Mixed strain preparation and application thereof in alcohol damage

Abstract

The invention discloses a mixed strain preparation and application thereof in alcohol damage, and belongs to the technical field of microorganisms. The strain A1H11 provided by the invention has the capability of degrading ethanol; the strain I3D12 has the capability of degrading acetaldehyde. The two lactobacillus plantarum strains are mixed to prepare a mixed strain preparation, so that the weight decrease trend of mice is relatively mild, and the weight gradually recovers to be close to the normal level; and the mixed strain preparation can be used for remarkably reducing the AST level of the model hour and remarkably improving the SOD activity of the mouse, so that the fatty degeneration degree of the mouse is relatively light, and the intestinal villus is relatively dense. Therefore, the alcoholic liver injury is improved from the aspects of efficiently metabolizing ethanol, relieving oxidative injury of the liver, relieving alcoholic fatty liver and the like, and the ethanol is effectively metabolized and the injury of alcohol to the body is relieved by utilizing the coordination effect of the ethanol degrading strain and the acetaldehyde degrading strain.

Description

Technical Field

[0001] The present invention relates to a mixed strain preparation and its use in alcohol injury, belonging to the field of microbial technology. Background Art

[0002] Drinking alcohol is inevitable in daily life, but the harm caused by alcohol to the human body cannot be ignored. The water solubility of ethanol allows it to freely enter and exit cells, causing toxic effects on cells. Alcohol and its metabolite acetaldehyde can cause permanent damage to DNA in cells and inhibit the DNA repair process. Long-term excessive drinking can lead to intestinal ecological disorders, anemia, pancreatitis, cancer, brain diseases, etc. Data shows that approximately 3 million people died globally in 2016 due to the harmful use of alcohol. Excessive drinking can also cause social problems such as traffic accidents, violence, and crime.

[0003] After alcohol is ingested by the human body, it diffuses passively into the blood in the gastrointestinal tract. In the stomach, alcohol undergoes its first metabolism (about 10% is absorbed), and most of the alcohol is absorbed into the blood in large amounts in the duodenum and then enters the liver for metabolism (70% is metabolized). After alcohol enters the liver, it is mainly metabolized in two steps by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) into non-toxic acetate and water. Due to different internal and external motivations and social cognitive reasons, there may be significant resistance to alcohol abstinence; therefore, developing anti-alcoholism products that help the body metabolize alcohol may help reduce health problems caused by alcohol intake. Some foods (fruits and vegetables, livestock products, grains, oils and fats), traditional Chinese herbal medicines (Pueraria lobata, Flos Puerariae, Hovenia dulcis), and functional drugs (polysaccharides, polypeptides) have been developed as anti-alcoholism products. Most of these anti-alcoholism products act to actively increase the activity of anti-alcohol enzymes in the intestine and liver, promoting the body's autonomous metabolism to excrete alcohol. Chemical drugs are also used in clinical treatment of alcoholism, such as benzodiazepines, naltrexone, disulfiram, topiramate, but they are not suitable for repeated use due to their addictive properties and side effects. Therefore, there is a need to develop a product that can help the human body metabolize alcohol. The increasing proportion of people who use alcohol harmfully makes the development of anti-alcoholism products more urgent.

[0004] Alcohol degradation mainly depends on two enzymes, ADH and ALDH. Currently, at least 5 classes and 7 genotypes of ADH are known (ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7), among which ADH1B is an important ethanol-metabolizing enzyme in the body and there are functional single nucleotide polymorphism sites in the population. The expression level and allelic diversity of different individuals can affect the activity of alcohol dehydrogenase. There may be certain deficiencies in the alcohol-detoxifying pathway based on the human body itself. On the basis of the body's own alcohol-detoxifying ability, exogenous supplementation of ADH and ALDH enzymes can help the body metabolize a part of alcohol, thereby reducing alcohol absorption. However, pure enzyme preparations cannot be successfully delivered to the intestine and thus cannot quickly decompose alcohol. Therefore, it is urgent to develop a new type of active preparation that can decompose alcohol, reduce the body burden, and relieve alcohol damage. Summary of the Invention

[0005] In view of the above prior art, the present invention provides a mixed strain preparation and its use in alcohol damage, aiming to solve the technical problem in the prior art that there is a lack of a bioactive preparation that can decompose alcohol, reduce the body burden, relieve alcohol damage and directly act on the intestine.

[0006] The first technical solution provided by the present invention is a Lactobacillus plantarum I3D12, which was deposited at the China Center for Type Culture Collection on January 2, 2024, and the strain deposit number is CCTCC NO: M2025004.

[0007] The second technical solution provided by the present invention is a microbial preparation, and the microbial preparation contains the Lactobacillus plantarum I3D12 described in the first technical solution.

[0008] In some embodiments, the bacterial concentration of the Lactobacillus plantarum in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0009] The third technical solution provided by the present invention is a mixed strain preparation, and the mixed strain preparation includes Lactobacillus plantarum A1H11 and the Lactobacillus plantarum I3D12 described in the first technical solution; the Lactobacillus plantarum A1H11 was deposited at the China Center for Type Culture Collection on January 2, 2025, and the strain deposit number is CCTCC NO: M 2025003.

[0010] In some embodiments, the total bacterial concentration of Lactobacillus plantarum A1H11 and Lactobacillus plantarum I3D12 in the mixed strain preparation is at least 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0011] In some embodiments, in the mixed strain preparation, the viable bacteria number ratio of Lactobacillus plantarum A1H11 and Lactobacillus plantarum I3D12 is 0.5:1 to 4:1; preferably, the viable bacteria number ratio of Lactobacillus plantarum A1H11 and Lactobacillus plantarum I3D12 is 1:1.

[0012] The fourth technical solution provided by the present invention is a product, which contains Lactobacillus plantarum I3D12 described in the first technical solution, the microbial preparation described in the second technical solution or the mixed strain preparation described in the third technical solution.

[0013] In some embodiments, the bacterial concentration of the Lactobacillus plantarum in the product is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0014] Furthermore, the bacterial concentration of the Lactobacillus plantarum in the product is at least 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0015] In some embodiments, the product is a food, a drug or a health product.

[0016] In some embodiments, the food is a fermented food containing Lactobacillus plantarum I3D12 described in the first technical solution, the microbial preparation described in the second technical solution, or the mixed strain preparation described in the third technical solution, or the food is a fermented food obtained by fermenting with Lactobacillus plantarum I3D12 described in the first technical solution, the microbial preparation described in the second technical solution or the mixed strain preparation described in the third technical solution.

[0017] In some embodiments, the fermented food includes dairy products, soy products, fruit and vegetable products or other fermented foods containing Lactobacillus plantarum I3D12 or the mixed strain preparation.

[0018] In some embodiments, the dairy products include milk, sour cream, cheese.

[0019] In some embodiments, the fruit and vegetable products include products of one or more of cucumbers, carrots, beets, celery, cabbages and other edible fruits and vegetables.

[0020] In certain embodiments, the fermented food further contains additives selected from one or a combination of two or more of spices, fruit and vegetable juices, flower tea juices, colorants, acidity regulators, preservatives, antioxidants, thickeners, and sweeteners.

[0021] In certain embodiments, the processed form of the fermented food includes solid food, liquid food, or semi-solid food.

[0022] The fifth technical solution provided by the present invention is the application of Lactobacillus plantarum I3D12 described in the first technical solution, the microbial preparation described in the second technical solution, or the mixed strain preparation described in the third technical solution in the preparation of products for relieving and / or treating chronic and / or acute alcoholic liver injury.

[0023] In certain embodiments, the application includes at least one of the following effects:

[0024] (1) Reducing the blood ethanol concentration of an individual;

[0025] (2) Reducing the level of aspartate aminotransferase (AST) in the blood of an individual;

[0026] (3) Increasing the levels of glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and high-density lipoprotein cholesterol (HDL-L) in the liver tissue of an individual;

[0027] (4) Relieving hepatic steatosis in an individual;

[0028] (5) Relieving intestinal injury in an individual.

[0029] In certain embodiments, the product includes food, medicine, or health products.

[0030] In certain embodiments, the medicine further contains a drug carrier and / or pharmaceutical excipients.

[0031] In certain embodiments, the drug carrier includes microcapsules, microspheres, nanoparticles, and / or liposomes.

[0032] In certain embodiments, the pharmaceutical excipients include excipients and / or additives.

[0033] In certain embodiments, the excipients include binders, fillers, disintegrants, and / or lubricants.

[0034] In certain embodiments, the additives include solubilizers, cosolvents, latent solvents, and / or preservatives.

[0035] In certain embodiments, the dosage form of the medicine is powder, granule, capsule, tablet, pill, or oral liquid.

[0036] The sixth technical solution provided by the present invention is the use of Lactobacillus plantarum I3D12 described in the first technical solution, the microbial preparation described in the second technical solution, or the mixed strain preparation described in the third technical solution in the preparation of hangover and liver protection products.

[0037] In some embodiments, the product includes food, medicine or health products.

[0038] In some embodiments, the medicine further contains a pharmaceutical carrier and / or pharmaceutical excipients.

[0039] In some embodiments, the pharmaceutical carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

[0040] In some embodiments, the pharmaceutical excipients include excipients and / or additives.

[0041] In some embodiments, the excipients include binders, fillers, disintegrants and / or lubricants.

[0042] In some embodiments, the additives include solubilizers, cosolvents, latent solvents and / or preservatives.

[0043] In some embodiments, the dosage form of the medicine is powder, granule, capsule, tablet, pill or oral liquid.

[0044] The present invention provides a seventh technical solution, a method for degrading acetaldehyde, which is to introduce Lactobacillus plantarum I3D122 described in the first technical solution, the microbial preparation described in the second technical solution, or the mixed strain preparation described in the third technical solution into an environment containing acetaldehyde to degrade acetaldehyde.

[0045] The eighth technical solution provided by the present invention is the use of Lactobacillus plantarum I3D122 described in the first technical solution, the microbial preparation described in the second technical solution, or the mixed strain preparation described in the third technical solution in the degradation of acetaldehyde.

[0046] The technical effects of the present invention are as follows:

[0047] The present invention provides strains. At alcohol concentrations of 10% and 20% (v / v), the in vitro degradation rates of strain A1H11 at 6 h are 33.7% and 24.2% respectively; at an acetaldehyde concentration of 100 mg / ml, the in vitro degradation rate of strain I3D12 at 6 h is 98.9%. The above two Lactobacillus plantarum strains are mixed to prepare a mixed strain preparation. The weight loss trend of the mice ingesting the mixed strain preparation is relatively mild, and the weight gradually recovers to near the normal level; the blood ethanol level of the mice ingesting the mixed strain preparation is the lowest, showing a significant difference compared with the control group, and it is reduced by 40.8% compared with the control group; the mixed strain preparation significantly reduces the AST level by 43.1%; the SOD activities of the mice ingesting the mixed strain preparation are significantly increased by 36.3% and 47.9% respectively; the degree of steatosis of the mice ingesting the mixed strain preparation is lighter, and the intestinal villi are denser. Therefore, the present invention improves alcoholic liver injury in terms of efficiently metabolizing ethanol, alleviating liver oxidative damage, and alleviating alcoholic fatty liver, and effectively metabolizes ethanol by utilizing the synergistic effect of the above ethanol-degrading strain and acetaldehyde-degrading strain, thereby alleviating the harm of alcohol to the body.

[0048] Biological preservation material

[0049] Lactobacillus plantarum A1H11, with the taxonomic name of Lactobacillus plantarum, was deposited at the China Center for Type Culture Collection on January 2, 2025. The deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2025003.

[0050] Lactobacillus plantarum I3D12, with the taxonomic name of Lactobacillus plantarum, was deposited at the China Center for Type Culture Collection on January 2, 2025. The deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2025004. Description of the drawings

[0051] Figure 1 Shows the ethanol degradation ability of the strain at 10% and 20% (v / v);

[0052] Figure 2 Shows the acetaldehyde degradation ability, autoagglutination ability, and hydrophobicity ability of the strain;

[0053] Figure 3 Shows the animal experiment process;

[0054] Figure 4 Shows the daily food intake and daily weight change of the mice;

[0055] Figure 5 is the blood ethanol content of mice;

[0056] Figure 6 are the ALT content, AST content, triglyceride content, high-density lipoprotein cholesterol (HDLC) content, SOD content, total antioxidant capacity, MDA content, and GSH content in the blood of mice;

[0057] Figure 7 are the HE staining, oil red staining of mouse liver sections, and HE staining of intestinal sections. Detailed implementation mode

[0058] The following is an illustration of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0059] Technical terms:

[0060] The "mixed strain preparation" in the present invention refers to a preparation that is obtained by fully mixing Lactobacillus plantarum A1H11 and Lactobacillus plantarum I3D12 and then administering them to mice before gavage. That is, it is a preparation that is mixed and prepared on-site using "Lactobacillus plantarum A1H11" and "Lactobacillus plantarum I3D12".

[0061] The "slow plus acute alcoholic liver injury" in the present invention refers to the acute deterioration of liver function caused by a large amount of alcohol consumption in a short period or other acute factors on the basis of chronic alcoholic liver disease (such as alcoholic hepatitis, alcoholic cirrhosis). Its clinical manifestations include rapidly progressive jaundice, coagulation dysfunction, ascites, hepatic encephalopathy, etc., and in severe cases, it can develop into acute-on-chronic liver failure (ACLF).

[0062] The "treatment" in the present invention refers to preventing, curing, reversing, weakening, alleviating, minimizing, inhibiting, stopping, and / or halting one or more clinical symptoms of a disease after the onset of the disease.

[0063] The "alcohol detoxification and liver protection" in the present invention refers to helping to accelerate alcohol metabolism, reduce the damage of alcohol to the liver, relieve the discomfort symptoms after drunkenness, and thus protect liver function and physical health through certain substances or methods.

[0064] The "hydrophobicity of strains" in the present invention refers to the ability of the surface of microbial cells to interact with water molecules, specifically manifested as the property of the cell surface to repel water molecules. This property makes the cell more likely to interact with non-polar substances (such as hydrocarbons).

[0065] The "auto-aggregation rate" in the present invention refers to the ability of microbial cells to spontaneously aggregate to form multicellular clumps under static conditions, and is usually used to evaluate the surface characteristics of strains and their functions in aspects such as biofilm formation, adhesion, and colonization.

[0066] Test method:

[0067] 1. The ethanol content is detected by GC-MS

[0068] Gas chromatography conditions: Incubate at 70°C for 5 min, and the stirring speed is 500 rpm. Chromatographic column: TG-WAXMS (30m x 0.25μm x 0.25mm); injection port temperature is 220°C, carrier gas is high-purity helium, flow rate is 1.0 mL / min, split injection, split ratio is 10:1; temperature programming: initial temperature is 35°C, hold for 4 min, then increase to 240°C at a rate of 40°C / min, and hold for 2 min. Mass spectrometry conditions: Ionization mode: E1; emission current: 50 A; electron energy: 70 eV; ion source temperature: 250°C; transfer line temperature: 230°C; ion scan range: 29, 44, 43, 31, 45, 46 m / z.

[0069] 2. The content of acetaldehyde is determined by the 3-methyl-2-benzothiazolinone hydrazone (MBTH) colorimetric method

[0070] After the strain is cultured in MRS liquid medium for 24 h, the bacterial solution is washed and resuspended with normal saline, and the OD600nm of the bacterial solution is adjusted to about 1.3 - 1.5, so that the bacterial solution concentration is 1×10 9 CFU / ml, add a 4.4 mg / L acetaldehyde solution, adjust the acetaldehyde concentration to 100 mg / L, culture at 37°C, take 100 μL of samples at 0, 2, 4, and 6 h respectively, centrifuge with a tabletop mini centrifuge and take the supernatant for detection. Pipette 20 μL of the supernatant sample into a 96-well cell culture plate, add 40 μL of 0.04 g·L-1 MBTH solution, mix and let stand for 20 min; add 40 μL of 0.1 g·L -1 ferric chloride solution, mix and let stand for 10 min; add 100 μL of deionized water, mix well, take the absorbance value at 660 nm and measure it with an enzyme-linked immunosorbent assay (ELISA) reader.

[0071] 3. Refer to the group standard T / CNHFA435—2024 "Test Method for Gastric Juice Tolerance of Probiotic Agents" issued by the China Nutrition and Health Food Association to prepare simulated gastric juice and evaluate the gastric juice tolerance of the strain.

[0072] Take 8.0 mL of electrolyte A solution and 1.0 mL of electrolyte B solution in a beaker, add pepsin equivalent to 4000 U, adjust the pH value with 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution (fasting: 2.0, full stomach: 4.0), make up the volume to 10 mL, mix well and filter through a 0.22 μm sterile filter membrane to prepare simulated gastric juice, which should be used immediately after preparation. The strain is cultured in MRS liquid medium under anaerobic conditions at 37 °C for 24 h, centrifuged at 3000 rpm for 5 min, the supernatant is discarded, and the cells are resuspended in 20 mL of 0.85% normal saline, and the total viable cell count of the bacterial suspension is determined. Take 2.0 mL of simulated gastric juice and 2.0 mL of the sample bacterial suspension in a 5 mL centrifuge tube, vortex and mix well, place the centrifuge tube in a 37 °C constant temperature water bath for incubation (fasting: 0.5 h, full stomach: 3.0 h), and determine the total viable cell count.

[0073] 4. Detection of hydrophobicity and auto-aggregation ability

[0074] Hydrophobicity: Add 1 ml of xylene to 3 ml of the bacterial suspension, mix well. Set up three parallel groups. Let it stand at room temperature for 20 min to separate the organic phase and the aqueous phase. Remove the organic phase, and measure the OD value of the aqueous phase at 600 nm with PBS buffer as the control, denoted as At. Repeat the experiment at least three times.

[0075] Hydrophobicity(%) = [(A0 - At) / A0] × 100%; (Hydrophobicity is divided into low: 0 - 29%, medium: 30% - 59% and high: 60 - 100%). A0 is the initial absorbance, and At is the absorbance after treatment.

[0076] Auto-aggregation ability: Take 4 ml of the adjusted lactic acid bacteria suspension, mix well, and incubate at room temperature for 5 h. Pipette 200 μL of the surface bacterial suspension, do not pipette up and down, and keep the bacterial suspension still. Measure the OD value at 600 nm with PBS buffer as the control, denoted as At. Repeat the experiment three times.

[0077] Auto-aggregation(%) = 1 - At / A0 × 100%. A0 is the initial absorbance, and At is the absorbance after treatment.

[0078] Raw materials used in the examples:

[0079] 1. MRS medium: Tryptone 10 g / L, Beef extract 5 g / L, Yeast extract 4 g / L, Glucose 20 g / L, Tween-80 1 mL / L, Dipotassium hydrogen phosphate 2 g / L, Sodium acetate 5 g / L, Diammonium citrate 2 g / L, Magnesium sulfate 0.2 g / L, Manganese sulfate 0.05 g / L. Make up the volume with deionized water and sterilize at 115 °C for 20 min.

[0080] 2. Electrolyte A solution: Weigh 0.064 g of potassium chloride, 0.015 g of potassium dihydrogen phosphate, 0.263 g of sodium bicarbonate, 0.345 g of sodium chloride, 0.003 g of magnesium chloride hexahydrate, 0.006 g of ammonium carbonate, 1.5 g of tryptone, and 0.05 g of L-cysteine hydrochloride monohydrate. Add 95 mL of distilled water and dissolve thoroughly. Adjust the pH to 3.0 with concentrated hydrochloric acid or 1 mol / L sodium hydroxide solution, make up the volume to 100 mL, and sterilize at 121 °C under high pressure for 15 min.

[0081] Electrolyte B solution: Weigh 0.022 g of calcium chloride dihydrate, dissolve it in water and make up the volume to 100 mL, and sterilize at 121 °C under high pressure for 15 min.

[0082] 3. C57BL / 6 male mice are sourced from SPF (Beijing) Biotechnology Co., Ltd.

[0083] 4. Lieber-DeCarli liquid diet is sourced from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.

[0084] Example 1 Screening and Obtaining of Strains

[0085] Enrich strains from pickled Chinese cabbage, pickles, enzyme fermented vegetables, and aged altar yellow rice wine. With the help of a high-throughput fully automatic strain screening workstation (Freedom EVO-2 200BASE, Tecan Group Ltd., Switzerland), dispense the high-throughput detection medium into 96-well plates, 200 μL per well. Use a high-throughput fully automatic microbial colony picking workstation (QPix 420, Molecular Devices, USA) to take pictures and automatically identify the colonies on the plate, and pick single colonies on the plate into the high-throughput detection medium in the 96-well plates (the detection medium contains 5% (v / v) ethanol, 7.5% (v / v) WST-8, 6.8 g / L of disodium hydrogen phosphate, 3.0 g / L of potassium dihydrogen phosphate, 1.0 g / L of ammonium chloride, 0.5 g / L of sodium chloride, and 5.0 g / L of glucose). The inoculated 96-well plates are anaerobically cultured at 37 °C for 4 h, 8 h, and 12 h, and the absorbance is measured with an enzyme-linked immunosorbent assay (ELISA) reader (Epoch2, BioTek, USA) (dual wavelengths Lm1 = 450, Lm2 = 600 nm). After 12 h, the strains grow appropriately. Take 100 μL of the bacterial liquid from the wells with higher absorbance and spread it on MRS solid plates, anaerobically culture at 37 °C, and after single colonies grow, streak and purify, and store at -80 °C by the glycerol method.

[0086] By setting specific absorbance thresholds - the absorbance value ≥ 0.6 at 4 h, the absorbance value ≥ 1.0 at 8 h, and the absorbance value ≥ 1.5 at 12 h, 34 strains were successfully screened out and preserved.

[0087] I. Obtaining Ethanol-Degrading Strains

[0088] The strain was cultured in MRS liquid medium to obtain a seed solution. The OD600nm of the seed solution was adjusted to 1.0, and it was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), and cultured at 37 °C for 24 h. The bacterial solution was centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and it was resuspended with 0.85% physiological saline, and the concentration of the bacterial solution was adjusted to 1×10 9 CFU / ml to obtain a bacterial solution.

[0089] The above-mentioned bacterial solution was added with 10% (v / v) and 20% (v / v) ethanol and cultured at 37 °C for 6 h. After mixing, 1 ml of the mixed solution was centrifuged at 4000 rpm for 5 min, and the supernatant was taken to detect the remaining ethanol content.

[0090] As Figure 1 (a) shows that A1H11 shows a strong degradation ability for 10% (v / v) ethanol. After culturing under anaerobic conditions for 6 hours, the degradation ability is 33.7%. Figure 1 (b) Further tests showed that A1H11 also has a strong degradation ability for 20% (v / v) ethanol. After culturing under anaerobic conditions for 6 hours, their degradation abilities are 24.2% respectively.

[0091] II. Obtaining of acetaldehyde-degrading strains

[0092] After culturing 16 strains to be screened in MRS liquid medium for 24 h, the bacterial solution was washed and resuspended with physiological saline, and the OD600nm of the bacterial solution was adjusted to about 1.3 - 1.5, so that the concentration of the bacterial solution was 1×10 9 CFU / ml, 4.4 mg / L acetaldehyde solution was added, the acetaldehyde concentration was adjusted to 100 mg / L, and it was cultured at 37 °C. 100 μL of samples were taken at 0, 2, 4, and 6 h respectively, and after centrifuging with a desktop mini-centrifuge, the supernatant was taken to detect the acetaldehyde content. As Figure 2 (a) shows the degradation ability of I3D12 for 100 mg / ml acetaldehyde. The remaining amount of acetaldehyde after 2 hours is 25.8%, it drops to 1.5% after 4 hours, and only 1.1% remains after 6 hours.

[0093] Example 2 Evaluation of gastric juice tolerance of strains

[0094] The A1H11 and I3D12 strains obtained by screening in the examples were cultured in MRS liquid medium under anaerobic conditions at 37 °C for 24 h, centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and it was resuspended with 20 mL of 0.85% physiological saline to determine the total viable count of the bacterial solution. Take 2.0 mL of simulated gastric juice and 2.0 mL of the sample bacterial solution into a 5 mL centrifuge tube, vortex and mix well, place the centrifuge tube in a 37 °C constant temperature water bath for culture (fasting for 0.5 h, full stomach for 3.0 h), and determine the total viable count. The results are shown in Table 1 and Figure 4 as shown.

[0095] Table 1 Resistance to gastric acid

[0096]

[0097] As shown in Table 1, A1H11 exhibited resistance to gastric juice and was able to achieve a certain degree of proliferation (the survival rate reached 106%). In contrast, the numbers of I3D12 both decreased in the fed state. As Figure 2 shown, the auto-aggregation ability of A1H11 first increased and then decreased within 1 to 4 hours, while the auto-aggregation ability of I3D12 gradually increased with time. Among the strains, A1H11 had the strongest auto-aggregation ability, and the auto-aggregation rates at 3 hours and 4 hours reached 17.88 ± 1.59% and 14.45 ± 2.24% respectively. The hydrophobicity of the two strains was relatively weak (both below 20%).

[0098] Example 3: Effects of strains on acute-on-chronic alcoholic liver injury

[0099] I. Construction and grouping of animal models

[0100] Healthy SPF-grade C57BL / 6 male mice aged 8 - 10 weeks and weighing over 20 g were selected and housed in an SPF-grade barrier environment. The breeding temperature was (24.0 ± 2.0) °C, the environmental relative humidity was 40% - 60%, and the 12h light cycle was carried out according to the actual situation of the animal center. The mice were randomly numbered by ear punching. After the animals were housed, they were adaptively fed for 3 - 5 days. The mice were randomly divided into 5 groups, with 10 mice in each group. They were the blank group, the control group, probiotic group I (A1H11 group), probiotic group II (I3D12 group), and probiotic group III (mixed strain preparation: A1H11 + I3D12 (1:1) group).

[0101] An acute-on-chronic alcoholic liver injury mouse model (Gao-bing model) was constructed as Figure 3 shown. First, a 5-day Lieber-DeCarli liquid diet adaptation period was given, and the alcohol transition period in the experimental groups (probiotic group I, probiotic group II, probiotic group III) was from 0% to 5%. Then, the control group and the experimental groups were fed with a liquid diet containing 5% (V / V) alcohol for 10 days, and the blank group was fed with an isocaloric Lieber-DeCarli liquid control diet. The control group and the experimental groups were respectively gavaged with 0.85% (w / v) normal saline and probiotics every day, with a gavage dose of 0.2 mL / 25 g, and the blank group was not treated. On the last day, at 7 - 9 am, 0.85% (w / v) normal saline and probiotics were gavaged respectively, and 1 hour later, a high-concentration alcohol (31.5% v / v) gavage was carried out once, with a dose of 5 g / kg. 1 hour after alcohol gavage, the mice were anesthetized with isoflurane, and 1 mL of blood was taken from the eye socket into a 1.5 mL EP tube, and then the mice were sacrificed by cervical dislocation after blood collection.

[0102] Control group liquid diet formula (g / L DIET): casein 41.4, L-cystine 0.5, DL-methionine 0.3, corn oil 8.5, olive oil 8.4, safflower oil 2.7, maltodextrin 135.2, cellulose 10, salt mixture 8.75, vitamin mixture 2.5, choline bitartrate 0.53, yellow pigment gum 3. The control group liquid diet contains 1.0 Kcal / ml, of which 16.7% comes from fat, 64% comes from carbohydrates, and 19.3% comes from protein. Weigh 221.8 g of the diet, make up to 1 L with water, store at 4 °C, and use it up within three days.

[0103] Alcohol group liquid diet (Lieber-DeCarli liquid diet of the experimental group) formula (g / L DIET): casein 41.4, L-cystine 0.5, DL-methionine 0.3, corn oil 8.5, olive oil 28.4, safflower oil 2.7, maltodextrin 25.6, cellulose 8.75, salt mixture 2.5, vitamin mixture 0.53, choline bitartrate 3, yellow pigment gum 41.4. The alcohol group liquid diet contains 1.0 Kcal / ml, of which 35% comes from fat, 11% comes from carbohydrates, 18% comes from protein, and 36% comes from alcohol. Weigh 132.18 g of the alcohol group liquid diet, add 57.3 mL of 95% ethanol, make up to 1 L with water, store at 4 °C, and use it up within three days.

[0104] II. Detection of indicators of slow plus acute alcoholic liver injury

[0105] Centrifuge the blood sample at 3000 rpm for 15 min, take the upper plasma, and store it at -80 °C. The blood ethanol content of mice was detected by GC-MS. The blood ethanol content of mice was detected by GC-MS, and the results are as Figure 5 shown. The significant difference in blood ethanol levels between the blank group and the control group (P < 0.0001) confirmed the successful construction of the model. The blood ethanol level of the probiotic group III mice was the lowest, with a significant difference compared with the control group (P = 0.0011), and it decreased by 40.8% compared with the control group.

[0106] The AST and ALT contents in the blood of mice were detected according to the test kits for aspartate aminotransferase (glutamic oxaloacetic transaminase / AST / GOT) and alanine aminotransferase (glutamic pyruvic transaminase / ALT / GPT).

[0107] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive markers for evaluating liver injury. There was no significant difference in the ALT levels between the probiotic group and the control group. However, compared with the control group, the AST level in the probiotic group III was significantly reduced (P < 0.05), with a decrease of 43.1%.

[0108] After the mice were sacrificed, the liver and small intestine were dissected. 0.1 g of liver tissue was weighed and placed in 1.0 mL of ice-cold physiological saline. It was homogenized thoroughly in a homogenizer to prepare a 10% liver tissue homogenate. According to the instructions of the triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-L), total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD) kits and glutathione (GSH) kits of Nanjing Jiancheng Bioengineering Co., Ltd., the liver tissue indexes of each group of mice were measured, and the results were as Figure 6 shown.

[0109] Compared with the control group, the GSH levels in the probiotic II group (P < 0.01) and the probiotic III group (P < 0.05) were significantly increased, while no significant difference was observed in the probiotic I group; superoxide dismutase (SOD) activity is an important index reflecting the antioxidant capacity of the body. Compared with the control group, the SOD activities in the probiotic I group (P < 0.05) and the probiotic III group (P < 0.005) were significantly increased, with increases of 36.3% and 47.9% respectively; malondialdehyde (MDA) is an index reflecting the degree of lipid peroxidation in the body. Although the MDA level in the probiotic treatment group was relatively low, there was no significant difference compared with the blank group and the control group; the probiotic III group effectively improved the level of high-density lipoprotein cholesterol in the liver (P < 0.05), and there was no significant difference compared with the blank group. Probiotic treatment did not significantly improve the accumulation of cholesterol and triglyceride in the liver.

[0110] The fixed liver and intestinal tissues with 4% paraformaldehyde solution were gradually dehydrated through a series of alcohol concentration treatments, then treated with xylene and embedded in paraffin, and cut with a microtome. The prepared 5-μm-thick paraffin sections were stained with hematoxylin-eosin (HE), Oil Red O and Masson staining respectively, and the damage of liver and intestinal tissue cells of each group of mice was observed under a microscope. The results were as Figure 7 shown.

[0111] Significant macrovesicular steatosis occurred in the liver of the control group mice. Lipid droplets were obvious in the cytoplasm of hepatocytes. A large number of orange-red lipid droplets were diffused in the Oil Red sections. The arrangement of hepatocytes was disordered, the cytoplasm was reduced, the nucleus was squeezed to the eccentric position by lipid droplets, the boundaries of hepatocytes were blurred, the number of hepatic sinusoids was reduced, and there was infiltration of inflammatory cells. The alcoholic fatty liver model was successfully constructed. Under the intervention of probiotics, the liver damage of the experimental group mice was alleviated, and the steatosis changed from macrovesicular to microvesicular. At the same time, the disordered arrangement of liver cells in the experimental group mice was improved, and the hepatic sinusoids were clearer. Compared with the other two groups, the probiotic III group showed a more obvious protective effect and a lighter degree of steatosis.

[0112] The pathological damage of the mouse intestine is as Figure 7 shown in the figure. In the blank group, the intestinal glands of the mice were arranged orderly, the epithelial layer was intact, and the mucosal surface was normal. The intestinal tissues of the alcohol control group of mice showed obvious morphological disorders, and the intestinal villi and crypt structures were severely damaged. The ability of the intestinal mucosa to clear acetaldehyde was weak, and the high concentration of acetaldehyde further aggravated the damage of the intestinal barrier, including the disintegration of tight junctions and adhesion junctions between epithelial cells. The intake of probiotics showed a certain protective effect on the intestinal damage of mice. In the probiotic group I, compared with the blank group, some goblet cells were missing on the upper part of the intestinal villi of the mice, but compared with the control group, the crypt structure remained relatively intact. Although the probiotic group III also showed certain intestinal villi damage, the intestinal villi were denser, which might be because the supplementation of probiotics promoted the increase in the height and depth of the villi. Due to the significant degradation effect on acetaldehyde in the probiotic group II, the intestinal mucosa was less damaged by acetaldehyde, so the intestinal morphology was clearer, and the intestinal villi and crypt structures were also more complete.

[0113] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Lactobacillus plantarum I3D12, characterized in that, It was deposited at the China Center for Type Culture Collection on January 2, 2024, and the strain deposit number is CCTCC NO: M 2025004.

2. A microbial preparation containing the Lactobacillus plantarum I3D12 described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The bacterial concentration of the Lactobacillus plantarum in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A mixed strain preparation, characterized in that The mixed strain preparation includes Lactobacillus plantarum A1H11 and the Lactobacillus plantarum I3D12 described in claim 1; the Lactobacillus plantarum A1H11 was deposited at the China Center for Type Culture Collection on January 2, 2025, and the strain deposit number is CCTCC NO: M 2025003.

5. The mixed strain preparation according to claim 4, wherein, The total bacterial concentration of Lactobacillus plantarum A1H11 and Lactobacillus plantarum I3D12 in the mixed strain preparation is at least 1×10 9 CFU / mL or 1×10 9 CFU / g.

6. The mixed strain preparation according to claim 4, wherein In the mixed strain preparation, the viable count ratio of Lactobacillus plantarum A1H11 to Lactobacillus plantarum I3D12 is 0.5:1 to 4:

1.

7. A product, characterized in that, The product contains the Lactobacillus plantarum I3D12 described in claim 1, the microbial preparation described in claim 2 or 3, or the mixed strain preparation described in any one of claims 4 to 6.

8. Use of the Lactobacillus plantarum I3D12 described in claim 1, the microbial preparation described in claim 2 or 3, or the mixed strain preparation described in any one of claims 4 to 6 in the preparation of a medicament for relieving and / or treating chronic and / or acute alcoholic liver injury.

9. The application according to claim 8, characterized in that, The use includes at least one of the following effects: (1) Reducing the blood ethanol concentration of an individual; (2) Reducing the level of aspartate aminotransferase in the blood of an individual; (3) Increasing the levels of glutathione, superoxide dismutase, malondialdehyde, and high-density lipoprotein cholesterol in the liver tissue of an individual; (4) Relieving hepatic steatosis in an individual; (5) Relieving intestinal injury in an individual.

10. Use of the Lactobacillus plantarum I3D12 described in claim 1, the microbial preparation described in claim 2 or 3, or the mixed strain preparation described in any one of claims 4 to 6 in the preparation of an anti-hangover and liver-protecting medicament.

11. A method for degrading acetaldehyde, which is to inoculate the Lactobacillus plantarum I3D12 described in claim 1, the microbial preparation described in claim 2 or 3, or the mixed strain preparation described in any one of claims 4 to 6 into an environment containing acetaldehyde to degrade acetaldehyde.

12. Use of the Lactobacillus plantarum I3D12 described in claim 1, the microbial preparation described in claim 2 or 3, or the mixed strain preparation described in any one of claims 4 to 6 in the degradation of acetaldehyde.

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