Plant lactobacillus with effects of dispelling effects of alcohol, protecting liver and restoring consciousness and application of plant lactobacillus

Abstract

The invention relates to a plant lactobacillus with effects of dispelling effects of alcohol, protecting the liver and restoring consciousness and application of the plant lactobacillus. The plant lactobacillus is a plant lactobacillus BGI-J9 strain, and the preservation number of the plant lactobacillus is CCTCC NO: M 20242630. The invention further discloses a preparation method of the plant lactobacillus with the effects of dispelling effects of alcohol, protecting the liver and restoring consciousness and application of the plant lactobacillus with the effects of dispelling effects of alcohol, protecting the liver and restoring consciousness. The strain has excellent tolerance to gastric acid, intestinal juice and cholate in a gastrointestinal tract system, can survive in the gastrointestinal tract, and provides a basis for continuously playing a probiotic effect; the compound has excellent inhibition capability on common intestinal pathogens; free radicals can be removed, and oxidative damage to the body is reduced; good alcohol metabolism enzyme synthesis capability and alcohol metabolism promoting potential are realized; the strain also has an excellent improvement effect on alcohol-damaged mice, specifically, the metabolism of ethanol and acetaldehyde in a body is accelerated, the occurrence time of drunkenness is obviously delayed, and the sobering time is shortened; intestinal injury and systemic inflammation caused by alcohol are improved; liver oxidative stress caused by alcohol is improved, and the occurrence and development process of alcoholic fatty liver is delayed; and cognitive function impairment caused by alcohol is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of new strain development and application, and relates to a Lactobacillus plantarum with alcohol-relieving, liver-protecting and brain-awakening effects and an application thereof. Background Art

[0002] Excessive drinking can lead to acute alcohol poisoning, which may cause multiple organ damage, such as liver damage, intestinal barrier damage, and neuroinflammation. Under normal metabolic conditions, ethanol is first converted into acetaldehyde by alcohol dehydrogenase (ADH) in the liver. Acetaldehyde is further converted into acetic acid by acetaldehyde dehydrogenase (ALDH), and finally decomposed into carbon dioxide and water and excreted from the body.

[0003] However, people who carry the ALDH2 gene mutation have significantly reduced acetaldehyde detoxification efficiency. As a primary carcinogen, excessive accumulation of acetaldehyde can cause vasodilation and tachycardia, and in the long term increase the risk of esophageal cancer and liver cancer. Drinking a lot of alcohol in a short period of time or drinking on an empty stomach, the rapid absorption of ethanol exceeds the body's ALDH metabolic load, which can also directly lead to acetaldehyde accumulation. On the other hand, the alcohol metabolism process produces a large amount of reactive oxygen species (ROS), which damage lipids, proteins and DNA, leading to liver cell death or abnormal proliferation, aggravating liver damage, and may cause fatty liver and cirrhosis in the long run, further increasing the risk of liver cancer. At the same time, ethanol and its metabolites, acetaldehyde, reactive oxygen species, etc., can also cause damage to other organs and systems in the body, such as damaging the intestinal barrier and causing neuroinflammation. Effective prevention or control measures are urgently needed to reduce alcohol damage.

[0004] In order to prevent and improve the damage to the body caused by drinking, current medical research and clinical practice have formed a multi-dimensional intervention strategy. In terms of drug intervention, for people carrying ALDH2 gene mutations, the efficiency of acetaldehyde metabolism can be improved by supplementing ALDH2 coenzymes (such as NAD+ precursors) or small molecule activators (such as Alda-1). Metadoxine, as an ethanol metabolism regulating drug, can accelerate the metabolism of ethanol and acetaldehyde, while naloxone, commonly used in emergency departments, shortens the coma time by antagonizing central nervous system inhibition. At the organ protection level, antioxidants such as silymarin and glutathione can significantly reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of alcoholic liver damage. In terms of dietary intervention, flavonoids in kudzu extract (such as puerarin) can inhibit ADH activity and prolong the incubation period of drunkenness. Curcumin enhances the activity of liver detoxification enzymes by activating the Nrf2 pathway and reduces the generation of harmful products such as free oxygen (ROS). Furthermore, some genetically engineered probiotics are designed to express human alcohol dehydrogenase (hADH) in the intestine. This highly efficient expression of the alcohol-degrading enzyme can achieve beneficial effects such as reduced alcohol absorption, prolonged alcohol tolerance, and shortened recovery time after drinking. These interventions work synergistically from multiple perspectives, including metabolic regulation, organ protection, and nutritional support, providing targeted solutions for individuals with different genetic backgrounds and levels of impairment.

[0005] While existing technologies have improved the damage caused by alcohol consumption to some extent, they still have many shortcomings. Metadoxine, a drug with a clear function of regulating alcohol metabolism, can accelerate ethanol metabolism, but it may increase the liver burden in patients with chronic liver disease. ALDH2 agonists (such as Alda-1) are only effective in people with ALDH2 mutations and are ineffective in those without mutations. Their long-term safety is insufficiently supported, posing safety risks.

[0006] While botanical ingredients are popular in the hangover treatment market, their dose-response relationships remain understudied. For example, kudzu root extract requires a specific blood concentration to inhibit ADH. Furthermore, there are no standardized dose-response specifications for kudzu root extracts due to varying origins and extraction methods. Excessive use can even lead to abnormal liver enzymes. Curcumin also has numerous drawbacks, including low bioavailability and gastrointestinal irritation.

[0007] As a breakthrough achievement of synthetic biology technology, there are currently no clear laws and regulations for the use of genetically engineered probiotics in dietary supplements, and there is a lack of specific basis for related manufacturing and supervision, which greatly restricts their transformation from scientific research to industry. In terms of application, genetically engineered probiotics also have many limitations, including unstable wake-up effects on people with different genetic differences. For example, the intestinal colonization efficiency of acetaldehyde dehydrogenase-activating engineered probiotics is significantly affected by individual bacterial flora differences, and their functional expression is also easily affected by fluctuations in the host intestinal environment (such as pH and oxygen content), which may cause abnormal activation of metabolic pathways. It is worth noting that the unexpected metabolites produced by engineered bacteria may induce metabolic poisoning, and there is still a lack of long-term follow-up data on their safety of use.

[0008] Therefore, a safe and effective method for preventing or treating body damage caused by drinking is urgently needed. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the present invention aims to provide a Lactobacillus plantarum having the effects of sobering up, protecting the liver and refreshing the brain, and its application.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a Lactobacillus plantarum having the effects of sobering up, protecting the liver and refreshing the brain. The Lactobacillus plantarum is the Lactobacillus plantarum BGI-J9 strain, with a preservation number of CCTCC NO: M20242630 and a preservation date of November 25, 2024 (the strain is currently named Lactiplantibacillus plantarum using a new classification).

[0012] The present invention isolates and preserves a new Lactobacillus plantarum strain from traditional fermented yogurt in Inner Mongolia, and names it the BGI-J9 strain. The strain has excellent tolerance to gastric acid, intestinal fluid, and bile salts in the gastrointestinal system and can survive in the gastrointestinal tract, providing a basis for continuously exerting its probiotic effects. The strain has excellent inhibitory ability against common intestinal pathogens, that is, it has the potential to regulate the flora by reducing harmful intestinal bacteria. The strain has strong antioxidant capacity, can scavenge free radicals, and reduce oxidative damage to the body. The strain has good ability to synthesize alcohol metabolic enzymes (ethanol dehydrogenase and acetaldehyde dehydrogenase), and therefore also has good potential to promote alcohol metabolism.

[0013] The BGI-J9 strain can achieve the beneficial effects of alcohol detoxification, liver protection, and brain refreshment through multiple pathways, including synthesizing and expressing alcohol-detoxifying enzymes, improving the body's antioxidant capacity, improving intestinal permeability, and inhibiting inflammation. This beneficial effect has been verified in studies of alcohol-damaged mouse models, specifically as follows: (1) effectively reducing the levels of ethanol and acetaldehyde in the blood, accelerating the metabolism of ethanol and acetaldehyde in the body, significantly delaying the onset of drunkenness, and shortening the time to sobering up; (2) improving intestinal damage and systemic inflammation caused by alcohol; (3) improving alcohol-induced liver oxidative stress and delaying the development of alcoholic fatty liver; and (4) improving alcohol-induced cognitive impairment.

[0014] In addition, Lactobacillus plantarum has a long history of application both domestically and internationally and is included in the "List of Bacteria Suitable for Food Use." It has good safety and no side effects when taken long-term. Therefore, the BGI-J9 strain of the present invention is highly safe when used to prepare products with related efficacy. This invention provides a new direction for preventing or treating alcohol-related body damage, and its application in food, health products, and pharmaceuticals holds broad industrial prospects.

[0015] In a second aspect, the present invention provides a bacterial agent having the effects of sobering up, protecting the liver and refreshing the brain, wherein the strain in the bacterial agent includes the BGI-J9 strain described in the first aspect.

[0016] Preferably, the dosage form of the bacterial agent includes liquid preparation, powder, tablet, granule or capsule.

[0017] The dosage form of the bacterial agent involved in the present invention is not limited, including the most commonly used liquid preparations, powders, or further prepared capsules, tablets or granules.

[0018] Preferably, the number of viable bacteria of the BGI-J9 strain in the microbial agent is not less than 1×10 7 CFU / mL or 1×10 7 CFU / g, for example 1×10 7 CFU / mL, 5×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL, 5×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 5×10 9 CFU / mL, 1×10 10 CFU / mL, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0019] Preferably, the bacterial agent further contains excipients, which include any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants or buffers.

[0020] Preferably, the bacterial agent further contains a protective agent, which includes any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, sodium alginate, sucrose, lactose, trehalose, sorbitol or xylitol.

[0021] In the present invention, when the bacterial agent is a liquid preparation, it is prepared by a method comprising the following steps:

[0022] The BGI-J9 strain is inoculated into a culture medium and activated and fermented in sequence to obtain a fermentation broth; the fermentation broth is centrifuged and resuspended in a solvent to obtain a BGI-J9 bacterial suspension, namely the liquid preparation.

[0023] In the present invention, when the bacterial agent is a powder, it is prepared by a method comprising the following steps:

[0024] The BGI-J9 strain is inoculated into a culture medium and activated and fermented in sequence to obtain a fermentation liquid; the fermentation liquid is centrifuged, mixed with a protective agent, and then freeze-dried to obtain BGI-J9 bacterial powder, namely the powder.

[0025] In a third aspect, the present invention provides use of the Lactobacillus plantarum described in the first aspect or the bacterial agent described in the second aspect in the preparation of a microecological preparation for repairing alcoholic damage.

[0026] Preferably, the alcoholic injury includes any one of alcoholic liver injury, alcoholic intestinal injury or alcoholic brain nerve injury, or a combination of at least two of them.

[0027] Preferably, the alcoholic liver damage includes alcohol-induced liver oxidative stress and liver fat accumulation.

[0028] Preferably, the alcoholic intestinal damage includes alcohol-induced intestinal mucosal damage, increased intestinal permeability and systemic inflammation induced by alcohol.

[0029] Preferably, the alcoholic brain nerve damage includes alcohol-induced neurocognitive dysfunction.

[0030] In a fourth aspect, the present invention provides use of the Lactobacillus plantarum described in the first aspect or the bacterial agent described in the second aspect in the preparation of an intestinal pathogen inhibitor.

[0031] Preferably, the intestinal pathogens include any one or a combination of at least two of Escherichia coli, Staphylococcus aureus, Enterobacter cloacae or Pseudomonas aeruginosa.

[0032] According to the research results of the present invention, the BGI-J9 strain can inhibit Escherichia coli, Staphylococcus aureus, Enterobacter cloacae or Pseudomonas aeruginosa at the in vitro level, that is, the BGI-J9 strain or its bacterial agent can be made into an intestinal pathogen inhibitor for use in related experimental research.

[0033] In a fifth aspect, the present invention provides use of the Lactobacillus plantarum described in the first aspect or the bacterial agent described in the second aspect in the preparation of a free radical scavenger.

[0034] Preferably, the free radical includes any one of DPPH free radical, ABTS free radical or hydroxyl free radical, or a combination of at least two of them.

[0035] According to the research results of the present invention, the BGI-J9 strain can scavenge DPPH free radicals, ABTS free radicals or hydroxyl free radicals at the in vitro level, that is, the BGI-J9 strain or its bacterial agent can be made into a free radical scavenger for use in related experimental research.

[0036] In a sixth aspect, the present invention provides use of the Lactobacillus plantarum described in the first aspect or the bacterial agent described in the second aspect in the preparation of a product that improves alcohol metabolic activity in the body.

[0037] Preferably, the product comprises food, health product or medicine.

[0038] In a seventh aspect, the present invention provides use of the Lactobacillus plantarum described in the first aspect or the bacterial agent described in the second aspect in the preparation of alcohol dehydrogenase and / or acetaldehyde dehydrogenase.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The Lactobacillus plantarum BGI-J9 strain isolated, obtained and preserved by the present invention has excellent tolerance to gastric acid, intestinal fluid and bile salts in the gastrointestinal system and can survive in the gastrointestinal tract, providing a basis for continuously exerting its probiotic effects; it has excellent inhibitory ability against common intestinal pathogens; it can scavenge free radicals and reduce oxidative damage to the body; and it has good alcohol metabolism enzyme (alcohol dehydrogenase and acetaldehyde dehydrogenase) synthesis ability and the potential to promote alcohol metabolism.

[0041] The beneficial effects of the BGI-J9 strain in sobering up, protecting the liver and refreshing the brain have been verified in studies on alcohol-damaged mouse models. Specifically, it is manifested as follows: (1) effectively reducing the levels of ethanol and acetaldehyde in the blood, accelerating the metabolism of ethanol and acetaldehyde in the body, significantly delaying the onset of drunkenness, and shortening the time to sobering up; (2) improving intestinal damage and systemic inflammation caused by alcohol; (3) improving alcohol-induced oxidative stress in the liver and delaying the development of alcoholic fatty liver; and (4) improving alcohol-induced cognitive damage to the brain. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a graph showing the evaluation results of the ability of the BGI-J9 strain and the control strain 299v to synthesize alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH);

[0043] Figure 2 This is a graph showing the evaluation results of the alcohol degradation ability of the BGI-J9 strain and the control strain 299v;

[0044] Figure 3 This is a statistical result graph of the duration of the intoxication latency of each group of mice in the anti-alcohol damage efficacy test;

[0045] Figure 4 This is a statistical result graph of the duration of drunkenness of mice in each group in the anti-alcohol damage efficacy test;

[0046] Figure 5 This is a statistical result graph of the area under the curve (AUC) of blood ethanol content in each group of mice in the anti-alcohol damage efficacy test;

[0047] Figure 6 This is a statistical graph of the area under the curve (AUC) of blood acetaldehyde content in each group of mice in the anti-alcohol damage efficacy test;

[0048] Figure 7 This is a statistical graph showing the expression levels of occludin, tight junction protein (ZO-1), and vascular endothelial growth factor A (VEGFA) in the intestinal tissues of mice in each group in the anti-alcohol damage efficacy test;

[0049] Figure 8 This is a statistical result graph of the expression levels of lipopolysaccharide (LPS) and tumor necrosis factor α (TNF-α) in the serum of each group of mice in the anti-alcohol damage efficacy test;

[0050] Figure 9 This is a diagram showing the analysis results of alcoholic liver injury markers in the liver tissues of mice in each group in the anti-alcohol injury efficacy test;

[0051] Figure 10 This is a statistical result chart of the expression levels of regulatory genes related to neurocognitive function in each group of mice in the anti-alcohol damage efficacy test;

[0052] Figure 11 This is the Oil Red O staining image of the liver tissue of each group of mice in the anti-alcohol damage efficacy test (100×);

[0053] The BGI-J9 strain involved in the present invention is classified and named Lactobacillus plantarum, the preservation time is November 25, 2024, the preservation number is CCTCC NO: M 20242630, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0055] The BGI-J9 strain involved in the following experiment is classified as Lactobacillus plantarum, deposited on November 25, 2024, and has a deposit number of CCTCC NO: M 20242630.

[0056] The Lactobacillus plantarum 299v strain involved in the following experiments is a commercially available strain purchased from Shanghai Jianyi Supply Chain Management Co., Ltd.

[0057] Example 1

[0058] Isolation, identification and preservation of BGI-J9 strain:

[0059] (1) Isolation of strains

[0060] Samples of traditional fermented yogurt from Inner Mongolia were collected and placed in an anaerobic chamber. 0.2 g of yogurt sample was mixed with 1 mL of sterile phosphate buffered saline (PBS, product number B640011, purchased from Shanghai Sangon Biotechnology Co., Ltd.) and then graded diluted. 4 Take 100 μL of the sample dilution (100 μL) and spread it on MRS agar (Cat. No. 027315, Guangdong Huankai Microbiology Technology Co., Ltd.) for 72 hours of anaerobically incubating at 37°C. Then, select a single colony suspected of being a lactic acid bacteria and streak it to obtain a pure culture strain.

[0061] (2) Identification of strains

[0062] DNA was extracted from the isolated pure culture strain using a bacterial genomic DNA extraction kit (Cat. No. DP302, purchased from Tiangen Biochemical Technology Beijing Co., Ltd.). The extracted genomic DNA was used as a template for PCR amplification of the 16S rDNA gene. The primer sequences used are shown in SEQ ID NO: 2 and SEQ ID NO: 3 (SEQ ID NO: 2, 27F-AGAGTTTGATCATGGCTCAG, SEQ ID NO: 3, 1492R-TAGGGTTACCTTGTTACGACTT). The PCR amplification program is shown in Table 1, and 30 cycles were performed. The PCR amplification product was sequenced using the Sanger method to obtain the 16S rDNA sequence of the strain (SEQ ID NO: 1). Comparison with the NCBI database revealed that the strain with the highest homology was Lactiplantibacillus plantarum (formerly named Lactobacillus plantarum), with a similarity of 100.00%. The strain was named BGI-J9.

[0063] Table 1

[0064]

[0065] (3) Preserved strains

[0066] BGI-J9 (Lactiplantibacillus plantarum BGI-J9) was deposited with the China Center for Type Culture Collection on November 25, 2024, under the accession number CCTCC No. M 20242630 at Wuhan University, Wuhan, China. (This strain has now been given the newly taxonomic name Lactiplantibacillus plantarum.)

[0067] Preparation Example 1

[0068] Preparation of BGI-J9 bacterial suspension:

[0069] The Lactobacillus plantarum BGI-J9 strain was inoculated into MRS liquid medium, cultured at 37°C for 20 h after activation of the second generation, and the BGI-J9 bacterial solution was obtained. The solution was centrifuged at 4000 rpm for 10 min at 4°C, and the bacterial slurry was collected, resuspended in PBS, and adjusted to the required concentration for the test.

[0070] Example 2

[0071] Evaluation of the gastrointestinal environment tolerance of BGI-J9 strain:

[0072] This example evaluates the tolerance of BGI-J9 by simulating the human gastrointestinal environment. 9 100 CFU / mL) were placed in artificial gastric fluid (pH = 3.0), artificial intestinal fluid (pH = 6.8), and MRS liquid medium (Cat. No. 027319, purchased from Huankai Microbiology Technology Co., Ltd.) containing 0.3% bile salts and incubated at 37°C for 2 hours. The initial viable count was considered 100% survival. At the end of the experiment, viable counts were performed for each group: survival rate (%) = 100% × number of viable cells after 2 hours / initial number of viable cells. The gastrointestinal environmental tolerance of the BGI-J9 strain is shown in Table 2.

[0073] Table 2

[0074]

[0075] As shown in Table 2, the survival rate of the BGI-J9 strain after 2 hours in gastric acid was (91.20±2.82)%, the survival rate after 2 hours in simulated small intestinal fluid was (81.32±5.16)%, and the survival rate at 0.3% bile salt concentration was (85.44±4.61)%. These results demonstrate that the BGI-J9 strain of the present invention has excellent tolerance to gastric acid, intestinal fluid, and bile salts in the gastrointestinal system, and its ability to survive in the gastrointestinal tract is a prerequisite for its probiotic effects.

[0076] Example 3

[0077] Evaluation of the inhibitory ability of BGI-J9 strain against intestinal pathogens:

[0078] This example evaluates the ability of BGI-J9 to inhibit common intestinal pathogens in vitro, which helps to understand its regulatory effect on intestinal flora.

[0079] Four common enteric pathogens, including Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, Enterobacter cloacae ATCC13047, and Pseudomonas aeruginosa ATCC27853, were activated using brain heart infusion broth (BHI, product number HB8297-1, purchased from Qingdao Haibo Biotechnology Co., Ltd.). After activation, the bacterial concentration was adjusted to OD600 nm = 0.7 using sterile phosphate buffered saline (PBS, product number HB8507-1, purchased from Qingdao Haibo Biotechnology Co., Ltd.) and used as the respective pathogenic bacterial broth. The BGI-J9 strain was activated and fermented using conventional methods. The BGI-J9 fermentation broth (1×10 9 The supernatant was centrifuged at 7000 rpm for 5 min to obtain the probiotic fermentation supernatant, which was then filtered through a 0.22 μm filter membrane for later use.

[0080] The ability of BGI-J9 to inhibit common enteric pathogens was assessed using a co-culture inhibition assay. In the experimental group, 50 μL of each pathogen solution (adjusted concentration), 50 μL of probiotic fermentation supernatant, and 50 μL of double-strength BHI broth were added to a 96-well plate. In the control group, sterile saline was used instead of probiotic fermentation supernatant. After addition, the 96-well plate was incubated at 37°C for 24 hours. The growth of the various pathogens was assessed by OD600 nm absorbance. The experiment was repeated three times, and the results are expressed as mean ± SD. Pathogen inhibition rate (%) = [1 - (OD600 experimental group / OD600 control group)] × 100%. The results are shown in Table 3.

[0081] Table 3

[0082]

[0083] As shown in Table 3, the BGI-J9 strain exhibited a growth inhibition rate of (92.47±3.64)% against Escherichia coli, (87.60±5.53)% against Staphylococcus aureus, (75.18±5.94)% against Enterobacter cloacae, and (85.89±3.15)% against Pseudomonas aeruginosa. These results demonstrate that the BGI-J9 strain exhibits a high inhibitory capacity against common intestinal pathogens and possesses the potential to regulate the microbiome by reducing harmful bacteria.

[0084] Example 4

[0085] Evaluation of the antioxidant capacity of BGI-J9 strain:

[0086] In this example, the antioxidant capacity of BGI-J9 fermentation broth was determined. The BGI-J9 strain was activated and fermented using conventional methods, and the bacterial cell concentration OD was adjusted using sterile phosphate buffered saline (PBS, product number E607008-0500, purchased from Sangon Biotechnology). 600 =0.8 for later use, and the adjusted bacteria are used as test samples (hereinafter referred to as: samples).

[0087] (1) Determination of 2,2-biphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability: Prepare a 0.2 mmol / L DPPH solution with anhydrous ethanol. Take 0.5 mL of sample and add 0.5 mL of DPPH solution, mix well, and react in the dark at room temperature for 30 min. Then centrifuge and take 200 μL of the reaction solution to measure the absorbance value at a wavelength of 517 nm (sample A). At the same time, set up a sample control well with the sample to be tested + anhydrous ethanol (sample A blank), a blank well with anhydrous ethanol + distilled water (A0), and a control well with DPPH solution + distilled water (A1). The calculation formula is: DPPH free radical scavenging rate = [1-(sample A-sample A blank) / (A1-A0)] × 100%. The higher the DPPH free radical scavenging rate of the sample, the stronger its antioxidant ability.

[0088] (2) Determination of ABTS free radical scavenging ability: Prepare potassium persulfate solution (2.6 mmol / L, 1 mg + 1.43 mL water) and ABTS solution (7.4 mmol / L, 8.12 mg + 2 mL water) for use. Mix the potassium persulfate solution and an equal volume of ABTS solution, keep in the dark overnight for 16 hours to obtain ABTS+ stock solution, dilute 30 times with 1×PBS, and measure OD734nm = 0.7 ± 0.03 to obtain ABTS+ working solution. Take 0.1 mL of sample solution or PBS solution and add 3.9 mL of ABTS+ working solution, mix well, and react at room temperature in the dark for 8 minutes before measuring OD 734 Absorbance. ABTS radical scavenging rate (%) = [1 - (Sample A - Sample A blank) / (A1 - A0)] × 100. Where: Sample A is the absorbance of the test sample solution plus ABTS solution, Sample A blank is the absorbance of the test sample solution plus PBS, A0 is the absorbance of PBS, and A1 is the absorbance of water plus ABTS solution. The higher the sample's ABTS radical scavenging rate, the stronger the sample's antioxidant capacity.

[0089] (3) Determination of hydroxyl radical scavenging ability: Prepare 0.01 mol / L phosphate buffer, 2.5 mmol / L o-phenanthroline solution, 2.5 mmol / L ferrous sulfate solution and 0.1% hydrogen peroxide solution respectively. Take 1 mL of phosphate buffer, 1 mL of o-phenanthroline solution, 1 mL of deionized distilled water and 1 mL of ferrous sulfate solution, mix them evenly, then add 0.5 mL of sample and mix them evenly, finally add 1 mL of hydrogen peroxide solution and mix them evenly, and place them in a 37°C water bath for 1 hour. Take 200 μL of the reaction solution and measure the absorbance value at a wavelength of 536 nm (sample A). At the same time, set up a control well (A1) without adding hydrogen peroxide and a blank well (A0) without adding sample. The calculation formula is: hydroxyl radical scavenging rate = (sample A - A0) / (A1 - A0) × 100%. The higher the hydroxyl radical scavenging rate of the sample, the stronger the antioxidant ability of the sample.

[0090] Free radicals produced by the body (such as reactive oxygen species) trigger oxidative stress and lead to damage to tissues such as the liver and intestines. By measuring antioxidant capacity, it is possible to evaluate whether probiotic metabolites can neutralize free radicals and reduce oxidative damage, thereby protecting the body from alcohol-induced inflammation and cell damage. The experimental results of the free radical scavenging ability of the BGI-J9 strain are shown in Table 4.

[0091] Table 4

[0092]

[0093] As shown in Table 4, the BGI-J9 strain had a scavenging rate of (10.03±1.07)% for DPPH radicals, (18.60±0.79)% for ABTS radicals, and (40.02±4.98)% for OH radicals. These results suggest that the BGI-J9 strain has strong antioxidant capacity and can reduce oxidative damage by scavenging free radicals.

[0094] Example 5

[0095] Evaluation of the alcohol metabolism enzyme synthesis ability of BGI-J9 strain:

[0096] In this example, the enzyme activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were determined for BGI-J9. The BGI-J9 strain was activated and fermented using conventional methods. The BGI-J9 fermentation broth (1×10 9 CFU / mL) was centrifuged at 10000rpm for 10 minutes to collect the bacterial precipitate, 200μL cell lysis buffer (0.05mol / L) and 20μL lysozyme solution (20mg / mL) were added to the precipitate, and then β-mercaptoethanol was added to a final concentration of 0.005mol / L. Subsequently, it was incubated at 37°C for 1h. The incubated sample was frozen at -20°C for 5min, then centrifuged at 10000rpm for 15min, and the supernatant was discarded. The precipitate was resuspended in PBS buffer (pH 8.8) at a solid-liquid ratio of 1:5, and then centrifuged at 10000rpm for 20min. The supernatant was collected as a crude extract of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). The activities of ADH and ALDH were determined using an alcohol dehydrogenase (ADH) detection kit and an aldehyde dehydrogenase (ALDH) detection kit (purchased from Sangon Biotech Co., Ltd., catalog numbers D799222 and D799210) according to the instructions. The commercially available Lactobacillus plantarum 299v was used as a control strain.

[0097] ADH and ALDH are the core enzymes of alcohol metabolism. The former converts ethanol into toxic acetaldehyde, while the latter further converts it into non-toxic acetic acid. By measuring the activity of these two enzymes in probiotics or their metabolites, their ability to directly degrade alcohol and acetaldehyde can be evaluated, thereby reducing the accumulation of toxic substances. Figure 1 As shown (** indicates significant difference between groups, p < 0.01), the ADH enzyme activity of BGI-J9 fermentation broth was as high as 392.67 ± 47.35 U / mL, and the ALDH enzyme activity was as high as 30.43 ± 1.54 U / mL, both of which were significantly higher than those of the control strain 299v, indicating good alcohol metabolism enzyme synthesis ability, suggesting that the BGI-J9 strain has good alcohol metabolism potential.

[0098] Example 6

[0099] Evaluation of alcohol degradation ability of BGI-J9 strain:

[0100] This example evaluates the in vitro alcohol degradation ability of BGI-J9 through a co-cultivation experiment. The BGI-J9 strain was activated using conventional methods, and an MRS culture medium containing 5% (v / v) anhydrous ethanol was prepared. The BGI-J9 activation solution was inoculated into the ethanol-containing MRS culture medium at a 1% inoculum size and cultured at 37°C for 24 hours. The ethanol content (concentration) in each group of MRS culture medium was detected using an ethanol content detection kit at 0h, 6h, 12h, 18h, and 24h of culture. An MRS culture medium containing 5% (v / v) ethanol without the BGI-J9 activation solution was used as a blank control to eliminate the effects of alcohol volatilization. The experiment used Lactobacillus plantarum 299v as the control strain.

[0101] By simulating an alcohol environment in vitro, we can directly test the ethanol clearance efficiency of probiotics and their metabolites and evaluate the alcohol metabolism ability of probiotics. Figure 2 As shown (* indicates significant differences between groups, p < 0.05), the 6-hour alcohol degradation rate of BGI-J9 was 9.38%, the 12-hour alcohol degradation rate was 18.57%, and the 24-hour alcohol degradation rate was 28.79%. Its alcohol degradation efficiency was significantly better than that of the control strain 299v, showing excellent alcohol metabolism ability.

[0102] Preparation Example 2

[0103] Preparation of BGI-J9 active freeze-dried bacterial powder:

[0104] High-density fermentation: Take a frozen tube of BGI-J9 strain and inoculate it into 300 mL of MRS liquid medium (product number 027312, purchased from Guangdong Huankai Microbiology Technology Co., Ltd.) at a 1% inoculum volume. Incubate it anaerobically at 37°C for 24 h. Then inoculate it into 5 L of MRS liquid medium at a 5% inoculum volume. Incubate it anaerobically at 37°C until the cell density in the fermentation broth reaches 2.0×10 9 CFU / mL, the fermentation broth was collected for subsequent steps.

[0105] Freeze drying: The fermentation broth was centrifuged at 4000 rpm for 10 min at 4°C, and the bacterial sludge was collected. 60 g of skim milk powder, 120 g of trehalose, 20 g of maltodextrin, 8 g of glycerol, and 1 g of sodium ascorbate were added as freeze-drying protective agents. 800 mL of sterile water was added to dissolve and mix, and the mixture was pre-frozen at -60°C for 3 h. The mixture was then placed in a freeze dryer and freeze-dried according to the instructions of the freeze dryer. Finally, BGI-J9 active probiotic powder was obtained, and its active probiotic content was 2 × 10 11 CFU / g.

[0106] Example 7

[0107] Evaluation of the BGI-J9 strain in improving alcohol-damaged mice:

[0108] (1) Preparation of low-, medium-, and high-dose probiotic preparations: Accurately weigh 0.01 g, 0.1 g, and 1 g of the BGI-J9 freeze-dried bacterial powder obtained in Preparation Example 2, and dissolve them in 199.99 mL, 199.90 mL, and 199.00 mL of sterile saline, respectively. Stir the solutions until completely mixed to obtain bacterial cell concentrations of 1.0 × 10 7 CFU / mL (low dose), 1.0×10 8 CFU / mL (medium dose), 1.0×10 9 CFU / mL (high dose) of probiotics, ready for use.

[0109] (2) Preparation of positive control agent: Accurately weigh 1.5 g of bifendate (product number ZH10970410, purchased from Dongfeng Pharmaceutical), dissolve it in 10 mL of sterile saline, and continue stirring until the solution is evenly mixed, finally obtaining an alcohol injury-improving agent with a drug concentration of 150 mg / mL.

[0110] (3) Experimental animals and groups: 36 SPF-grade C57BL / 6 male mice, weighing 20±2g. They were group-housed, 6 mice / cage, with a temperature and humidity of 20-26°C, 40%-70%, and a 12h:12h daylight cycle. The conditions in the housing room were kept stable to ensure the reliability of the experimental results. The experiment was divided into 6 groups, including a normal control group (A), a model control group (B), a positive control group (bifendate, C), and a low-dose (D), medium-dose (E), and high-dose (F) intervention group of Lactobacillus plantarum BGI-J9, with 6 mice in each group. Detailed grouping is shown in Table 5.

[0111] Table 5

[0112]

[0113] (4) Experimental process: The stages are divided into three phases: adaptation period (7 days), intervention period (30 days), and alcohol exposure period (31 days). (a) Adaptation period: After completing animal quarantine, the mice were fed maintenance feed under the barrier system and observed for 7 days; (b) Intervention period: The BGI-J9 intervention group was gavaged with probiotics at low, medium, and high doses, the normal control group and the model control group were gavaged with normal saline, and the positive control group was gavaged with diphenyl ester; (c) Alcohol exposure period: After gavage of samples for 30 days in each dose group, all groups were strictly fasted for 24 hours (water was not allowed). Except for the normal control group, all mice were given 56% Red Star Erguotou, and the animals' behavior of drunkenness and soberness was observed.

[0114] (5) Sampling: Blood samples were collected at 0.5 h, 1 h, 2 h, and 4 h after gavage with 56% Red Star Erguotou. Blood ethanol and acetaldehyde concentrations were determined by gas chromatography. Ten hours after gavage, the animals were sacrificed by cervical dislocation, and blood samples were collected. Liver, hippocampal, and colon tissues were obtained after autopsy.

[0115] (6) The following indicators were tested and analyzed on mice:

[0116] (6.1) Alcohol sobering performance: The time of gavage administration of alcohol to each mouse was recorded, marked as the drinking point; the time of disappearance of the righting reflex of each mouse was recorded, marked as the drunken point; the time of recovery of the righting reflex of each mouse was recorded, marked as the sobering point. The duration of the intoxication latency and the duration of the drunken state were calculated based on the above times;

[0117] (6.2) Blood Indicators: Ethanol and acetaldehyde concentrations in mouse blood were determined according to the national standard GB / T 42430-2023, "Test for Ethanol, Methanol, n-Propanol, Acetone, Isopropanol, and n-Butanol in Blood and Urine." Lipopolysaccharide (LPS, Catalog No. E-EL-0180, purchased from Eliruite Biotechnology) and tumor necrosis factor-α (TNF-α, Catalog No. E-EL-M3063, purchased from Eliruite Biotechnology) levels were determined according to the instructions for the corresponding kits.

[0118] (6.3) Liver indicators: The left lobe of the mouse liver was fixed and stained with Oil Red O. The slides were examined under an optical microscope to assess lipid droplet infiltration. The right lobe of the mouse liver was rinsed with pre-chilled PBS (0.01 M, pH = 7.4) to fully remove residual blood. The tissue was weighed and minced. The minced tissue was mixed with PBS at a weight-to-volume ratio of 1:9 and rapidly frozen in liquid nitrogen for later use. During the test, liver tissue homogenate was prepared according to the instructions of the kit, and the levels of aspartate aminotransferase (AST, product number E-BC-K236-M, purchased from Elerite Biotechnology), alkaline phosphatase (ALP, product number P0321M, purchased from Biyuntian Biotechnology), superoxide dismutase (SOD, product number A001-3-2, purchased from Jiancheng Biotechnology), reduced glutathione (GSH, product number A006-1-1, purchased from Jiancheng Biotechnology), malondialdehyde (MDA, product number A003-1-2, purchased from Jiancheng Biotechnology), and triglyceride (TG, product number A110-1-1, purchased from Jiancheng Biotechnology) were measured.

[0119] (6.4) Brain Indicators: Mice were fixed, the brain was exposed by craniotomy, and the hippocampus was carefully isolated and stored on ice. The hippocampal tissue was ground and lysed with Trizol reagent. After adding chloroform to separate the layers, the upper aqueous phase was removed and RNA was precipitated with isopropanol. The RNA was washed with ethanol and dissolved in DEPC water. RNA quality control and analysis were then performed. qPCR was used to detect the expression levels of functional genes related to memory and cognitive ability in hippocampal tissue, including brain-derived neurotrophic factor (BDNF), neural cell adhesion molecule (NCAM), and immediate early gene (c-Fos).

[0120] (6.5) Intestinal markers: After killing mice, the colon was isolated, cleaned, and kept on ice. The colon was cut into small pieces and lysed with Trizol reagent. After separation by adding chloroform, the upper aqueous phase was collected and RNA was precipitated with isopropanol. The RNA was washed with ethanol and dissolved in DEPC water. RNA quality control and analysis were then performed. qPCR was used to measure the expression levels of genes related to intestinal mucosal barrier and intestinal inflammation in colonic tissue, including vascular endothelial growth factor (VEGF), occludin, and tight junction protein (ZO-1).

[0121] (7) Experimental data processing: All data were expressed as mean ± standard deviation, and the above experimental data were statistically analyzed using the one-way ANOVA test. When p < 0.05, it indicated that the difference between the groups was significant (* indicates p < 0.05 relative to the model control group; ** indicates p < 0.01 relative to the model control group; *** indicates p < 0.001 relative to the model control group).

[0122] (8) In vivo test results of anti-alcohol damage efficacy:

[0123] (8.1) BGI-J9 accelerates alcohol metabolism and shortens the drunken state:

[0124] The duration of the intoxication latency period can reflect the rate of alcohol absorption and metabolic capacity, and the duration of the drunken state measures the efficiency of the body in clearing alcohol; the blood ethanol and acetaldehyde levels reflect the concentration of unmetabolized alcohol and the accumulation level of toxic intermediates, respectively.

[0125] The duration of intoxication latency and intoxication state of each group of mice were as follows: Figure 3 and Figure 4 As shown, the duration of the intoxication latency of mice in the BGI-J9 intervention group was significantly prolonged, while the duration of the intoxication state was significantly shortened compared with the model control group.

[0126] Further analysis of blood ethanol and acetaldehyde levels, such as Figure 5 and Figure 6 As shown in the results, the area under the curve (AUC) of blood ethanol and acetaldehyde levels in the BGI-J9 intervention group was significantly lower than that in the model control group. These results suggest that BGI-J9 may improve alcohol tolerance by enhancing the efficiency of ethanol metabolism.

[0127] (8.2) BGI-J9 repairs intestinal mucosa and improves systemic inflammatory response:

[0128] Occludin and ZO-1 are core proteins in the junctions between intestinal epithelial cells, and their expression levels determine the integrity of the barrier. Overexpression of vascular endothelial growth factor A (VEGFA) may aggravate inflammation-related vascular leakage and disrupt the balance of mucosal repair. Figure 7 As shown in the results, BGI-J9 was found to significantly upregulate the expression of tight junction proteins Occludin and ZO-1, while downregulating the expression level of vascular endothelial growth factor (VEGF).

[0129] Lipopolysaccharide (LPS) is a marker of intestinal barrier integrity, and its entry into the blood indicates intestinal mucosal damage. Tumor necrosis factor α (TNF-α) is a pro-inflammatory factor and a marker of systemic inflammatory response. The results of its serum content test are as follows: Figure 8 As shown, BGI-J9 was found to significantly inhibit the alcohol-induced systemic inflammatory response.

[0130] This result suggests that BGI-J9 can inhibit systemic inflammatory response by reducing intestinal permeability and reducing the entry of endotoxin into the blood.

[0131] (8.3) BGI-J9 reduces liver oxidative stress and functional damage:

[0132] Aspartate aminotransferase (AST) and alkaline phosphatase (ALP) indicate liver cell damage and liver inflammation; superoxide dismutase (SOD) and reduced glutathione (GSH) reflect antioxidant capacity; malondialdehyde (MDA) is the end product of lipid peroxidation and indicates the level of oxidative damage; elevated triglycerides (TG) indicate liver lipid metabolism disorders and fat accumulation.

[0133] The analysis results of the above alcoholic liver injury markers are as follows Figure 9 As shown, BGI-J9 intervention significantly suppressed the alcohol-induced increase in serum aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels, indicating improved liver damage and inflammation. Regarding oxidative stress, the activities of superoxide dismutase (SOD) and reduced glutathione (GSH) in the livers of mice in the BGI-J9 intervention group were significantly increased, while the levels of malondialdehyde (MDA), the end product of lipid peroxidation, and alcoholic fatty liver markers (TG) were reduced. These results suggest that BGI-J9 alleviates alcohol-induced oxidative stress and liver fat accumulation through multiple pathways, including free radical scavenging and lipid metabolism regulation, thereby inhibiting the development of alcoholic fatty liver disease.

[0134] (8.4) BGI-J9 improves alcohol-induced neurocognitive dysfunction:

[0135] Brain-derived neurotrophic factor (BDNF) regulates neuronal survival and synaptic plasticity, and neural cell adhesion molecule (NCAM) participates in neural network reconstruction. Together, they maintain cognitive function. Immediate-early gene (c-Fos) expression reflects the degree of neuronal stress activation, and abnormal increases indicate neural damage.

[0136] The results of the measurement of the expression levels of the above-mentioned neurocognitive function-related regulatory genes are as follows Figure 10 As shown in the results, BGI-J9 was found to improve the alcohol-induced decrease in the expression of brain-derived neurotrophic factor (BDNF) and neural cell adhesion molecule (NCAM) in the hippocampus, while inhibiting the abnormal increase in the expression of the immediate early gene c-Fos. This finding provides a basis for BGI-J9 to regulate alcohol-induced neurodegeneration through the "gut-brain axis."

[0137] (8.5) BGI-J9 inhibits the pathological process of alcoholic fatty liver disease:

[0138] Liver Oil Red O staining visually displays the area of ​​lipid droplets by labeling neutral lipids. The size of lipid droplets directly reflects the degree of hepatic steatosis and is the core morphological evidence of alcoholic fatty liver. Figure 11As shown in the data, the liver cells of mice in the model control group showed typical macrovesicular fatty degeneration, while in the BGI-J9 intervention group, only scattered small lipid droplets were observed, and the overall lipid droplet area was significantly reduced. This result was consistent with the aforementioned decrease in liver TG content, suggesting that BGI-J9 inhibited alcohol-induced fat accumulation in mouse liver cells, providing an important basis for the prevention and treatment of alcoholic fatty liver.

[0139] In summary, this study reveals the core mechanism by which BGI-J9 improves alcohol exposure-induced damage through multi-target, cross-organ synergistic effects. BGI-J9 can accelerate alcohol metabolism and reduce the accumulation of harmful substances; it can also enhance free radical scavenging ability and reduce multi-organ oxidative stress; at the same time, BGI-J9 can effectively repair the intestinal barrier and reduce the entry of endotoxins into the blood, thereby inhibiting systemic inflammatory responses and liver oxidative stress; it can also affect the regulation of lipid metabolism and significantly alleviate alcoholic fatty liver. In addition, BGI-J9 improves cognitive function by upregulating neurotrophic factors. This "gut-liver-brain" multi-cascade protection network clarifies the mechanism of action of BGI-J9 in systemically intervening in multi-organ damage caused by drinking, providing a theoretical basis for further research and development.

[0140] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0141] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0142] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A plant lactobacillus having the effects of sobering up, protecting the liver and refreshing the mind, characterized in that: The Lactobacillus plantarum is the Lactobacillus plantarum BGI-J9 strain, with a preservation number of CCTCC NO: M 20242630 and a preservation date of November 25, 2024.

2. A bacterial agent with the effects of sobering up, protecting the liver and refreshing the mind, characterized in that: The strain in the bacterial agent includes the BGI-J9 strain according to claim 1.

3. The microbial agent according to claim 2, characterized in that The dosage form of the bacterial agent includes liquid preparation, powder, tablet, granule or capsule; Preferably, the number of viable bacteria of the BGI-J9 strain in the microbial agent is not less than 1×10 7 CFU / mL or 1×10 7 CFU / g; Preferably, the bacterial agent further contains excipients, which include any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants or buffers; Preferably, the bacterial agent further contains a protective agent, which includes any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, sodium alginate, sucrose, lactose, trehalose, sorbitol or xylitol.

4. The microbial agent according to claim 3, characterized in that The bacterial agent is a liquid preparation, which is prepared by a method comprising the following steps: The BGI-J9 strain is inoculated into a culture medium and activated and fermented in sequence to obtain a fermentation broth; the fermentation broth is centrifuged and resuspended in a solvent to obtain a BGI-J9 bacterial suspension, i.e., the liquid preparation; The bacterial agent is a powder, which is prepared by a method comprising the following steps: The BGI-J9 strain is inoculated into a culture medium and activated and fermented in sequence to obtain a fermentation liquid; the fermentation liquid is centrifuged, mixed with a protective agent, and then freeze-dried to obtain BGI-J9 bacterial powder, namely the powder.

5. Use of the plant lactobacillus according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of a microecological preparation for repairing alcoholic injuries.

6. The use according to claim 5, wherein the alcoholic injury comprises any one of alcoholic liver injury, alcoholic intestinal injury, or alcoholic brain nerve injury, or a combination of at least two thereof; Preferably, the alcoholic liver damage includes alcohol-induced liver oxidative stress and liver fat accumulation; Preferably, the alcoholic intestinal damage includes alcohol-induced intestinal mucosal damage, increased intestinal permeability and systemic inflammation induced by alcohol; Preferably, the alcoholic brain nerve damage includes alcohol-induced neurocognitive dysfunction.

7. Use of the Lactobacillus plantarum according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of an intestinal pathogen inhibitor; Preferably, the intestinal pathogens include any one or a combination of at least two of Escherichia coli, Staphylococcus aureus, Enterobacter cloacae or Pseudomonas aeruginosa.

8. Use of the Lactobacillus plantarum according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of a free radical scavenger; Preferably, the free radical includes any one of DPPH free radical, ABTS free radical or hydroxyl free radical, or a combination of at least two of them.

9. Use of the Lactobacillus plantarum according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of a product that improves alcohol metabolism activity in the body; Preferably, the product comprises food, health product or medicine.

10. Use of the Lactobacillus plantarum according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of alcohol dehydrogenase and / or acetaldehyde dehydrogenase.

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