Lactobacillus mucosus for accelerating alcohol metabolism and improving hangover state and application thereof

Abstract

The application provides a fermented Lactobacillus muci-dei capable of accelerating alcohol metabolism and improving hangover state and an application thereof.The strain has high safety, does not contain resistance genes and pathogenic virulence genes, and has strong tolerance to bile salts, artificial gastric juice and artificial intestinal juice.The strain has strong antioxidant capacity and has strong scavenging capacity for ABTS, DPPH and hydroxyl radicals.The fermented Lactobacillus muci-dei can improve the content of ethanol dehydrogenase of an alcoholic liver disease model mouse, and has significant application value in terms of antioxidant, alcohol elimination and hangover prevention.Meanwhile, the strain can produce melatonin and can convert NR into nicotinic acid.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a fermenting Lactobacillus mucinus and its application in antioxidation, hangover relief, and degradation of hangover reactions. Background Technology

[0002] The mechanism of hangovers is complex. Firstly, the large amount of ethanol and its metabolites in alcohol are the primary triggers for hangovers. Ethanol is metabolized in the liver through multiple pathways, including ADH and MEOS, into toxic acetaldehyde, which is then converted into acetic acid. Animal experiments further show that histamine and other specific homologues may exacerbate hangovers by reducing the levels of neurotransmitters such as serotonin. At the biochemical and neurochemical levels, excessive alcohol consumption damages the intestinal barrier, allowing endotoxins such as LPS to enter the bloodstream. This activates immune cells such as microglia, releasing large amounts of pro-inflammatory cytokines such as IL-6, TNF-α, and MCP-1 through the TLR4 / NF-κB pathway, triggering typical hangover symptoms such as nausea, headache, fatigue, and depression. Simultaneously, alcohol and its metabolites disrupt the balance of key neurotransmitters such as dopamine and serotonin, inducing headaches and low mood. Ethanol metabolism via the CYP2E1 pathway produces large amounts of reactive oxygen species (ROS) and depletes the body's antioxidants, triggering oxidative stress, directly causing cell damage, and amplifying the inflammatory response, further worsening the hangover. In addition, ethanol exposure can damage the function of mitochondria in brain neurons, inhibit ATP synthesis, and exacerbate ROS accumulation. This bioenergy metabolism crisis is directly related to the decline in motor coordination that accompanies hangovers.

[0003] Current traditional hangover remedies mainly consist of chemically synthesized drugs and traditional Chinese medicine extracts, which generally suffer from problems such as unclear ingredients, unstable effects, and risks of side effects. Consumers urgently need a safe, effective, and long-term-suitable hangover and liver-protection solution that covers the entire cycle of "pre-drinking prevention, in-drinking protection, and post-drinking repair." Probiotics, due to their high safety and well-defined probiotic functions, have become a research hotspot in the field of hangover relief and improvement, but current technologies still have significant shortcomings.

[0004] *Lactobacillus fermentum* is currently considered a particularly suitable strain of probiotics for hangover relief, liver protection, and hangover improvement. This strain possesses natural gastrointestinal tolerance, allowing it to successfully colonize the intestines through the acidic and bile-salt environment of the stomach and stably perform its physiological functions. Simultaneously, *Lactobacillus fermentum* can accelerate alcohol metabolism by increasing the activity of alcohol dehydrogenase and aldehyde dehydrogenase, reducing the accumulation of toxic intermediates. It also possesses strong free radical scavenging capabilities, mitigating alcohol-induced oxidative stress and liver cell damage. Some strains can even synthesize melatonin and regulate neurotransmitters and energy metabolism, improving hangover-related fatigue, sleep disorders, and low mood from multiple targets, meeting the full-cycle needs of hangover relief and repair. Based on these unique advantages, *Lactobacillus fermentum* has become an ideal strain for developing functional hangover-relieving probiotics.

[0005] Lactobacillus fermentum, a probiotic, has attracted attention for its potential in areas such as hangover relief, anti-oxidation, and gut health. For example, patent CN 116769656 B discloses a strain of Lactobacillus fermentum YYS-K2, which verifies its potential for hangover relief and liver protection through in vitro ethanol tolerance and alcohol dehydrogenase activity. Patent CN 116656542 A discloses a strain of Lactobacillus fermentum that can alleviate alcoholic liver damage. However, these existing technologies often focus only on detecting a single indicator of hangover relief and liver protection, without addressing key probiotic functions closely related to hangover improvement, such as hydrogen peroxide tolerance, nicotinamide conversion, and melatonin synthesis. Their limited functional scope makes it difficult to comprehensively improve the fatigue, sleep disturbances, and mood disorders associated with hangovers.

[0006] Meanwhile, traditional probiotic fermentation primarily uses glucose as the carbon source, resulting in high raw material costs and significant grain resource consumption. Substituting non-grain carbon sources and developing efficient fermentation processes have become crucial directions for the industrialization of probiotics. However, most existing publicly available strains are not compatible with low-cost, high-viable-count non-grain carbon source fermentation systems, further limiting their industrial application and market penetration. Fermentation of *Lactobacillus mucilaginosus* (…) Limosilactobacillus fermentum *Lactobacillus fermentans* belongs to the lactic acid bacteria group. It is facultatively anaerobic, tolerant of certain aerobic disturbances, suitable for non-strictly anaerobic industrial environments, tolerant of low pH (4.0–5.5), adaptable to organic acid accumulation systems, and exhibits strong tolerance to substances such as acetic acid. *Lactobacillus fermentans* possesses a complete metabolic pathway for fermentation using non-grain carbon sources, making it well-suited for such fermentation. Regarding ethanol utilization, *Lactobacillus fermentans* possesses two complete metabolic enzymes: alcohol dehydrogenase and acetaldehyde dehydrogenase, which can convert ethanol to acetaldehyde, and then acetaldehyde to acetic acid, thus utilizing ethanol energy. Patent CN119587589A discloses a strain of *Lactobacillus fermentans* A21196, demonstrating its ability to utilize and degrade ethanol and acetaldehyde by detecting the content of alcohol dehydrogenase and acetaldehyde dehydrogenase in the fermentation broth. Regarding acetic acid metabolism, GapMind analysis by Berkeley Lab clearly shows that *Lactobacillus fermentans* strain DSM 20052 has the ability to degrade acetic acid and possesses the ybhL, ackA, and pta-related genes for acetic acid degradation. The KEGG database shows that *Lactobacillus fermentum* CECT 5716 possesses a complete one-carbon folate metabolic pathway (One carbon pool by folate, lfr00670). The existence of this pathway means that this bacterium can assimilate formic acid into the one-carbon metabolic network, converting formic acid into other folate derivatives (5,10-methylenetetrahydrofolate, 5-methyltetrahydrofolate, etc.), participating in the biosynthesis of purines, thymidine, methionine, etc. Based on the above evidence, *Lactobacillus fermentum* has the ability to utilize multiple non-food carbon sources.

[0007] In summary, existing hangover remedies generally suffer from drawbacks such as limited functionality, insufficient evidence, high fermentation costs, and unclear hangover-reducing effects. The market urgently needs a novel fermented Lactobacillus mucosa that is highly safe, resilient, multifunctional, adaptable to non-grain carbon sources for efficient fermentation, and possesses multiple functions including hangover relief, liver protection, anti-oxidation, melatonin synthesis, and NR conversion, to meet the application needs of full-cycle alcohol health management. Summary of the Invention

[0008] The technical problem this invention aims to solve is to provide a strain of *Lactobacillus fermentum* LT0119 with antioxidant functions, capable of accelerating alcohol metabolism and alleviating hangover symptoms. This bacterium was isolated from farm-raised pickled vegetables in Ankang City, Shaanxi Province. It can grow normally in a culture medium containing 10% 53% vol alcohol. This probiotic exhibits high tolerance in simulated gastric juice, simulated intestinal juice, and bile salts, particularly in simulated intestinal juice containing 0.3% bile salts, where its 24-hour tolerance rate reaches 73.72%. This indicates that the bacterium can effectively colonize and function in the intestines. In terms of antioxidant activity, compared to other *Lactobacillus fermentum* strains, this bacterium demonstrates superior ABTS, DPPH, and hydroxyl radical scavenging abilities, exhibiting excellent antioxidant capacity. Mouse animal experiments verified the bacterium's ability to detoxify and protect the liver. Experimental results showed that *Lactobacillus fermentum* LT0119 can reduce indicators related to alcoholic liver disease in mice and alleviate its pathological signs. Furthermore, this bacterium also has the ability to produce melatonin and convert NR to niacin.

[0009] This invention provides a strain of *Lactobacillus fermentatus* LT0119, which was screened from pickled vegetables grown in Ankang City, Shaanxi Province. This bacterium was deposited on December 16, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, with accession number GDMCC No. 67490. The classification and naming are as follows: Limosilactobacillus fermentum .

[0010] Furthermore, the 16S nucleotide sequence of the fermenting Lactobacillus mucinus LT0119 is shown in SEQ ID NO.1.

[0011] The present invention also provides a lyophilized powder formulation comprising the aforementioned *Lactobacillus fermentans* LT0119.

[0012] This invention also provides a method for producing the above-mentioned fermented Lactobacillus mucinus LT0119 by lyophilization, comprising: The above-mentioned fermented Lactobacillus mucinus LT0119 was inoculated into the fermentation medium at an inoculation rate of 2%-5% and cultured at 37°C for 12-24 hours to obtain a culture solution. The culture solution was centrifuged and the wet bacterial precipitate was collected. The wet bacterial precipitate was resuspended and mixed with the lyophilization protectant solution and then freeze-dried to obtain a lyophilized powder preparation.

[0013] The fermentation medium consists of 10g yeast powder, 10g peptone, 5g beef extract, 30g glucose, 1g dipotassium hydrogen phosphate, 0.1g magnesium sulfate, 0.01g manganese sulfate, 1g Tween 80, 1000ml water, and pH 7.0 before sterilization.

[0014] The freeze-drying protectant consists of 150g trehalose, 50g sucrose, 10g sorbitol, 1g ascorbic acid, and 1000mL water.

[0015] The present invention also provides a microbial preparation comprising the above-mentioned *Lactobacillus fermentatus* LT0119, wherein the viable count of the above-mentioned *Lactobacillus fermentatus* LT0119 is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0016] The microbial preparation also contains other probiotics, such as Lactobacillus plantarum and Lactobacillus rhamnosus, and may also contain other auxiliary ingredients, such as vitamins and minerals.

[0017] The present invention also provides a medicine with antioxidant function, which can accelerate alcohol metabolism and improve hangover state, comprising the above-mentioned microbial preparation.

[0018] The preferred dosage form of the medicine is tablets, capsules, granules, oral liquid, etc.

[0019] The drug also includes a carrier.

[0020] This invention has the following beneficial technical effects. The *Lactobacillus mucinus* LT0119, which possesses antioxidant, hangover-relieving, and anti-hangover functions, exhibits survival rates of 1.26%, 89.78%, and 67.33% under simulated satiety, starvation, and standard conditions in artificial gastric juice, respectively. After 24 hours in artificial intestinal juice containing 0.3% bile salts, the survival rate remains at 73.72%. Regarding antioxidant properties, the scavenging capacity of this bacterium for ABTS free radicals, DPPH free radicals, and hydroxyl free radicals in cell-free contents is 67.10%, 45.16%, and 12.68%, respectively, demonstrating strong free radical scavenging ability. Animal experiments show that in an alcoholic liver disease model, *Lactobacillus mucinus* LT0119 significantly improves the reaction time from alcohol intake to intoxication and significantly reduces the sobering-up time in mice. Serum results show that in an alcoholic liver disease model, *Lactobacillus mucinus* LT0119 reduces serum ALT, AST, TG, and LDL levels in mice. Pathological results showed that the fermenting Lactobacillus mucinus LT0119 could repair alcoholic liver damage caused by alcohol.

[0021] The fermented Lactobacillus mucinus LT0119 can increase the level of alcohol dehydrogenase in a mouse model of alcoholic liver disease, demonstrating significant application value in antioxidation, alcohol detoxification, and hangover prevention. Simultaneously, this bacterium can also produce melatonin and convert NR into niacin. Attached Figure Description

[0022] Figure 1 The growth inhibition rate of fermented Lactobacillus mucilaginosus LT0119 in this invention; Figure 2 Results of blood agar streak plate experiments using Lactobacillus mucilaginosus LT0119 fermented in this invention; Figure 3 The liquid chromatogram for nicotinic acid detection in *Lactobacillus mucilaginosus* LT0119 of this invention; Figure 4 The liquid chromatogram for melatonin detection in fermented Lactobacillus mucilage LT0119 of this invention; Figure 5 Bar chart of serum ALT content in mice fermented with Lactobacillus mucinus LT0119 according to this invention; Figure 6 Bar chart of serum AST levels in mice fermented with Lactobacillus mucinus LT0119 according to this invention; Figure 7 Bar chart of serum TG content in mice fermented with Lactobacillus mucinus LT0119 according to this invention; Figure 8 Bar chart of LDL content in mouse serum from fermented Lactobacillus mucinus LT0119 according to this invention; Figure 9 Bar chart showing the content of alcohol dehydrogenase in mouse liver from fermented Lactobacillus mucinus LT0119 according to this invention; Figure 10 HE staining results of various groups in mouse animal experiments using fermented Lactobacillus mucinus LT0119 according to this invention. Detailed Implementation

[0023] The following embodiments and accompanying drawings are used to describe in detail the implementation of the present invention, so that the process of how the present invention uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0024] Example 1: Screening and Identification of Strains with Alcohol-Detoxifying Ability 1.1 Initial screening of strains Thirty samples of fresh milk and sauerkraut were collected from various parts of the country. One mL of fresh milk or one mL of sauerkraut juice from each sample was placed in a test tube containing 9 mL of sterile physiological saline (0.85%, %w / v) and diluted thoroughly. The dilution gradient was recorded as 10. -1 Then, pipette 1 mL into a test tube containing 9 mL of sterile physiological saline and dilute and mix well. Record the dilution gradient as 10. -2The samples were then serially diluted to 10⁻⁶. -6 The original sample solution and the above-mentioned dilution were spread onto MRS selection medium plates and incubated at 37°C in an anaerobic workstation for 48 h. Single colonies with different morphology, size, and color and obvious calcium dissolution zones were selected from the plates and streaked onto MRS plates for purification to obtain single colonies. The obtained single colony strains were subjected to Gram staining microscopy and catalase test. Gram-positive and catalase-negative strains were preliminarily identified as lactic acid bacteria, numbered, and stored for later use.

[0025] The MRS medium consisted of: 10 g peptone, 5 g yeast extract, 10 g beef extract, 20 g glucose, 5 g sodium acetate, 1.5 g diammonium citrate, 1 mL Tween 80, 0.5 g magnesium sulfate, 0.05 g manganese sulfate, and 2 g dipotassium hydrogen phosphate, brought to a final volume of 1 L and sterilized at 121°C for 15 min. MRS plate culture was prepared by adding 1.5-2% (w / v) agar powder to the MRS medium. MRS screening medium was prepared by adding 1.5% (w / v) CaCO3 and 1.5-2% (w / v) agar powder to the MRS medium.

[0026] 1.2 Secondary screening and strain identification of microorganisms 1.2.1 Determination of the growth capacity of alcohol-containing MRS Commercially available 56-degree Hongxing Erguotou (a type of Chinese liquor) was added to sterilized MRS medium, resulting in an alcohol concentration of 5 degrees. The selected bacterial strain was inoculated into MRS medium and cultured overnight to obtain seed culture. The seed culture was then inoculated at a 3% inoculation rate into both alcohol-containing MRS and MRS medium, and cultured for 24 hours. The OD (octane rating) of the fermentation broth was measured. 600 Calculate the growth inhibition rate: Growth inhibition rate % = (M1 - M2) / M1 × 100% (M1: MRS medium). Results are as follows. Figure 1 As shown, strain A2 exhibited the lowest growth inhibition rate and a high tolerance to alcohol.

[0027] 1.2.2 Microscopic examination results and 16s identification of the strain A pure culture of A2 bacteria, which showed low MRS growth inhibition rate in the presence of Erguotou (a type of Chinese liquor), was selected and sent for 16S testing. After analysis using the NCBI BLAST database, the bacterium was confirmed as *Lactobacillus fermentatus*, named LT0119. This bacterium was deposited on December 16, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 67490.

[0028] SEQ ID NO.1:

[0029] Example 2: Safety Analysis of the Strains 2.1 Genome Analysis The complete genome sequence of *Lactobacillus fermentatus* LT0119 was obtained by submitting the sample for testing. The assembled genome sequence was then analyzed using the ABRicate tool to detect virulence and resistance genes. Comparisons were performed using locally installed databases: Megares, CARD, VFDB, PlasmidFinder, Ecoh, NCBI, ecoli_vf, ResFinder, and ARGANNOT. The comparison criteria were: similarity ≥70% and coverage ≥70%. The results showed that the bacterium did not contain any anti-biotin resistance genes or virulence genes, indicating that it is a safe strain.

[0030] 2.2 Drug sensitivity and safety analysis The antimicrobial resistance of *Lactobacillus fermentum* LT0119 was determined using the micro-broth dilution method according to GB 31615.2-2025, National Food Safety Standard for Safety Evaluation of Microbial Strains for Food Use. *Lactobacillus fermentum* LT0119 was streaked from a glycerol tube onto an MRS plate and anaerobically cultured at 36±1℃ for 20 h. Simultaneously, the quality control strain *Lactobacillus paracasei* ATCC334 was streaked onto an MRS plate and anaerobically cultured for 20 h. Well-grown bacterial colonies were transferred from the plates to 15 mL centrifuge tubes containing 5 mL of sterile physiological saline, and the OD was measured using a spectrophotometer. 625 The bacterial concentration was adjusted with physiological saline to maintain an OD value between 0.16 and 0.2. LSM medium was used to dilute *Lactobacillus fermentans* LT0119 and *Lactobacillus paracasei* ATCC334 500-fold. 50 μL of each solution was added to an antimicrobial microdilution plate and anaerobically cultured at 28±1℃ for 48 h. The MIC value of the antimicrobial agent for *Lactobacillus fermentans* LT0119 was obtained, with each well containing 50 μL of antibiotic solution.

[0031] Experimental analysis results showed that the breakpoint values ​​of different antibiotics against *Lactobacillus fermentum* LT0119 were all equal to or less than the breakpoint values ​​of *Lactobacillus fermentum* required in Table E.6 of GB 31615.2-2025. This indicates that *Lactobacillus fermentum* LT0119 is not resistant to ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, and chloramphenicol, demonstrating its good safety profile.

[0032] 2.3 Hemolysis Verification Lactobacillus fermentum LT0119 was streaked onto Columbia blood agar plates and cultured at 37°C for 48 h in an anaerobic workstation. The results are as follows: Figure 2The results indicate that *Lactobacillus mucinus* LT0119 does not exhibit hemolytic activity, while *Staphylococcus aureus* ATCC6538 shows β-hemolysis. As a positive control to verify the plate's effectiveness, *Staphylococcus aureus* ATCC6538 was purchased from the Guangdong Provincial Microbial Culture Collection Center.

[0033] Example 3: Fermentation experiment using non-grain carbon sources Single colonies of *Lactobacillus fermentum* LT0119, activated by streak plating, were inoculated into MRS medium supplemented with a non-grain mixed carbon source. The mass ratio of the non-grain carbon source components was ethanol:sodium formate:sodium acetate = 20:3:8. In one group of MRS medium, the non-grain carbon source replaced 30% of the glucose, while in another group, an additional 30% carbon source was added to the MRS medium. Specific formulations are shown in Table 1 below. The control group used MRS medium. After culturing at 37℃ for 16 h in an anaerobic incubator, the viable count of bacteria in the medium was measured. The results are shown in Table 2 below. After replacing 30% of the glucose in the medium with a non-grain carbon source, the viable count of *Lactobacillus fermentum* LT0119 was 4.31 ± 0.25 × 10⁻⁶. 9 CFU / mL, the effective viable count in the control group MRS medium was 4.39±0.21×10⁻⁶. 9 With an additional 30% non-grain carbon source, the viable cell count during fermentation was 4.91 ± 0.27 × 10⁻⁶ CFU / mL. 9 CFU / mL. The results show that replacing glucose with a non-grain carbon source did not significantly change the number of viable fermentation cells, but adding an additional non-grain carbon source significantly increased the number of viable fermentation cells.

[0034] Table 1. Culture medium composition table

[0035] Table 2. Count of viable bacteria in each group of fermented Lactobacillus mucinus LT0119

[0036] Note: 'a' indicates that compared with the control group, P > 0.05, n = 3.

[0037] Example 4: High-density fermentation of bacterial strains and preparation of freeze-dried powder The *Lactobacillus mucinus* LT0119 glycerol culture was streaked onto MRS plates and anaerobically cultured at 37°C for 24 h. Colonies were then picked and inoculated into 12 ml shake tubes containing 5 ml of MRS medium and anaerobically cultured at 37°C for 20 h. 0.5 mL of the culture was then transferred to a 150 mL Erlenmeyer flask containing 30 mL of MRS medium and anaerobically cultured at 37°C for 10 h. The culture from the Erlenmeyer flask was then inoculated into a 5 L fermenter containing 3 L of fermentation medium and incubated at 37°C, 50 rpm, and aeration for 5 h to obtain the primary seed culture. This primary seed culture was then inoculated into a 100 L fermenter containing 70 L of fermentation medium and fermented at 37°C, 20 rpm, and aeration at a constant pH of 5.5 for 12 h. The pH was adjusted using 20% ​​sodium carbonate. The fermentation endpoint was determined by pH measurement; fermentation ended when the pH rose. At the end of fermentation, the viable cell count on MRS plates reached 11 billion CFU / mL. After fermentation, the fermentation broth was centrifuged at 8000 rpm and 4℃ for 15 min, the supernatant was discarded, and the bacterial sludge and freeze-drying protectant were mixed at a 1:1 volume ratio to form an emulsion. The emulsion was poured into a stainless steel freeze-drying tray, with a thickness not exceeding 10 mm. The emulsion was pre-frozen in a freeze dryer at -45℃ for 4 hours. The freeze-drying curves were as follows: -40℃ for 2 hours, -30℃ for 2 hours, -20℃ for 8 hours, -10℃ for 12 hours, 0℃ for 8 hours, 10℃ for 6 hours, and 25℃ for 10 hours. The moisture content at the freeze-drying endpoint was less than 3%. 1.5 kg of freeze-dried Lactobacillus LT0119 was obtained, and the viable count of the freeze-dried powder reached 460 billion CFU / g.

[0038] Fermentation medium: 10g yeast powder, 10g peptone, 5g beef extract, 30g glucose, 1g dipotassium hydrogen phosphate, 0.1g magnesium sulfate, 0.01g manganese sulfate, 1g Tween 80, 1000ml water, pH 7.0 before sterilization, sterilize at 121℃ for 30min.

[0039] Freeze-drying protectant: 150g trehalose, 50g sucrose, 10g sorbitol, 1g ascorbic acid, 1000mL water.

[0040] Example 5: Detection of strain resistance 5.1 Simulated Gastric and Intestinal Fluid Tolerance Test Sterile simulated gastric fluid was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Glycerol-coated bacterial cultures were streaked onto MRS plates and anaerobically cultured at 37°C for 24 h. Colonies were then picked and inoculated into 12 ml shake tubes containing 5 ml of MRS medium, and anaerobically cultured at 37°C for 20 h. 0.5 mL of the culture was then transferred to a 150 mL Erlenmeyer flask containing 30 mL of MRS medium and anaerobically cultured at 37°C for 16 h. 5 mL of the bacterial culture was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the culture was resuspended in sterile physiological saline. The resuspended culture was mixed at a 1:1 ratio with simulated gastric fluid at pH 2.0, 3.0, and 4.0, and incubated in a water bath at 37°C. Counts were performed at serial dilutions at 0 h, 0.5 h, 2 h, and 3 h, with three replicates for each bacterial culture. The survival rate was calculated as (number of viable cells after culture / number of viable cells at 0 h) × 100%. pH 2.0 simulated fasting, pH 3.0 simulated normal conditions, and pH 4.0 simulated satiated conditions. Table 3 shows that the survival rate of fermenting Lactobacillus mucinus LT0119 was high under all three simulated conditions.

[0041] Table 3. Viable count of *Lactobacillus fermentum* LT0119 in simulated gastric fluid.

[0042] 5.2 Simulated artificial intestinal fluid bile salt tolerance test The resuspended bacterial solution was mixed with simulated artificial intestinal fluid at a ratio of 1:9, with 0.3% bovine bile salts added to the intestinal fluid. The mixture was incubated at 37°C, and samples were taken at 0 h, 3 h, and 24 h for serial dilution and counting. Each strain was counted in triplicate. The survival rate of the strain was calculated as (number of viable bacteria at sampling time / number of viable bacteria at 0 h) × 100%. As shown in Table 4, *Lactobacillus fermentans* LT0119 exhibited good tolerance to intestinal fluid and bile salts.

[0043] Table 4. Viable count of *Lactobacillus fermentum* LT0119 in simulated intestinal fluid

[0044] 5.3 Salinity tolerance MRS culture media with sodium chloride concentrations of 0‰, 20‰, 40‰, 60‰, 80‰, and 100‰ were prepared. 20 mL of MRS culture medium was placed in a 50 mL Erlenmeyer flask, and inoculated at a 2.5% inoculum. The culture was then incubated at 37℃ for 18 hours, and the viable count was determined.

[0045] The results are shown in Table 5 below. Lactobacillus fermentum LT0119 can grow normally in MRS with a sodium chloride concentration of 40‰, and can even survive in MRS with a sodium chloride concentration of 100‰, indicating that Lactobacillus fermentum LT0119 has a strong salt tolerance.

[0046] Table 5. Viable count of *Lactobacillus fermentum* LT0119 in MRS media with different sodium chloride concentrations.

[0047] 5.4 Oxidative stress tolerance Prepare physiological saline solutions containing 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM hydrogen peroxide (hydrogen peroxide was added after sterilization of the physiological saline). Fermenting *Lactobacillus mucinus* LT0119 cultured for 16 h was centrifuged at 8000 rpm for 5 min, resuspended in physiological saline, and the resuspended solution was added to the hydrogen peroxide-containing physiological saline solution at a 1:1 ratio. After standing at 37℃ for 1 h, the viable cell count and OD600 were measured.

[0048] The results are shown in Table 6 below. The survival rate of Lactobacillus fermentum LT0119 after standing in physiological saline containing 10 mM hydrogen peroxide for 1 hour was still 24.57%, indicating that Lactobacillus fermentum LT0119 has high antioxidant activity and can still survive under high concentration of oxygen stimulation.

[0049] Table 6. Survival rate of *Lactobacillus fermentatus* LT0119 in different concentrations of hydrogen peroxide.

[0050] 5.5 Determination of Acid Production Capacity The titration method was used to determine the acid value. 5g of *Lactobacillus fermentans* LT0119 cultured for 16 hours was taken, 100mL of pure water was added, and 3-4 drops of phenolphthalein indicator were added. Titration was then performed with 0.1M / L sodium hydroxide until the solution turned pink and did not fade after 30 seconds. X = ((V-V0)×c×40) / m. Where X = acid value, in milligrams per gram (mg / g); V = volume of standard titrant consumed in the sample solution titration, in milliliters (mL); V0 = volume of standard titrant consumed in the blank test, in milliliters (mL); c = concentration of the standard titrant used, in mol / L; 40 = molar mass of sodium hydroxide, in grams per mole (g / mol); m = mass of the bacterial culture used in the test. The results showed that the fermentation acid value of *Lactobacillus fermentans* LT0119 was approximately 5.74.

[0051] 5.6 Antibacterial test Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC25922 were cultured overnight on NB medium. The bacteria were diluted to 1-5 × 10^8 CFU / mL with physiological saline. The diluted bacteria were added at 1% to NA medium at 45℃, shaken well, and 20 mL was accurately pipetted into agar plates. After the plates solidified, 100 μL of the probiotic cultured for 16 h was added to the plates, and the plates were incubated at 4℃ for 6 h, followed by overnight incubation at 36℃. The size of the inhibition zone was measured using calipers. Table 7 shows that *Lactobacillus mucinus* LT0119 exhibits certain antibacterial activity.

[0052] Table 7. Inhibition diameter of *Lactobacillus fermentum* LT0119

[0053] 5.7 Self-aggregation and co-aggregation Lactobacillus fermentum LT0119, Staphylococcus aureus ATCC 6538 (purchased from Guangdong Provincial Microbial Culture Collection Center), and Escherichia coli ATCC25922 (purchased from Guangdong Provincial Microbial Culture Collection Center), cultured for 18 h, were centrifuged at 3000×g for 10 min, and the supernatant was discarded. An equal volume of sterile PBS buffer was added, and the mixture was resuspended and mixed. The mixture was centrifuged at 3000×g for 10 min, and this step was repeated twice. A small amount of PBS buffer was added and the mixture was resuspended and mixed. The bacterial suspension to be tested was mixed with PBS buffer at a certain ratio so that the OD value of the mixed solution at a wavelength of 600 nm was about 0.8, which was recorded as A0. 4 ml of the bacterial suspension after adjusting the concentration was taken and thoroughly mixed, and incubated at room temperature for 5 h. 1 ml of the upper bacterial suspension was taken, and the OD value was measured at 600 nm with PBS buffer as a control, which was recorded as At. The self-aggregation rate (%) was calculated as: 1 - At / A0 × 100%.

[0054] Equal volumes (2 ml) of *Lactobacillus myxobolus* LT0119 suspension and *Staphylococcus aureus* ATCC 6538 or *Escherichia coli* ATCC25922 suspension were mixed and incubated at room temperature for 5 h. A control group containing 4 ml of single-bacterial suspension was cultured under the same growth conditions, and the OD value was measured at 600 nm. The experiment was repeated three times, and the co-aggregation rate (%) was calculated as follows: = [(Ax+Ay) / 2 - A(x+y)] / (Ax+Ay) × 100%. (Ax and Ay represent the absorbance of the two bacterial suspensions, and A(x+y) represents the absorbance of the mixed bacterial suspension.) The test results are shown in Table 8 below. The results indicate that Lactobacillus fermentans LT0119 can effectively aggregate Staphylococcus aureus and Escherichia coli.

[0055] Table 8. Determination of autoaggregation and coagulation of *Lactobacillus fermentatus* LT0119

[0056] Comparative Example 1 The *Lactobacillus fermentatus* CICC 21838 was used to replace *Lactobacillus fermentatus* LT0119 in Example 3. The other detection methods were the same as in Example 3. *Lactobacillus fermentatus* CICC 21838 was purchased from the China Industrial Microbial Culture Collection Center. The detection results are shown in Table 9. Under the three conditions of fasting simulated gastric juice, standard simulated gastric juice, and satiated simulated gastric juice, the survival rate of *Lactobacillus fermentatus* CICC 21838 was lower than that of *Lactobacillus fermentatus* LT0119. *Lactobacillus fermentatus* LT0119 has a stronger gastric juice tolerance.

[0057] Table 9. Number of viable bacteria of *Lactobacillus fermentum* CICC 21838 in simulated gastric juice.

[0058] Example 6: Determination of Free Radical Scavenging Ability 6.1 Determination of ABTS free radical scavenging ability After activation on agar plates, the culture was incubated in shake flasks for 16 hours. The fermentation broth was then centrifuged at 8000 rpm for 10 minutes. The supernatant was collected and set aside. The precipitate was resuspended in PBS, and the cell concentration was adjusted to 10⁻⁶. 9 CFU / mL. The resuspended solution was sonicated in an ice bath at 300 W for 3 seconds, with a 7-second interval, for a total of 3 min. The supernatant was collected and placed on ice for testing. The ABTS free radical scavenging capacity of the fermentation broth supernatant and cell-free intracellular contents was simultaneously measured. 3.6 mL of ABTS solution and 0.4 mL of glutathione solution were added to tube 1 as the experimental group (As); 3.6 mL of ABTS solution and 0.4 mL of sample solvent solution were added to tube 2 as the blank group (Ab). After thorough mixing, the mixture was reacted at room temperature in the dark for 5 min, centrifuged at 1000 rpm for 5 min at room temperature, and the absorbance of the supernatant was measured using a UV spectrophotometer at a wavelength of 734 nm (sample solvent was used for zeroing calibration). The ABTS scavenging rate was calculated as follows: P = (Ab - As) / Ab × 100%. P: Scavenging rate; Ab: Absorbance of the mixture of ABTS solution and sample solvent solution; As: Absorbance of the mixture of test solution and ABTS solution.

[0059] 6.2 Determination of DPPH free radical scavenging ability After activation on agar plates, the culture was incubated in shake flasks for 16 hours. The fermentation broth was then centrifuged at 8000 rpm for 10 minutes. The supernatant was collected and set aside. The precipitate was resuspended in PBS, and the cell concentration was adjusted to 10⁻⁶.9 CFU / mL. The resuspension was sonicated in an ice bath at 300 W for 3 seconds, with a 7-second interval, for a total of 3 min. The supernatant was collected and placed on ice for testing. The DPPH free radical scavenging capacity of the fermentation broth supernatant and cell-free intracellular contents was simultaneously measured. Glutathione standard solutions were prepared at concentrations of 0.03125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5.0 mg / mL. 3.0 mL of 50 μg / mL DPPH solution and 1.0 mL of the above glutathione standard solution were added to a test tube as the experimental group; 1.0 mL of glutathione solution and 3.0 mL of anhydrous ethanol solution were added to a test tube as the control group; and 3.0 mL of DPPH solution and 1.0 mL of sample solvent solution were added to a test tube as the blank group. After thorough mixing, the mixture was reacted at room temperature in the dark for 30 min, centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was collected. The absorbance was measured using a UV spectrophotometer at a wavelength of 517 nm (zeroing was performed using the sample solvent). The DPPH scavenging rate was calculated as follows: P = (1 - (AS - AC) / AB)) × 100%. P: scavenging rate; AS: absorbance of the mixture of the test solution and DPPH solution; AC: absorbance of the mixture of the test solution and anhydrous ethanol solution; AB: absorbance of the mixture of DPPH solution and sample solvent solution.

[0060] 6.3 Hydroxyl radical scavenging capacity determination The hydroxyl radical scavenging capacity was determined using a kit from Beijing Solarbio Science & Technology Co., Ltd. After activation of the bacterial culture on agar plates, single colonies were inoculated into MRS medium and incubated statically in shake flasks for 16 h. The fermentation broth was then aspirated and centrifuged at 8000 rpm for 10 min. The supernatant was collected as the test sample. The fermentation broth was centrifuged at 8000 rpm for 10 min, and the precipitate was resuspended in PBS to adjust the cell concentration to 10-1. 9CFU / mL. The resuspended solution was sonicated in an ice bath at 200 W for 3 seconds, with 10-second intervals, for a total of 6.5 min. Then, it was centrifuged at 8000 rpm at 4℃ for 10 min. The supernatant was collected and placed on ice for testing (no cell contents). The hydroxyl radical scavenging capacity of the fermentation broth supernatant and cell-free intracellular contents was simultaneously measured. The spectrophotometer or microplate reader was preheated for at least 30 min, and the wavelength was adjusted to 536 nm. The spectrophotometer was zeroed with distilled water. The reagents were added sequentially to 1.5 mL EP tubes according to the instructions, vortexed, and reacted accurately in a 37℃ water bath for 60 min. Afterward, the sample was centrifuged at 10000 rpm at room temperature for 10 min. 200 μL of the sample was measured at 536 nm in a microcup. Hydroxyl radical scavenging rate (%) = (Ameasured - Acontrol) / (Ablank - Acontrol) × 100%. The test results are shown in Table 10. The experimental results show that compared with Lactobacillus fermentum CICC 21838 and Lactobacillus fermentum CICC 22719, Lactobacillus fermentum LT0119 has a better ability to scavenge ABTS free radicals, DPPH free radicals and hydroxyl free radicals, indicating that it has certain potential in anti-oxidation and liver repair.

[0061] Table 10 Free radical scavenging ability of Lactobacillus fermentans LT0119

[0062] Example 7 Verification of nicotinic acid and nicotinamide production After activation of the bacterial culture on plates, colonies were picked and transferred to MRS precursor medium, and incubated statically at 37°C for 16 hours. The MRS precursor medium was prepared as follows: MRS medium was prepared, sterilized at 121°C for 15 minutes, cooled, and then 500 mg / L of NR malate was added via filtration sterilization. The cultured bacterial solution was centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was collected, filtered through a 0.22 μm filter, and analyzed by liquid chromatography. The detection method was as follows: Mobile phase A: 1 ml phosphoric acid, 150 ml acetonitrile, 850 ml water, 1.22 g sodium 1-decanesulfonate; 100% A was eluted isocratically; flow rate: 1.0 mL / min; injection: 10 µL; detection time: 40 min. Experimental results are as follows: Figure 3 As shown, the nicotinic acid yield of fermented Lactobacillus mucin LT0119 was 20.25 mg / L.

[0063] Example 8: Verification of Indole Derivative Production After activation of the bacterial culture on agar plates, colonies were picked and transferred to MRS precursor medium, and incubated statically at 37°C for 16 h. The cultured bacterial solution was then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected, filtered through a 0.22 μm filter, and analyzed by liquid chromatography. The detection method was as follows: using a C18 4.6 × 250 mm, 5 µm (40°C) mobile phase A: water; B: acetonitrile, gradient elution: 0–1 min 10% B; 1–8 min 10–95% B; 8–9.5 min 95% B; 9.6–12 min 10% B; 12–15 min 10% B, flow rate: 1.0 mL / min, injection volume: 10 µL. Figure 4 As shown, liquid chromatography analysis revealed that the melatonin production of fermented Lactobacillus mucinus LT0119 was 12.924 mg / L.

[0064] Example 9 Animal Experiment SPF-grade male mice aged 4-6 weeks were used. Animals were housed in an environment of 21-25°C and 48-55% humidity, maintaining a 12-hour light / 12-hour dark cycle. Mice were randomly divided into four groups of 10 mice each: Negative control group (NC): administered only saline without alcohol; Model control group (AD): administered only alcohol; Lactobacillus fermentum LT0119 group: administered alcohol and 10... 8 CFU / kg of probiotics. Positive drug group: Metadoxine capsules - Xinlide 200mg / kg·d. After one week of acclimatization, the model group and control group were administered 0.2 mL of physiological saline by gavage at 9:30 AM daily, while the experimental group was administered 0.2 mL of probiotic solution by gavage, for 19 consecutive days. Starting from day 15, the model group and experimental group were administered 53% ABV Erguotou (a type of Chinese liquor) by gavage 1 hour after the administration of probiotics and positive control drugs, at a dose of 8 mL / kg body weight, adjusted to 14 mL / kg body weight on the last day. The control group was administered the same volume of physiological saline by gavage to establish an acute alcohol poisoning model. After the experiment, the animals were fasted for 6 hours but allowed free access to water. Blood was collected under inhalation anesthesia to test ALT, AST, TG, LDL, and carbon dioxide levels. The animals were euthanized by asphyxiation, and their livers were harvested to test for alcohol dehydrogenase. The livers were then used for pathological hematologic staining.

[0065] After the final gavage, six mice were selected from each group (excluding the control group) for testing. The mice were placed back-down in their cages, and their position was adjusted every 5 seconds until they could no longer correct themselves immediately. If a mouse could not roll over on its own within 30 seconds, the reflex was considered lost (intoxicated); if the mouse could roll over again on its own, the reflex was considered restored (sober). The latency period of intoxication (time from alcohol intake to the loss of the reflex) and the sobering time (time from the loss of the reflex to its recovery) were recorded after the mice were given alcohol via gavage. like Figures 5-8 The results showed that AST, ALT, TG and LDL levels in the Lactobacillus fermentum LT0119 group decreased compared with those in the model group, indicating that Lactobacillus fermentum LT0119 has a certain repair ability in liver injury repair in mice and can alleviate liver damage caused by ethanol.

[0066] like Figure 9 As shown, compared with the model group, the group fermented with Lactobacillus mucin LT0119 showed an increase in alcohol dehydrogenase, indicating that oral administration of fermented Lactobacillus mucin LT0119 can promote the activity of alcohol dehydrogenase in mice after alcohol stimulation and help improve the mice's ability to metabolize alcohol.

[0067] The results in Table 11 show that, compared with the model group, the positive drug group and the Lactobacillus fermentation LT-0119 group had significantly reduced alcohol intake, intoxication reaction time, and sobering-up time.

[0068] Table 11 Comparison of alcohol intake time to intoxication reaction time and sobering time in different groups of mice

[0069] Note: 'a' indicates a comparison with the model group. P < 0.05, n = 6.

[0070] Based on HE staining results ( Figure 10 It was found that the liver histological structure of the control group sections basically maintained the normal lobular structure. Specific pathological features: the lobular structure was relatively clear, hepatocytes were arranged in cords, and the hepatic cords and sinusoidal structures were distinguishable. Central veins (or vascular structures) and portal structures were visible, including a large luminal structure (possibly an intrahepatic vessel or bile duct, with no obvious contents or only a blank area). The surrounding hepatocytes were generally normal in morphology, with round nuclei and normal nucleoplasm ratio. No obvious ballooning degeneration or fatty degeneration of hepatocytes was observed. Inflammatory cell infiltration was not obvious, and there were no obvious areas of hepatocyte necrosis. There was no obvious fibrosis in the lobules and portal areas.

[0071] Pathological results of liver tissue from the model group showed numerous vacuoles in the hepatocyte cytoplasm, appearing as round vacuoles of varying sizes, with some areas showing vacuolation, suggesting moderate to severe fatty degeneration. Hepatocyte arrangement was slightly disordered, and the hepatic lamina structure was locally blurred. Fatty degeneration was evident in hepatocytes around the central vein, and the sinusoids were narrowed due to hepatocyte swelling. Inflammatory cell infiltration within the hepatic lobules was predominantly lymphocytes and monocytes, with a small number of neutrophils. There was no significant large-scale hepatocyte necrosis, but localized apoptosis (nuclear pyknosis, increased eosinophilicity) of individual hepatocytes was observed. No significant fibrosis was observed in the portal areas, and the vascular structure showed no obvious abnormalities. The study presented with moderate to severe hepatocyte fatty degeneration accompanied by an inflammatory response.

[0072] In the positive drug group, the liver lobule structure was basically intact, and the arrangement of hepatocyte cords was not significantly disordered. A small number of small vacuoles were visible in the hepatocyte cytoplasm, which was significantly reduced compared with the model group. The nuclei were normal in morphology and the nucleocytoplasmic ratio was normal. There was no obvious inflammatory cell infiltration, no signs of hepatocyte necrosis, and no fibrosis in the liver lobules or portal areas. Mild fatty degeneration of hepatocytes and local vascular congestion were the main characteristics.

[0073] The liver histological structure of the *Lactobacillus fermentum* LT-0119 group was generally clear, and the outlines of the liver lobules were discernible, but some minor local abnormalities were observed. Specific pathological features: Hepatocytes were polygonal, arranged in a roughly plate-like structure, and the sinusoidal spaces were visible. A small number of vacuolar changes were observed in the cytoplasm of hepatocytes in some areas, suggesting possible mild fatty degeneration; the central vein and portal structures were present, with no obvious inflammatory cell infiltration in the portal areas; inflammatory cells were rare in the liver lobules, and there were no obvious foci of hepatocyte necrosis. There was no obvious dilation or congestion of the sinusoids and vascular structures, and no obvious signs of fibrosis-related collagen deposition. The lesions were mild, belonging to mild pathological changes. Mild hepatocyte fatty degeneration was considered. The lesions showed significant improvement compared to the model group.

[0074] All of the foregoing primary implementations of this intellectual property right do not limit other forms of implementation of this new product and / or new method. Those skilled in the art will utilize this important information to modify the foregoing to achieve similar implementations. However, all modifications or alterations based on this new product invention are reserved rights.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Fermenting lactobacillus that can accelerate alcohol metabolism and improve hangover symptoms ( Limosilactobacillus fermentum ), characterized by: The fermenting *Lactobacillus mucinus* was named LT0119, with accession number GDMCC No. 67490, and its 16S nucleotide sequence is shown in SEQ ID NO.

1.

2. A lyophilized powder formulation comprising the *Lactobacillus fermentans* of claim 1.

3. The method for producing *Lactobacillus mucinus* by fermentation freeze-drying according to claim 1, characterized in that, include: The fermenting *Lactobacillus mucinus* was inoculated into a fermentation medium and cultured at 37°C for 12-24 hours to obtain a culture solution. The culture solution was centrifuged, and the wet bacterial precipitate was collected. The wet bacterial precipitate was resuspended and mixed with a lyophilization protectant solution, and then freeze-dried to obtain a lyophilized powder formulation.

4. The method for producing *Lactobacillus mucinus* by fermentation freeze-drying as described in claim 3, characterized in that: The fermentation medium consists of 10g yeast powder, 10g peptone, 5g beef extract, 30g glucose, 1g dipotassium hydrogen phosphate, 0.1g magnesium sulfate, 0.01g manganese sulfate, 1g Tween 80, and 1000ml water. The pH of the fermentation medium before sterilization is 7.

0.

5. The method for producing *Lactobacillus mucinus* by fermentation freeze-drying as described in claim 3, characterized in that: The freeze-drying protectant solution consists of 150g trehalose, 50g sucrose, 10g sorbitol, 1g ascorbic acid, and 1000mL water.

6. A microbial preparation comprising the *Lactobacillus fermentans* of claim 1, characterized in that: The viable count of the fermenting *Lactobacillus mucinus* is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

7. The microbial preparation according to claim 6, characterized in that: It also contains other probiotics.

8. A drug that can accelerate alcohol metabolism and improve hangover symptoms, characterized in that: It includes the microbial preparation described in claim 6 or 7.

9. The use of the fermented *Lactobacillus mucinus* according to claim 1 in the preparation of a pharmaceutical product that can accelerate alcohol metabolism and improve hangover symptoms.

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