and 1495r, and then bidirectional sequencing was performed. The splicing results were compared with the data in the NCBI database by Blast, and the results showed that the similarity between Clostridium butyricum JJ-100 and Clostridium butyricum was greater than 99%, so the strain JJ-100 was determined to be Clostridium butyricum.

[0055] The JJ-100 strain involved in the present invention is classified and named Clostridium butyricum, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration. The preservation time is January 3, 2025, the preservation number is CGMCC No.33288, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0056] Example 2 Isolation, Screening and Identification of Lactobacillus rhamnosus LR

[0057] 1. Isolation of strains

[0058] The collected 3g fecal samples of healthy young men who drank alcohol for a long time were diluted to 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 There are 5 gradients in total. Use a pipette to pipette 100 μl onto MRS solid medium and spread evenly. Set up 3 parallels for each group.

[0059] The culture medium was transferred to an anaerobic incubator and cultured anaerobically at 37°C for 24 hours. When colonies were formed, a single colony was picked for streaking on a plate and examined under a microscope to observe the morphological characteristics of the colony.

[0060] 2. Screening of strains

[0061] After multiple single colonies were subcultured, bacterial suspensions were prepared. The DPPH free radical scavenging ability, reducing activity and lipid peroxidation inhibition ability of each strain suspension were measured (the measurement method was the same as in Example 1). By analyzing various indicators, the antioxidant activity of each strain was comprehensively evaluated. Finally, it was found that strain LR had the highest antioxidant activity ( Figure 2 ).

[0062] The purified LR strain was subjected to physiological and biochemical studies. The colonies of the strain were slightly larger, smooth, milky white, and had neat and convex edges.

[0063] 3. Identification of strains

[0064] The strain was cultured in a 37°C incubator until mid-logarithmic phase, and sufficient bacteria were collected after washing with PBS. DNA was extracted using a bacterial DNA extraction kit and the quality of the extracted DNA was tested for bidirectional sequencing. The splicing results were compared with the data in the NCBI database by Blast, and the results showed that the similarity between strain LR and Lactobacillus rhamnosus was greater than 99%, so strain LR was determined to be Lactobacillus rhamnosus.

[0065] The LR strain involved in the present invention is classified and named as Lactobacillus rhamnosus, and the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation time is January 3, 2025, the preservation number is CGMCC No.33287, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0066] Example 3 Preparation of probiotics using Clostridium butyricum JJ-100 as strain

[0067] 1. Fermentation of Clostridium butyricum JJ-100:

[0068] (1) Activation of strains: Clostridium butyricum JJ-100 was inoculated into MRS solid medium and cultured anaerobically at 37°C for 24 h to obtain a single colony of the strain;

[0069] (2) Preparation of primary seed solution: a single colony of Clostridium butyricum JJ-100 was selected from a solid culture medium, inoculated into a modified MRS liquid culture medium, and cultured anaerobically at 37°C for 24 h to obtain a primary seed solution;

[0070] (3) Expansion culture of bacterial strains: The primary seed liquid was inoculated into the modified liquid MRS medium at a rate of 5%, and cultured anaerobically at 37° C. for 24 h to obtain the fermentation liquid.

[0071] 2. Preparation of freeze-dried Clostridium butyricum JJ-100 powder

[0072] Method 1:

[0073] (1) Collection of Bacteria: The fermentation broth was centrifuged at 6000 rpm for 10 min to collect the bacteria, which were washed with 0.05 M PBS buffer (pH 6.0-7.0), resuspended, centrifuged and the supernatant removed, and the washing was repeated once to obtain a bacterial slurry of Clostridium butyricum JJ-100.

[0074] (2) Freeze-drying of bacterial cells: Add skim milk powder to the collected bacterial slurry at a ratio of 1 part of Clostridium butyricum slurry to 3 parts of skim milk powder, stir evenly, and then freeze-dry at -50 to -60°C for 10 to 12 hours to obtain freeze-dried bacterial powder.

[0075] Method 2:

[0076] The fermentation liquid obtained by fermentation is added with 2.5% to 3.5% skimmed milk powder, mixed and then centrifuged and spray dried to obtain the original bacterial powder. The conditions are: air inlet temperature 180 to 200°C, air outlet temperature 80 to 95°C, centrifugal nozzle speed: 10000 to 13000rpm.

[0077] 3. Preparation of Clostridium butyricum JJ-100 probiotic agent (bacterial powder composition):

[0078] According to weight, 1 part of freeze-dried bacterial powder was mixed with 2 parts of anhydrous glucose and 5 parts of skim milk powder to obtain a bacterial count of 3.1×10 9 CFU / g of Clostridium butyricum JJ-100 powder composition.

[0079] Example 4 Preparation of probiotics obtained by compounding Clostridium butyricum JJ-100 and Lactobacillus rhamnosus LR

[0080] 1. Fermentation of Lactobacillus rhamnosus LR

[0081] (1) Activation of strains: Lactobacillus rhamnosus LR was inoculated into MRS solid medium and cultured anaerobically at 37°C for 24 h to obtain a single colony of the strain;

[0082] (2) Preparation of primary seed solution: a single colony of Lactobacillus rhamnosus LR was selected from a solid culture medium, inoculated into a modified MRS liquid culture medium, and cultured anaerobically at 37° C. for 24 h to obtain a primary seed solution;

[0083] (3) Expansion culture of the strain: inoculate the primary seed liquid into a modified liquid MRS medium at a 5% inoculum volume, and culture anaerobically at 37°C for 24 h to obtain a fermentation liquid;

[0084] (4) Collection of cells: The fermentation broth was centrifuged at 6000 rpm for 10 min to collect the cells, which were then washed with 0.05 M PBS buffer (pH 6.0-7.0), resuspended, centrifuged and the supernatant removed, and the washing was repeated once to obtain cells of Lactobacillus rhamnosus LR; the cell count was 4.2 × 10 11 CFU / g;

[0085] (5) Freeze-drying of bacteria: Add 2 times the weight of skim milk powder to the collected bacteria and freeze-dry them to obtain freeze-dried bacterial powder; the number of viable Lactobacillus rhamnosus LR in the freeze-dried bacterial powder was 1.2×10 11CFU / g;

[0086] (6) Preparation of bacterial powder composition: According to weight parts, 2 parts of Lactobacillus rhamnosus LR freeze-dried bacterial powder, 15 parts of glucose and 50 parts of skim milk powder were mixed to obtain a bacterial count of 3.8×10 9 CFU / g of Lactobacillus rhamnosus LR powder composition;

[0087] (7) The Clostridium butyricum JJ-100 powder composition prepared in Example 4 and the Lactobacillus rhamnosus LR powder composition obtained in step (6) are mixed at a ratio of 1:5 to 5:1 in terms of viable bacteria count to prepare a probiotic agent of Clostridium butyricum JJ-100 and Lactobacillus rhamnosus LR.

[0088] Example 5 Ability to improve acute alcoholic liver injury in mice

[0089] The bacterial suspension preparation method involved in this embodiment is as follows:

[0090] (1) Activation of bacterial strains: The desired strain was inoculated into MRS solid medium and cultured anaerobically at 37°C for 24 h to obtain a single colony of the desired strain;

[0091] (2) Preparation of primary seed solution: Pick a single colony of the desired strain from the MRS solid medium, inoculate it into the modified MRS liquid medium, and culture it anaerobically at 37°C for 24 h to obtain the primary seed solution;

[0092] (3) Expansion culture of bacterial strains: The primary seed solution was inoculated into a modified liquid MRS medium at a 5% (v / v) inoculation rate and cultured anaerobically at 37°C for 24 h to obtain a fermentation liquid of the desired strain;

[0093] (4) The fermentation broth of the desired strain was centrifuged at 6000 × g for 10 min, the cells were collected, and the cells were washed with 0.05 M PBS buffer (pH 6.0-7.0), resuspended, centrifuged and the supernatant was removed, and the washing was repeated once to obtain the cells of the desired strain; the cells were resuspended with PBS solution.

[0094] The MRS solid culture medium includes, by concentration, 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 5 g / L sodium acetate, 5 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 2 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.05 g / L MnSO4·7H2O, 80 mL Tween, and 20 g / L agar.

[0095] The modified MRS medium includes, by concentration, 26 g / L peptone, 5 g / L beef extract, 45 g / L glucose, 8.5 g / L sodium acetate, 3 g / L yeast powder, 2 g / L diammonium hydrogen citrate, 3.5 g / L K2HPO4, 0.35 g / L MgSO4·7H2O, 0.05 g / L MnSO4·7H2O, and 80 mL Tween.

[0096] The experimental method and results of this embodiment are as follows:

[0097] 1. Experimental Animals

[0098] 60 6-week-old SPF male BALB / c mice were placed in a constant temperature and humidity incubator for mice at 25±2℃, following a 12h light / dark cycle. The relative humidity was 40±10% during the entire study, and the room temperature was maintained at 21-23℃. Feed and water were fed without restriction 1 week before the start of the experiment. During the feeding period, the mice were free to find food and drink water, and the feed was changed every 2 days.

[0099] 2. Animal Grouping

[0100] After one week of adaptive feeding of mice as mentioned above, 35 mice were randomly divided into 5 groups, with 7 mice in each group: normal control group (Control), model group (Ethanol); probiotic CL group (mice were fed with a composite bacterial solution of JJ-100 and LR, with the ratio of the number of live bacteria of JJ-100 and LR being 1:1); probiotic CL2 group (mice were fed with a composite bacterial solution of JJ-100 and LR, with the ratio of the number of live bacteria of JJ-100 and LR being 5:1); probiotic CL3 group (mice were fed with a composite bacterial solution of JJ-100 and LR, with the ratio of the number of live bacteria of JJ-100 and LR being 1:5); probiotic CB group (mice were fed with Clostridium butyricum JJ-100 bacterial solution), and probiotic LR group (mice were fed with Lactobacillus rhamnosus LR bacterial solution).

[0101] 3. Animal modeling and intervention methods are as follows:

[0102] Control group: normal feeding + daily gavage of pure water 10ml / kg body weight for five consecutive weeks;

[0103] Model group: normal feeding + daily oral administration of 10 ml / kg body weight of pure water for five consecutive weeks;

[0104] Probiotic intervention group: normal feeding + daily intragastric administration of probiotic solution (total bacterial count of 5×10 7 CFU / day / animal), for five consecutive weeks;

[0105] From the second week, all mice except the control group were gavaged with 10 ml / kg 52% (v / v) ethanol once a day until the end of the experiment.

[0106] 4. Indicator analysis

[0107] After the experiment, all mice were fasted for 12 hours and then killed. Mouse serum was separated from blood by centrifugation and stored at -80°C; cecal contents were collected in sterile tubes and stored at -80°C; small intestinal tissue was removed and fixed in 4% paraformaldehyde solution; liver was separated and weighed, and its weight relative to body weight was calculated as follows: liver index (mg / g) = liver weight (mg) / body weight (g), and the liver was stored in a -80°C refrigerator.

[0108] Preparation of mouse serum: Collect blood samples and place them in a test tube without anticoagulant; place them at room temperature for 1 to 2 hours, wait for the blood to coagulate, and centrifuge them at 2000 to 3000 rpm for 5 to 10 minutes. The supernatant is the serum.

[0109] Preparation of 10% mouse liver tissue homogenate: Accurately weigh the liver tissue, add 9 times the volume of normal saline at a ratio of weight (g): volume (mL) = 1:9, mechanically homogenize in an ice water bath, centrifuge at 2500 rpm for 10 minutes, and the supernatant is the liver tissue homogenate.

[0110] The above supernatants were subjected to liver function tests (aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels), lipid content determination (determination of triglyceride (TG) and total cholesterol (T-CHO) levels), oxidative stress level detection (catalase (CAT) and superoxide dismutase (SOD), and inflammation detection (tumor necrosis factor (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6) and myeloperoxidase (MPO) activity).

[0111] The test results were statistically analyzed using GraphPad. Since the data of the probiotic group CL2 and the probiotic group CL3 were consistent with the probiotic group CL, only the data of the probiotic group CL were listed in this example. Compared with the control group or the model group, **** represents p<0.0001, *** represents p<0.001, ** represents p<0.01, and * represents p<0.05.

[0112] Liver function test results such as Figure 3 As shown in the figure, compared with the control group, the serum ALT level of the model group mice was higher ( Figure 3 A) and AST( Figure 3B) activity increased significantly, indicating that acute alcohol exposure can damage normal liver function (ALT and AST activity levels in mouse serum are often used to detect liver damage). However, after intervention with probiotics JJ-100 (CB group) or LR (LR group) or a combination of the two (CL group), ALT and AST activity in mouse serum decreased significantly, with the CL group showing the best effect. In addition, alcohol can damage the small intestinal barrier function, and lipopolysaccharide enters the blood, increasing the activity of lipopolysaccharide in the blood. Figure 3 As shown in C, compared with the control group, the level of lipopolysaccharide in the serum of the mice in the model group was significantly increased, and it tended to be normal after intervention with the probiotics JJ-100 or LR or a combination of the two, and the effect of the CL group was the best.

[0113] The damage of alcohol to the liver is also reflected in the accumulation of fat. The main manifestation of alcoholic fatty liver is the accumulation of triglycerides and cholesterol. Figure 4 As shown in the figure, compared with the control group, the serum TG and TC levels of mice in the model group were significantly increased, indicating that acute alcohol exposure caused fat accumulation in mice. After intervention with the probiotics JJ-100 or LR or a combination of the two (CL group), the serum TG levels of mice were significantly reduced, and the CL group had the best effect ( Figure 4 A). The serum TC level of mice in the single microbial preparation group treated with probiotics JJ-100 or LR did not decrease significantly, while the serum TC level of mice in the probiotic preparation group treated with JJ-100 and LR was significantly suppressed, indicating that the composite probiotic preparation of the present invention can effectively inhibit the increase in lipid levels in mice caused by acute alcohol, and play a role in reducing lipids and protecting the liver ( Figure 4 B).

[0114] Oxidative stress test results Figure 5 As shown in the figure, compared with the control group, the liver SOD ( Figure 5 A) and CAT( Figure 5 B) activity was significantly reduced (p<0.05), indicating that oxidative stress occurred in the liver. However, after intervention with probiotics JJ-100 or LR or a combination of JJ-100 and LR (CL group), there were improvements to varying degrees, among which the CL group was the best in alleviating liver oxidative stress in mice with acute alcohol poisoning.

[0115] The results of liver inflammation test are as follows Figure 6 As shown, the expression of TNF-α in mouse liver was measured. Figure 6 A) IL-4 ( Figure 6 B) IL-6 ( Figure 6 C) and MPO( Figure 6D) Levels. Compared with the control group, the activity levels of TNF-α, IL-4, IL-6 and MPO in the liver of the model group mice were significantly increased, but after intervention with the probiotics JJ-100 or LR or JJ-100 and the combination of the two (CL group), they were reduced to varying degrees, among which the CL group had the best inhibitory effect on liver inflammation.

[0116] The protein expression levels of tight junction protein-related genes in the ileum of each group of mice were further detected. The test results are as follows: Figure 7 Compared with the control group, the expression levels of ZO-1, claudin3 and Occludin proteins in the model group mice decreased significantly after acute alcohol exposure, while the CB group and CL group effectively alleviated this trend, among which the CL group had the best effect.

[0117] Microscopic images of liver sections of mice in each group after H&E staining are shown in the figure below. Figure 8 As shown. It can be seen that the livers of mice in the control group appeared normal, while the liver tissues of mice in the model group were significantly damaged, including fatty degeneration and vacuolar degeneration, and inflammatory cell infiltration, swelling, and deformation were observed in the hepatocytes. Some improvements were observed in the mice in the CB and LR groups, mainly manifested as reduced liver fat vacuoles and reduced inflammatory cell infiltration, and only slight liver fat deformation was observed in the CL group. Histopathological observations of liver sections showed that the composite probiotic agent of the present invention had the best effect in alleviating liver pathological damage in mice exposed to acute alcohol.

[0118] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A probiotic for alleviating acute alcoholic liver damage, characterized in that: The probiotics for alleviating acute alcoholic liver injury are Clostridium butyricum ( Clostridium butyricum ) JJ-100, deposited on January 3, 2025 in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33288.

2. A probiotic for alleviating acute alcoholic liver damage, characterized in that: The strain in the probiotic is the probiotic Clostridium butyricum ( Clostridium butyricum ) JJ-100.

3. The probiotic for alleviating acute alcoholic liver damage according to claim 2, characterized in that: In the probiotics, the total number of viable bacteria is not less than 2×10 9 CFU / g or 2×10 9 CFU / ml.

4. The probiotic for alleviating acute alcoholic liver damage according to claim 2, characterized in that: The probiotics also contain Lactobacillus rhamnosus (CGMCC No. 33287). Lacticaseibacillus rhamnosus )LR.

5. The probiotic for alleviating acute alcoholic liver damage according to claim 4, characterized in that: The ratio of the viable count of the JJ-100 strain to the LR strain is 1:5 to 5:

1.

6. The probiotic for alleviating acute alcoholic liver damage according to claim 4, characterized in that: The total number of viable bacteria of the JJ-100 strain and the LR strain is not less than 2×10 9 CFU / g or 2×10 9 CFU / ml.

7. The probiotic agent for alleviating acute alcoholic liver injury according to claim 2 or any one of claims 4 to 6, characterized in that: The probiotics also include anhydrous glucose and skim milk powder.

8. The probiotic according to any one of claims 2 to 7, characterized in that The dosage form of the probiotics includes probiotics obtained by freeze drying, spray drying, embedding, microencapsulation and other methods and their products.

9. Use of the probiotic according to any one of claims 2 to 7 in the preparation of a medicament for improving or treating acute alcoholic liver injury.

10. The use according to claim 9, characterized in that: The medicine also contains excipients; the excipients include any one of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

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