Lactobacillus muci genosus DACN611 and application thereof in preparation of products for protecting liver from alcohol

Abstract

The application discloses a fermenting limosilactobacillus DACN611 (Limosilactobacillus fermentum DACN611) with a preservation number of CCTCC NO: M 20232522 and application of the fermenting limosilactobacillus DACN611 in preparation of a product for relieving alcoholism and protecting liver, which not only expands the application range of the fermenting limosilactobacillus DACN611 and improves the utilization value, but also provides a new fermenting limosilactobacillus capable of significantly improving the symptoms of alcoholism of a host, significantly degrading ethanol and acetaldehyde in the host, having a rapid alcohol relief effect and having a wide market application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of fermenting Lactobacillus mucinus DACN611 and its application in the preparation of products for hangover relief and liver protection. Background Technology

[0002] Alcohol is a potentially addictive psychoactive drug that has long been widely used as a recreational beverage. The WHO (World Health Organization) annual "Global Status Report on Alcohol and Health" reported that in 2016, the number of alcoholics worldwide exceeded 200 million. Moderate drinking may have health benefits, but acute heavy drinking can lead to gastrointestinal damage, alcoholic liver damage, and even death. Studies have shown that some chemicals have anti-alcohol effects, such as disulfiram, naltrexone, and acampalic acid, but these drugs have certain side effects, such as nausea, dizziness, and vomiting. Therefore, it is necessary to develop natural and safe drugs to alleviate the harms of acute heavy drinking.

[0003] As is well known, after a host ingests alcohol, a small portion is directly metabolized in the stomach, while the majority is absorbed into the bloodstream via the stomach and upper intestines, and then metabolized in the liver. Alcohol metabolism primarily involves the microsomal oxidation pathway and the ethanol oxidation pathway. In the microsomal oxidation pathway, alcohol is metabolized into acetaldehyde by the cytochrome P450 enzyme system (CYP2E1), and then converted to acetic acid. Acetic acid enters the tricarboxylic acid cycle and continues to be metabolized, ultimately producing carbon dioxide and water, which are excreted from the body. In the ethanol oxidation pathway, alcohol is first metabolized into acetaldehyde by alcohol dehydrogenase (ADH), and then converted into acetic acid by aldehyde dehydrogenase (ALDH). Probiotics are live microorganisms that, when ingested in certain doses, are beneficial to the host's health. Generally speaking, probiotics are safe and have no significant side effects on the host. Studies have shown that some lactic acid bacteria that directly degrade ethanol can improve alcohol metabolism after drinking and alleviate alcohol-induced organ damage. Therefore, developing lactic acid bacteria capable of degrading ethanol into functional formulations has broad prospects and deserves further research. Summary of the Invention

[0004] One of the objectives of this invention is to provide a strain of *Limosilactobacillus fermentum* DACN611, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20232522.

[0005] Another object of the present invention is to provide a microbial agent whose active ingredient comprises the aforementioned *Lactobacillus fermentans* DACN611.

[0006] Preferably, the bacterial agent, Lactobacillus mucinus DACN611, is used alone.

[0007] Another object of the invention is to provide a method for preparing the above-mentioned microbial agent, wherein the above-mentioned fermenting Lactobacillus mucinus DACN611 is inoculated into a culture medium and cultured, preferably in MRS medium at 30-37 °C for 18-24 h.

[0008] The final objective of the invention is to provide the application of the aforementioned fermenting Lactobacillus mucinus DACN611 or the aforementioned bacterial agent in the preparation of products for degrading alcohol or for hangover relief and liver protection, wherein the products are functional foods or food additives.

[0009] Preferably, in the application technology solution, the product has at least one of the following functions:

[0010] (1) Degrades ethanol and / or acetaldehyde in serum;

[0011] (2) Shorten the time of intoxication and sobering up of the subjects;

[0012] (3) Increase the activity of ADH and ALDH enzymes in the subject; preferably increase the activity of ADH and ALDH enzymes in the stomach and liver of the subject;

[0013] (4) Remove ethanol and acetaldehyde from the subject’s body; the body includes blood, gastric juice, or intestinal fluid including duodenal fluid, small intestinal fluid or large intestinal fluid;

[0014] (5) Protect subjects from the effects of alcohol metabolism disorders caused by excessive alcohol intake;

[0015] (6) To prevent the rise in serum ALT and AST after the subjects ingested alcohol;

[0016] (7) Reduce liver damage caused by alcohol consumption in subjects.

[0017] Preferably, in the application technology solution, according to the source of raw materials of the functional food, the food includes dairy products, soy products, fruit and vegetable products, and fermented foods.

[0018] Preferably, in the application technology solution, according to the state of the functional food, the food includes solid food, liquid food, and semi-solid food.

[0019] The results of screening ethanol-degrading bacteria in this invention showed that among 102 lactic acid bacteria strains, *Lactobacillus fermentum* DACN611 had the highest in vitro ethanol degradation efficiency. After gastric acid and bile salt resistance and safety experiments, *Lactobacillus fermentum* DACN611 exhibited good gastric acid and bile salt resistance and antibiotic susceptibility characteristics, and showed no hemolytic activity. Results in an acute alcohol intoxication model mouse showed that *Lactobacillus fermentum* DACN611 significantly prolonged the loss of corrective reflex (LORR) delay time and significantly reduced the duration of LORR in mice; it significantly reduced serum ethanol and acetaldehyde levels in mice, significantly increased ADH and ALDH activities in the stomach and liver, reduced hepatic CYP2E1 protein expression, and improved ethanol-induced metabolic disorders; simultaneously, it significantly increased serum ALT and AST levels in mice, reduced hepatocyte vacuolation and inflammatory factor infiltration in mice, and improved alcoholic liver injury in mice.

[0020] The beneficial effects of this invention are as follows: Compared with other reported fermented *Lactobacillus* species with hangover-relieving functions, the *Lactobacillus* DACN611 of this invention has a particularly significant effect on improving the host's hangover symptoms. After the host ingests 56-degree baijiu (Chinese liquor), the use of a single strain of *Lactobacillus* DACN611 shortens the host's hangover time by 140.45% and the sobering-up time by 42.52%. Simultaneously, *Lactobacillus* DACN611 exhibits a significant rapid hangover-relieving effect. After the host ingests 56-degree baijiu, *Lactobacillus* DACN611 significantly increases the activity of ADH and ALDH enzymes in the host's stomach and liver (P < 0.05), significantly downregulates the expression of CYP2E1 protein in the liver (P < 0.05), and simultaneously increases the host's serum ethanol degradation rate to 77.04% and the host's serum acetaldehyde degradation rate to 77.66%. The *Lactobacillus* DACN611 of this invention has a significant rapid hangover-relieving effect. In addition, *Lactobacillus* DACN611 also has a liver-protective effect. Attached Figure Description

[0021] Figure 1 The results are shown in the following diagrams: strain development tree (a), gastric acid and bile salt resistance results (b), Gram staining results (c), strain colony diagram (d), and strain hemolysis plate results (e).

[0022] Figure 2 Mouse body weight change trend (a), mouse body weight on the last day (b), and liver index (c).

[0023] Figure 3 The corrected reflex delay time (a) and duration (b) in mice.

[0024] Figure 4 The results show the serum ethanol (a), serum acetaldehyde (b), gastric ADH (c), liver ADH (d), gastric ALDH (e), and liver ALDH (f) in mice.

[0025] Figure 5 Immunohistochemical image of CYP2E1 protein in mouse liver (a) and image of CYP2E1 protein positive area (b).

[0026] Figure 6 The results of serum ALT (a), serum AST (b), and liver histopathological observation (c) in mice are shown.

[0027] In the above figure, there were no significant differences among groups labeled with the same lowercase English letters (a, b, c) (P > 0.05); there were significant differences among groups labeled with different lowercase English letters (a, b, c) (P < 0.05). Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, the experimental methods in the following examples are conventional methods; unless otherwise specified, the biological and chemical reagents used are conventional reagents in the art and can be obtained commercially.

[0030] Example 1: Screening of ethanol-degrading strains

[0031] 1. Experimental Materials

[0032] Lactic acid bacteria strain DACN611, isolated from traditional Tibetan fermented yogurt, is from the strain library of the Food Microbiology and Fermentation Engineering Laboratory, College of Food Science, Southwest University.

[0033] 2 Experimental Methods

[0034] 2.1 Screening for lactic acid bacteria that degrade ethanol

[0035] Lactic acid bacteria were activated for three generations in MRS broth, and the bacterial cells were washed with 0.85% (v / v) sterile physiological saline, followed by centrifugation to obtain a bacterial precipitate (6000 g, 4℃). The bacterial concentration was adjusted to 5 × 10⁻⁶ with sterile physiological saline. 8 CFU mL -1 Bacterial suspensions were prepared. In the experimental group, 200 μL of bacterial suspension was inoculated into 10 mL of MRS broth containing 5% (v / v) ethanol. In the control group, 200 μL of sterile physiological saline was inoculated into 10 mL of MRS broth containing 5% (v / v) ethanol. Both groups were then cultured at 37°C for 8 h. The MRS broth culture medium was centrifuged (10 min, 8000 g) to collect the supernatant. 1 mL of the supernatant was mixed with 25 mL of potassium dichromate solution (0.136 mol / L). -1 ) and 4 mL concentrated sulfuric acid (18.3 mol L) -1Mix the solutions. Then place them in a boiling water bath for 10 min. After cooling, measure the absorbance of the solution at a wavelength of 610 nm. Select the strain with the highest ethanol degradation rate for further experiments. The ethanol degradation rate (%) was calculated using formula (1).

[0036]

[0037] Where A represents absorbance.

[0038] 2.2 Determination of ethanol degradation rate of strains under different concentration conditions

[0039] 5% (v / v) lactic acid bacteria suspension was inoculated into 10 mL of MRS broth culture medium with different ethanol concentration gradients (2.5%, 5%, 10%, 15% v / v). After incubation at 37℃ for 24 h, the ethanol degradation rate in the MRS supernatant was measured.

[0040] 2.3 PCR amplification of 16S rDNA sequence

[0041] DNA from the bacterial strain was extracted and purified using a bacterial genomic DNA extraction kit. PCR amplification was performed in a 25 µL reaction system: 1 µL template DNA, 1 µL upstream primer (10 µm), 1 µL downstream primer (10 μm), 12.5 µL 2×Taq PCR MasterMix, and sterile ultrapure water was added to a final volume of 25 µL. PCR amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1.5 min, 55℃ annealing for 1 min, 72℃ extension for 1.5 min, for a total of 30 cycles; and 72℃ final extension for 10 min. Finally, the qualified PCR amplification products were bidirectionally sequenced by Shanghai Sangon Biotech Co., Ltd., and the sequencing results were analyzed for homology using the BLAST program in NCBI.

[0042] 2.4 Strain resistance to gastric acid and bile salts and safety experiments

[0043] 2.4.1 Gastric acid resistance test

[0044] First, prepare artificial gastric fluid by mixing NaCl (0.2 wt%) and pepsin (0.35 wt%), adjusting the pH to 3.0 with 1 M HCl, and filtering under sterile conditions. Then, take 5 mL of the activated bacterial culture and pour it into a sterile 10 mL centrifuge tube. Centrifuge at 4000 g for 10 min at 4°C to collect the cells. Add 5 mL of sterile physiological saline (0.85%, v / v) and mix well to prepare a bacterial suspension. Take 1 mL of the bacterial suspension and mix it with 9 mL of pH 3.0 artificial gastric fluid. Incubate in a constant temperature shaker (37°C, 100 r). Take samples (10 μL) at 0 h and 3 h and pour them onto MRS agar medium (37°C) for 48 h. Determine the viable cell count using the plate count method and calculate the survival rate (%).

[0045] 2.4.2 Bile salt tolerance test

[0046] Five mL of the activated bacterial strain was inoculated at a 2% (v / v) inoculation rate (100 μL) into MRS-THIO medium (MRS broth medium with 0.2% (v / v) sodium thioglycolate) containing 0.3 wt% porcine bile salts. After incubation at 37°C for 24 h in a constant temperature shaker, using blank medium (uninoculated MRS-THIO medium) as a control, 200 μL of each medium was plated onto an ELISA plate to determine the OD600 value of the different concentrations of medium, and the strain's tolerance to bile salts was calculated.

[0047] 2.4.3 Hemolytic test

[0048] The bacterial strain was inoculated onto 5% (v / v) defibrinated whole sheep blood on scientific agar plates using the streak plating method and incubated at 37°C for 48 h. Staphylococcus aureus was selected as a positive control. A positive area was defined as the appearance of a clear or translucent area around the colony.

[0049] 2.4.4 Antibiotic susceptibility testing

[0050] Antibiotic susceptibility of the strains was determined using the disc diffusion method. We used 20 important antibiotics, including ampicillin (10 μg), penicillin (10 U), tetracycline (30 μg), erythromycin (15 μg), ceftriaxone (30 μg), cefuroxime sodium (30 μg), amikacin (30 μg), cefoperazone (75 μg), gentamicin (10 μg), vancomycin (30 μg), kanamycin (30 μg), minocycline (30 μg), polymyxin B (300 IU), streptomycin (10 μg), doxycycline (30 μg), cefazolin (30 μg), piperacillin sodium (100 μg), lincomycin (2 μg), cefazolin (30 μg), and cephalexin (30 μg). The experiments were repeated three times, and data are recorded as mean ± standard deviation.

[0051] 3. Experimental Results and Analysis

[0052] 3.1.1 Screening of ethanol-degrading lactic acid bacteria

[0053] To obtain strains with high ethanol degradation activity, we used 102 lactic acid bacteria as candidate strains. As shown in Table 1, *L. fermentum* DACN611 (serial number 11 in Table 1) exhibited the strongest ethanol degradation ability (ethanol degradation rate of 30.93% ± 2.80%). We selected *L. fermentum* DACN611, which had the strongest ethanol degradation ability, for further experiments.

[0054] Table 1. Results of ethanol degradation rate (%) of 102 lactic acid bacteria strains in 5% (v / v) ethanol MRS medium

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] 3.1.2 Degradation rate of different concentrations of ethanol by fermented Lactobacillus mucilaginosus DACN611

[0061] Table 2. Degradation rate of ethanol at different concentrations by fermenting Lactobacillus mucilaginosus DACN611

[0062]

[0063] The results are shown in Table 2. The degradation rate of 15% ethanol by fermenting Lactobacillus mucinus DACN611 was 26.77% ± 3.24%, which shows good ethanol degradation ability in vitro.

[0064] 3.2 Strain Identification

[0065] 3.2.1 Colony morphology and cell morphology of the isolated strains

[0066] After purification, the strain formed single colonies in MRS medium. The colonies were almost uniform in morphology, mostly round, white, and with a smooth, moist surface. Gram staining revealed purple cells under a microscope, confirming them as Gram-positive bacteria (G). + The colony morphology and Gram staining results of the strain are shown in [reference needed]. Figure 1 c.

[0067] 3.2.2 16S rDNA sequence analysis of the strain

[0068] Homology analysis of 16S rDNA (sequencing results shown in SEQ ID NO.1) showed 100% homology with *Limosilactobacillus fermentum* known in the Gene Bank database. The phylogenetic tree of *Limosilactobacillus fermentum* DACN611 is shown below. Figure 1 As shown in a.

[0069] The preservation information for *Lactobacillus fermentatus* DACN611 is as follows:

[0070] The *Limosilactobacillus fermentum* DACN611 was deposited at the China Center for Type Culture Collection (CCTCC) in December 2023. The address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province. The deposit date is December 11, 2023; the accession number is CCTCC NO:M 20232522; and the classification name is *Limosilactobacillus fermentum* DACN611.

[0071] The 16S rDNA (SEQ ID NO.1) sequence of the strain is as follows:

[0072]

[0073] 3.2.3 Gastric acid and bile salt resistance test

[0074] Probiotics can survive in the human gastrointestinal tract and produce beneficial effects. To evaluate the in vitro resistance of *Lactobacillus fermentum* DACN611, its survival rate in simulated gastric fluid at pH 3.0 and its growth efficiency in 0.3% (v / v) bile salts were determined. Figure 1 As shown in b, *Lactobacillus fermentum* DACN611 was less affected by artificial gastric fluid, with a survival rate of 84.02% ± 2.71%. Its growth rate in 0.3% (v / v) bile salts was 19.37% ± 2.04%. These results indicate that *Lactobacillus fermentum* DACN611 is capable of surviving and colonizing in the digestive tract.

[0075] 3.2.4 Hemolytic test

[0076] like Figure 1 As shown in Figure d, the positive control group exhibited significant hemolytic activity. In contrast, *Lactobacillus fermentum* DACN611 did not cause hemolysis.

[0077] 3.2.5 Antibiotic susceptibility testing

[0078] As shown in Table 3, among 20 drug-sensitive tablets, *Lactobacillus fermentum* DACN611 was sensitive to 14 antibiotics (ampicillin, penicillin, tetracycline, erythromycin, cefotaxime, cefuroxime sodium, cefoperazone, minocycline, doxycycline, cefazolin, piperacillin sodium, lincomycin, cefoperazone oxime, and cefalexin), moderately sensitive to one antibiotic (cefoperazone, cefuroxime sodium, cefoperazone), piperacillin sodium, lincomycin, cefazolin oxime, and cefalexin), moderately sensitive to one antibiotic (gentamicin), and resistant to five antibiotics (amikacin, vancomycin, kanamycin, polymyxin B, and streptomycin). The results indicate that *Lactobacillus fermentum* DACN611 is sensitive to most antibiotics and has a certain degree of safety in its application.

[0079] Table 3. Results of antibiotic susceptibility testing of Lactobacillus fermentum DACN611 against 20 antibiotics.

[0080]

[0081] Example 2: The alleviating effect of Lactobacillus fermentum DACN611 on alcohol poisoning in mice.

[0082] 1. Experimental Materials

[0083] The experimental strain was *Limosilactobacillus fermentum* DACN611, with accession number CCTCC NO: M 20232522. After activation, *Limosilactobacillus fermentum* DACN611 was inoculated into MRS broth at a 2% (v / v) inoculum and cultured at 37°C in a shaker for 18-24 h. A bacterial suspension was then prepared using 0.85% (wv%) sterile physiological saline and administered via gavage to mice in the Alcohol+DACN611 group.

[0084] The experimental animals were 8-week-old male Kunming mice, purchased from Chongqing Enswell Biotechnology Co., Ltd. They were housed in a standardized laboratory with a room temperature of 25±2℃, relative humidity of 50±5%, and a 12-hour light / 12-hour dark cycle. Experiments began after one week of acclimatization.

[0085] 2 Experimental Methods

[0086] 2.1 Grouping and treatment of experimental animals

[0087] Mice were randomly divided into four groups: a control group, a model group, a positive control group, and a group fermented with Lactobacillus mucin DACN611 (Alcohol + DACN611), with eight mice in each group. The experiment lasted for 8 days, during which mice were administered gavage twice daily. The control and model groups were administered 0.1 mL / 10 g saline via gavage for the first time each day, the positive control group was administered 150 mg / kg bw biphenyl diester via gavage for the first time each day, and the Alcohol + DACN611 group was administered 5 × 10 g of biphenyl diester via gavage for the first time each day. 10 CFU Kg -1 Fermented Lactobacillus mucinus DACN611 bacterial suspension. Except for the control group, the other three groups of mice underwent a second gavage administration of 0.12 mL / 10 g of Erguotou (a type of Chinese liquor). Mouse weight was monitored daily until the end of the experiment. The time to loss of righting reflex (LORR) was recorded on day 1. After gavage on day 8, mice were euthanized, and blood was collected from their eyes in sodium citrate anticoagulant tubes to determine alcohol concentration. Serum was collected on the last day, centrifuged at 3000 g for 10 min at 4℃, and stored at -80℃ for later use. After blood collection, mice were euthanized by cervical dislocation, and liver and stomach tissues were dissected and weighed immediately. Appropriate amounts of liver and stomach tissue were cut and immediately fixed in neutral paraformaldehyde. All tissues were then frozen in liquid nitrogen and finally stored at -80℃.

[0088] 2.2 Determination of the index of loss of righting reflex

[0089] Mice were fasted for 12 hours before the experiment. The activity of each group of mice was observed, and the time of loss and recovery of the righting reflex was recorded. The time of loss of the righting reflex was defined as the mouse remaining on its back for 30 seconds. Recovery of the righting reflex was indicated when the mouse was moving freely, agile, alert, and had smooth fur. After the righting reflex disappeared, the mice were placed in a V-shaped trough. (LORR delay time = time of righting reflex loss - time of alcohol administration; LORR duration = time of righting recovery - time of alcohol administration). The time of intoxication for the mice is the LORR duration, and the time to sober up is the sum of the LORR delay time and the LORR duration.

[0090] 2.3 Measurement of serum markers

[0091] Mice were sacrificed 5 h after oral administration of alcohol, and serum alcohol degradation rate was determined. Serum ethanol and acetaldehyde levels were determined by gas chromatography. 100 μL of serum was filtered through a 45 μm filter and then mixed with 500 μL of tert-butanol solution. The solution was centrifuged at 3000 g for 10 min at 4 °C, followed by GC analysis. Serum alcohol concentration analysis was performed using a GC-2010plus system equipped with an SH-Rtx-Wax capillary column (30 m × 0.25 mm, 0.25 μm, Shimadzu, Japan) and a flame ionization detector. The injector and detector temperatures were set to 200 °C, with a split ratio of 20:1. The column temperature was 65 °C for 3 min, followed by a decrease at 6 °C for 1 min. -1 Heat to 110°C at a rapid rate.

[0092] The levels of AST and ALT in mouse serum were measured according to the kit instructions.

[0093] 2.4 Determination of biochemical indicators in the liver and stomach

[0094] Liver and stomach tissues were each mixed with 9 volumes of physiological saline (m / V) and homogenized in an ice-water bath. The prepared 10% homogenate was centrifuged at 3500 g and 4℃ for 10 min, and the supernatant was collected. The levels of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in mouse liver and mouse stomach were measured using a commercial kit (Nanjing Jiancheng Biotechnology Institute, China).

[0095] 2.6 Histological and Immunohistochemical Analysis

[0096] Fresh liver tissue samples were extracted from the same lobe of each mouse, immediately fixed with 4% (v / v) paraformaldehyde, dehydrated in ethanol, removed with xylene, and then embedded in paraffin. Tissue sections with a thickness of 5 μm were prepared and stained with H&E. Pathological changes in the liver tissue were observed under an optical microscope.

[0097] Paraffin-embedded liver sections were dewaxed in an environmentally friendly dewaxing solution and then hydrated in fractionating ethanol and water. After blocking with bovine serum albumin (BSA), sections were incubated overnight at 4°C with CYP2E1 primary antibody (1:200). After rinsing with PBS, sections were incubated with secondary antibody (1:300) at room temperature for 50 min. Sections were stained using a polymer HRP detection system. Sections were developed with diaminobenzidine solution and reversed with hematoxylin. The staining was observed under a microscope and quantified using ImageJ software.

[0098] 3. Experimental Results and Analysis

[0099] 3.1 Effects of Lactobacillus fermentum DACN611 on body weight and liver index in mice

[0100] like Figure 2 As shown in b and 2c, after alcohol intervention, the body weight of mice in the model group was significantly lower than that in the control group (P < 0.05). There was no significant difference in body weight between the positive control group and the Alcohol + DACN611 group and the model group (P > 0.05). Liver indices can reflect the degree of liver damage to some extent. The results of liver index measurements in each group showed that the liver index of mice in the model group was significantly higher than that in the control group (P < 0.05). There was no significant difference in liver index between the positive control group and the Lactobacillus fermentum DACN611 group and the model group (P > 0.05). These results indicate that alcohol intervention can lead to weight loss and increased liver indices in mice, but dietary intervention had no significant effect on these changes.

[0101] 3.2 Effect of Lactobacillus fermentum DACN611 on Loss of Corrective Reflex (LORR)

[0102] Alcohol can affect the central nervous system, causing many behavioral problems, including the Loo-Rate Response (LORR). The LORR delay time represents the time from alcohol administration to intoxication in mice; the shorter the time, the deeper the intoxication. The LORR duration represents the time from intoxication to recovery; the longer the time, the deeper the intoxication. For example... Figure 3 As shown in a and b, the LORR latency and duration in the Alcohol+DACN611 group were 53.50 ± 7.48 min and 146.30 ± 12.35 min, respectively. Compared with the model group, *Lactobacillus fermentum* DACN611 shortened the intoxication time by 140.45% and the sobering-up time by 42.52%. Furthermore, there was no significant difference in LORR latency and latency between the positive control group and the Alcohol+DACN611 group (P > 0.05). These results further confirm our hypothesis that *Lactobacillus fermentum* DACN611 has a positive effect on alcohol metabolism in mice.

[0103] 3.3 Effects of Lactobacillus fermentum DACN611 on alcohol metabolism in mice

[0104] On day eight, serum ethanol and acetaldehyde levels were measured 5 hours after the last alcohol intake. Figure 4 As shown in ab, the in vivo ethanol degradation rate of fermenting Lactobacillus mucin DACN611 was 77.04%, and the in vivo acetaldehyde degradation rate was 77.66%, which were significantly different from those of the model group mice (P < 0.05).

[0105] Probiotics can lower blood concentrations of ethanol and acetaldehyde by increasing the activity of alcohol-metabolizing enzymes. The main enzymes involved in alcohol metabolism are ADH and ALDH. Figure 4 As shown in cf, alcohol intake significantly reduced ADH and ALDH activities in the stomach and liver (P < 0.05), while *Lactobacillus fermentum* DACN611 could restore these reductions. Meanwhile, there was no significant difference in ADH and ALDH levels between the Positive group and the Alcohol + DACN611 group (P > 0.05). Figure 5 As shown in b, the expression of CYP2E1 protein in mouse liver was detected by immunohistochemistry. The results showed that, compared with the control group, the expression of CYP2E1 protein in the liver of the model group mice was significantly increased, but this increase could be reversed by *Lactobacillus fermentum* DACN611. These results indicate that *Lactobacillus fermentum* DACN611 can protect mice from the effects of alcohol metabolism disorders caused by excessive alcohol intake.

[0106] 3.4 Effects of fermentation of Lactobacillus mucinus DACN611 on serum ALT and AST levels in mice, and on liver tissue morphology.

[0107] The liver is the largest detoxification organ and lipid metabolism center in the human body. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important indicators for diagnosing liver function and can reflect the degree of liver abnormalities. ALT and AST are mainly distributed in hepatocytes. When hepatocytes die, ALT and AST are released into the bloodstream, causing an increase in serum enzyme levels. The level of this increase is positively correlated with the degree of liver tissue abnormality. Figure 6 As shown in a and 6b, alcohol intervention increased serum ALT and AST levels in mice, but after treatment with *Lactobacillus fermentum*, serum ALT and AST levels in mice decreased significantly (P < 0.05). Meanwhile, there was no significant difference in efficacy between the positive control group and the *Lactobacillus fermentum* DACN611 treatment (P > 0.05).

[0108] 3.5 Effects of fermentation of Lactobacillus mucinus DACN611 on mouse liver tissue morphology

[0109] Acute heavy alcohol consumption can impair normal liver function. Excessive ethanol intake can lead to lipid metabolism disorders and inflammatory responses in the liver of mice. Therefore, pathological changes in mouse livers may include fat vacuoles and infiltration of inflammatory factors. Figure 6 As shown in Figure c, in the normal group mice, the hepatic cords were arranged radially around the central vein, and the hepatocyte structure was intact. In the model group mice, the hepatic cords were disordered, microvacuolization appeared in the hepatocyte cytoplasm, and there was infiltration of a large number of inflammatory factors. However, the degree of liver damage was significantly reduced after treatment with the positive control drug and Lactobacillus fermentum DACN611.

Claims

1. A strain of fermenting *Lactobacillus mucilaginosus* ( Limosilactobacillus fermentum DACN611 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20232522.

2. A microbial agent, the active ingredient of which comprises the fermenting Lactobacillus mucinus DACN611 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that: Fermented Lactobacillus mucinus DACN611 can be used alone.

4. The method for preparing the microbial agent according to claim 2 or 3, characterized in that: The fermenting Lactobacillus mucinus DACN611 of claim 1 was obtained by inoculating it into a culture medium.

5. The application of the fermenting Lactobacillus mucinus DACN611 of claim 1 or the bacterial agent of claim 2 or 3 in the preparation of products for degrading alcohol or for relieving hangovers and protecting the liver, wherein the product is a functional food or a food additive.

6. The application according to claim 5, characterized in that: The product has at least one of the following functions: (1) Degrades ethanol and / or acetaldehyde in serum; (2) Shorten the time of intoxication and sobering up of the subjects; (3) Increase the activity of ADH and ALDH enzymes in the subjects; (4) Remove ethanol and acetaldehyde from the subject’s body; the body includes blood, gastric juice, or intestinal fluid including duodenal fluid, small intestinal fluid or large intestinal fluid; (5) Protect subjects from the effects of alcohol metabolism disorders caused by excessive alcohol intake; (6) To prevent the rise in serum ALT and AST after the subjects ingested alcohol; (7) Reduce liver damage caused by alcohol consumption in subjects.

7. The application according to claim 5, characterized in that: According to the source of raw materials for the functional foods, the foods include dairy products, soy products, fruit and vegetable products, and fermented foods.

8. The application according to claim 5, characterized in that: According to the state of the functional food, the food includes solid food, liquid food, and semi-solid food.

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