Animal bifidobacterium lactis ba-c10 improving alcohol metabolism or liver damage and application thereof

Abstract

The application discloses animal bifidobacterium lactis BA-C10 for improving alcohol metabolism or liver injury and application thereof, wherein the animal bifidobacterium lactis BA-C10 has been preserved in the China Center for Type Culture Collection on March 11, 2024, and the preservation number is CCTCC NO: M 2024462. The product prepared from the animal bifidobacterium lactis BA-C10 has the effect of promoting alcohol metabolism and can reduce the damage of alcohol to the liver.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bifidobacterium lactis subspecies BA-C10 that improves alcohol metabolism or liver damage and its applications. Background Technology

[0002] With the improvement of living standards and increased stress, the frequency of alcohol consumption in daily social activities has increased significantly, leading to a year-on-year increase in alcohol-induced liver damage. Alcoholic liver disease (ALD) refers to a series of liver diseases caused by long-term heavy drinking, including alcoholic fatty liver disease (AFLD), alcoholic hepatitis (AH), and alcoholic cirrhosis (AC). The occurrence of these diseases is closely related to the toxic effects of alcohol on the liver, abnormal activation of the immune system, and the damaging mechanisms of oxidative stress.

[0003] During alcohol metabolism, ethanol is converted to acetaldehyde by alcohol dehydrogenase (ADH), which is then converted to acetic acid by aldehyde dehydrogenase (ALDH). Acetaldehyde, a highly reactive and toxic metabolite, can trigger significant oxidative stress, causing oxidative damage to intracellular lipids, proteins, and DNA, thereby inducing hepatocyte apoptosis and necrosis. Furthermore, alcohol prevents the effective removal of large amounts of intestinal toxins and harmful metabolites (such as ammonia, indole, and phenolic compounds), leading to intestinal microecological imbalance, promoting the overgrowth of harmful bacteria, increasing endotoxin absorption, and ultimately causing liver inflammation and damage. Therefore, developing effective intervention strategies and treatments is of significant scientific importance.

[0004] In recent years, probiotics, as beneficial microorganisms that regulate the gut microbiota, have received widespread attention. Studies have found that probiotics can protect against alcohol-induced liver damage by regulating the gut microbiota, enhancing intestinal barrier function, and reducing the absorption of endotoxins. These effects of probiotics can effectively reduce the toxicity of alcohol to the liver and slow the progression of alcoholic liver disease. Therefore, probiotics have potential application value as an adjunct therapy in the prevention and treatment of alcoholic liver disease. Summary of the Invention

[0005] The purpose of this invention is to provide a Bifidobacterium lactis subsp. BA-C10 that improves alcohol metabolism or liver damage, and its applications. Products prepared from Bifidobacterium lactis subsp. BA-C10 of this invention have the effect of promoting alcohol metabolism and reducing alcohol-induced liver damage.

[0006] The technical solution of the present invention is: a subspecies of Bifidobacterium lactis BA-C10, which was deposited at the China Center for Type Culture Collection on March 11, 2024, with accession number: CCTCC NO: M2024462.

[0007] The above-mentioned application of Bifidobacterium animalis subsp. lactis BA-C10 in the preparation of products that promote alcohol metabolism.

[0008] The aforementioned applications refer to products such as microbial agents, food, and / or pharmaceuticals.

[0009] In the aforementioned applications, the food is one or more of fermented milk, cheese, milk-containing beverages, solid beverages, and milk powder; the food contains live and / or inactivated solutions of Bifidobacterium animalis subsp. lactis BA-C10.

[0010] In the aforementioned applications, the drug comprises a live and / or inactivated solution of Bifidobacterium lactis subsp. BA-C10, and / or other drugs compatible with the live and / or inactivated solution of Bifidobacterium lactis subsp. BA-C10, as well as pharmaceutically acceptable carriers and / or excipients.

[0011] In the aforementioned applications, the Bifidobacterium animalis subsp. lactis BA-C10 is used to prepare microbial agents, foods, and / or pharmaceuticals that enhance the activity of alcohol dehydrogenase and glutathione peroxidase.

[0012] In the aforementioned applications, the Bifidobacterium animalis subsp. lactis BA-C10 is used to prepare microbial agents, foods, and / or pharmaceuticals that reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

[0013] In the aforementioned applications, the Bifidobacterium lactis subsp. BA-C10 is used to prepare bacterial agents, foods, and / or drugs that improve alcoholic liver injury.

[0014] The aforementioned application, and the preparation of the live bacterial solution of *Bifidobacterium animalis* subsp. *lactamase* BA-C10, are as follows:

[0015] The strain BA-C10, preserved in glycerol tubes, was first streaked 2-3 times on LB agar plates for activation. Then, single colonies were picked and cultured in LB liquid medium at 37°C for 18-24 hours until the bacterial concentration reached 10⁻⁶. 9 ~10 10 Approximately CFU / mL, used as a live bacterial solution.

[0016] The inactivated bacterial solution of Bifidobacterium animalis subsp. lactis BA-C10, as described above, is prepared as follows:

[0017] The live bacterial solution was inactivated by high temperature and high pressure at 121℃ for 30 minutes to obtain the inactivated bacterial solution.

[0018] Compared with existing technologies, the *Bifidobacterium lactis* subsp. BA-C10 of this invention promotes alcohol metabolism by reducing plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and increasing the activity of hepatic alcohol dehydrogenase (ALT) and glutathione peroxidase (GRP), thereby reducing alcohol-induced liver damage. Experiments have shown that both the live and inactivated bacterial solutions of *Bifidobacterium lactis* subsp. BA-C10 of this invention have excellent alcohol metabolism-promoting capabilities and are safe, effective, and have no significant side effects. Furthermore, *Bifidobacterium lactis* subsp. BA-C9 of this invention exhibits good gastrointestinal tolerance, thus surviving more easily in the stomach and small intestine after oral or edible administration. The development of the live and inactivated bacterial solutions of *Bifidobacterium lactis* subsp. BA-C10 of this invention opens up new directions for the research and improvement of products that promote alcohol metabolism. Attached Figure Description

[0019] Figure 1 Image of bacterial cell morphology after Gram staining with BA-C10;

[0020] Figure 2 The effects of BA-C10 on plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice;

[0021] Figure 3 The effect of BA-C10 on the rate of alcohol metabolism in mice;

[0022] Figure 4 The effect of BA-C10 on the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in mice;

[0023] Figure 5 Effects of inactivated BA-C10 on plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice;

[0024] Figure 6 The effect of inactivated BA-C10 on the rate of alcohol metabolism in mice;

[0025] Figure 7 Effects of inactivated BA-C10 on the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in mice. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0027] Example 1: Screening and identification of Bifidobacterium animalis subsp. lactis BA-C10:

[0028] 1.1 Sample source;

[0029] The strain used in this invention was isolated from the feces of a six-year-old child.

[0030] 1.2 Isolation and purification of bacterial strains;

[0031] Remove the strain from the -80℃ freezer and inoculate it onto fresh LB medium at pH 6 using a streak inoculation method. Incubate upside down in a 37℃ incubator for 2-3 days. Once single colonies have grown, pick up a single colony with an inoculation loop and inoculate it onto fresh LB medium at pH 6 using a streak inoculation method. After single colonies have grown, store them at 4℃ for later use.

[0032] 2. Identification of Bifidobacterium animalis subsp. lactis BA-C10;

[0033] 2.1 Colony characteristics;

[0034] After culturing the isolated and purified Bifidobacterium lactis subsp. BA-C10 on LB solid medium for 48 hours, the colonies were nearly round, white, uniform in size, with smooth and neat edges without wrinkles, and moist surface.

[0035] 2.2. Microscopic morphology;

[0036] Bifidobacterium animalis subsp. lactis BA-C10 colony smear: Gram-negative, non-spore-forming, short, round-ended rods, single or in short chains, see Figure 1 .

[0037] 2.3 Identification of 16S rDNA;

[0038] Genomic DNA was extracted from the target bacterial strain using the Ezup column-based bacterial genomic DNA extraction kit. This extracted genomic DNA was used as a template for PCR amplification. PCR experiments were performed using universal bacterial primers 27F and 1492R for 16S rDNA. After PCR amplification, the PCR products were analyzed and photographed on an agarose gel. The amplified fragment length was approximately 1.2 kbp. The PCR products were sent to Zhejiang Tianke High-Tech Development Co., Ltd. for sequencing. The results are as follows:

[0039]

[0040] BLAST sequence alignment was performed on the NCBI website, and the results showed that the sequence had more than 99% homology with the 16S rDNA sequence identified as a single bacterium of *Bifidobacterium animalis* subsp. *lactis*. Combining the sequence alignment results and physiological and biochemical results of strain *Bifidobacterium animalis* subsp. *lactis* BA-C10, the screened strain was confirmed as *Bifidobacterium animalis* subsp. *lactis*, and therefore named *Bifidobacterium animalis* subsp. *lactis* BA-C10. The strain of this invention is deposited under the name *Bifidobacterium animalis* subsp. *lactis* BA-C10, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2024462, deposited on March 11, 2024. The classification name of this invention is *Bifidobacterium animalis* subsp. *lactis* BA-C10.

[0041] In this invention:

[0042] The preparation method of BA-C10 bacterial suspension is as follows: The BA-C10 strain preserved in glycerol tubes is first streaked onto LB agar plates 2-3 times for activation. Then, single colonies are picked and cultured in LB liquid medium at 37°C for 18-24 hours until the bacterial suspension concentration reaches 10. 9 ~10 10 The concentration is approximately CFU / mL, forming a bacterial suspension (a live bacterial suspension). In practical use, the bacterial suspension can be adjusted to the desired concentration using physiological saline, for example, to 5 × 10⁻⁶ CFU / mL. 9 CFU / mL.

[0043] 2. The above-mentioned BA-C10 bacterial suspension was inactivated at 121℃ under high temperature and high pressure for 30 minutes to obtain the inactivated BA-C10 bacterial solution.

[0044] Example 2: Bifidobacterium animalis subsp. lactis BA-C10 promotes alcohol metabolism;

[0045] 1. Laboratory Animals: Forty-eight male C57BL / 6 mice were housed in an SPF (Special Purpose Free) environment at the Laboratory Animal Center of Zhejiang University of Traditional Chinese Medicine. Housing Environment: Room temperature 20-25℃, with alternating light and dark every 12 hours, relative humidity approximately 50%. Feed and water were uniformly provided, and the indoor environment was well-ventilated and cleaned daily to maintain a hygienic environment within the cages. All experiments were conducted in accordance with animal ethics guidelines and the relevant regulations of the Laboratory Animal Ethics Committee, specifically following IAC (Independent Animal Control) and other relevant standard operating procedures (SOPs). After the experiments, blood was drawn under abdominal aortic anesthesia, and the carcasses were disposed of harmlessly and uniformly.

[0046] 2. Reagents: Anhydrous ethanol (CAS-NO: 64-17-5, lichrosolv), ALT kit (Catalog No.: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (Catalog No.: C010-2 Nanjing Jiancheng Bioengineering Institute), glutathione peroxidase assay kit (Catalog No.: S0057S Beyotime Biotechnology), alcohol dehydrogenase assay kit (Catalog No.: BC1085 Solarbio).

[0047] 3. Method:

[0048] After one week of acclimatization in an SPF-grade animal laboratory, 8-week-old C57BL / 6 mice were randomly divided into two groups of 24 mice each. Intervention methods: The BA-C10 group received 0.2 ml of BA-C10 bacterial suspension (concentration 10⁹ CFU / mL) via gavage daily, while the CON group received 0.2 ml of physiological saline via gavage daily. After 4 weeks of continuous intervention, the mice were anesthetized by intraperitoneal injection of 1% pentobarbital, and blood was collected from the inferior vena cava to measure plasma ethanol and other indicators.

[0049] 3.1 Gas chromatography (GC) is used to detect plasma ethanol content via headspace vials;

[0050] Dilute the sample (mouse plasma) with ultrapure water (1:1). Gently shake; if the sample contains many impurities, centrifuge lightly (4℃, 3000×g, 5 minutes) and collect the supernatant. Mix the supernatant with chromatographic grade tert-butanol (1:5), filter through a 0.45μm disposable filter, and inject into a chromatographic vial. The gas chromatograph includes an AOC-20S autosampler, a flame ionization detector (FID), a GC-2010, and an Rtx-BAC1 column (30m×0.32mm×1.8μm). Injection method: split injection, split ratio 20:1; carrier gas control: constant linear velocity, carrier gas is nitrogen (2ml / min), injection volume 0.2μL. The column temperature program can be adjusted as follows: initial temperature 40℃, hold for 2 minutes, increase to 240℃ at a rate of 10℃ / min, hold for 1 minute, total time 23min.

[0051] 3.2 Plasma ALT detection;

[0052] Mouse plasma samples were directly collected for testing. The matrix solution was preheated to 37°C. 20 μL of matrix solution and 5 μL of the test sample were added to the sample wells and mixed thoroughly. 20 μL of matrix solution was added to the control wells, and the mixture was incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to both the test and control wells, and 5 μL of the test sample was added to the control wells. The mixture was then incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed thoroughly, and incubated at room temperature for 15 min. The OD value of each well was measured using a microplate reader at 510 nm. The corresponding ALT / GPT activity units were determined by referring to the standard curve.

[0053] 3.3 Plasma AST detection;

[0054] Mouse plasma samples were directly collected for testing. The matrix solution was preheated to 37°C. 20 μL of matrix solution and 5 μL of the test sample were added to the sample wells and mixed thoroughly. 20 μL of matrix solution was added to the control wells, and the mixture was incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to both the test and control wells, and 5 μL of the test sample was added to the control wells. The mixture was then incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed thoroughly, and incubated at room temperature for 15 min. The OD values ​​of each well were measured using a microplate reader at 510 nm. The corresponding AST / GPT activity units were determined by referring to the standard curve.

[0055] 3.4 Determination of Liver Alcohol Dehydrogenase (ADH) Content

[0056] To detect alcohol dehydrogenase (ADH) activity, the tissue mass (g) and extraction liquid volume (mL) were first homogenized under ice bath conditions at a ratio of 1:5-10 (approximately 0.1g of tissue was recommended to be added to 1mL of extraction liquid). The homogenate was then centrifuged at 16,000×g for 20 minutes at 4°C, and the supernatant was placed on ice for analysis. ADH catalyzes the reduction of acetaldehyde to ethanol by NADH. NADH exhibits an absorption peak at 340nm, while NAD+ shows no absorption. ADH activity was calculated by measuring the rate of decrease in absorbance at 340nm. A microplate reader was preheated for 30 minutes, set to a wavelength of 340nm, and zeroed with distilled water. Reagent 1 was preheated in a 25°C water bath for at least 30 minutes. Blank tube: Add 20 μL of distilled water, 8 μL of reagent II, 152 μL of reagent I, and 20 μL of reagent III to a 96-well plate. Mix well and measure the absorbance values ​​A1 and A2 at 340 nm after 15 seconds and 75 seconds, respectively. Calculate ΔAblank tube = A1 - A2. Test tube: Add 20 μL of supernatant instead of distilled water and repeat the above operation. Record the absorbance values ​​A3 and A4 at 15 seconds and 75 seconds, respectively. Calculate ΔAtest tube = A3 - A4. Calculate ADH activity based on the difference between ΔAtest tube and ΔAblank tube.

[0057] 3.5. Determination of liver glutathione reductase (GR) content;

[0058] To detect glutathione reductase (GR) activity, animals were first perfused with physiological saline containing 0.16 mg / mL heparin to remove blood. Subsequently, tissue samples were collected and homogenized at a ratio of 200 μL lysis buffer per 20 mg of tissue. Homogenization was performed using TissueMaster. TM The tissue homogenizer was used at 4°C. After homogenization, the sample was centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was used to detect GR activity. During the assay, the sample was reacted with oxidized glutathione (GSSG) and the chromogenic substrate DTNB. The generated yellow TNB showed a linear positive correlation with GR activity. The amount of TNB generated was measured at an A412 wavelength using a microplate reader to calculate GR activity. Since glutathione reductase is the rate-limiting factor in the reaction system, the amount of TNB generated reflects the activity of GR.

[0059] 4. Indicator measurement;

[0060] Figure 2 The effects of BA-C10 on plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice. Figure 2 The results showed that mice in the CON group were administered 0.2 ml of physiological saline by gavage daily, while mice in the BA-C10 group were administered 0.2 ml of BA-C10 bacterial culture at a concentration of 10⁹ CFU / mL by gavage daily. Plasma ALT and AST levels were measured after gavage administration of ethanol (5 g / kg) (p < 0.05). The results indicated that serum ALT and AST levels were significantly lower in the BA-C10 group compared to the CON group.

[0061] Figure 3 Effects of BA-C10 on alcohol metabolism rate in mice. Figure 3 The results showed that after oral administration of ethanol (5 g / kg), the plasma ethanol content in the BA-C10 group mice was lower than that in the CON group at 2 hours, 4 hours, and 8 hours (p<0.05). This indicates that the BA-C10 bacterial solution can accelerate the metabolism of ethanol.

[0062] The effects of BA-C10 on the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in mice. Figure 4 The results showed that after oral administration of ethanol (5 g / kg), the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in the BA-C10 group mice were significantly higher than those in the CON group (p<0.05). This indicates that the BA-C10 bacterial culture can enhance the metabolism and antioxidant capacity of ethanol.

[0063] Example 3: Preparation of inactivated Bifidobacterium lactis subsp. BA-C10 bacterial suspension

[0064] Dissolve 1 g of BA-C10 bacterial powder in 500 mL of LB liquid medium, incubate at 37°C for 24 h, then inactivate the bacterial suspension at 121°C under high temperature and high pressure for 30 min, centrifuge at 3000 rpm at room temperature for 15 min. After centrifugation, discard the supernatant, add an appropriate amount of PBS to wash off the residual medium, centrifuge again, discard the supernatant to obtain inactivated Bifidobacterium animalis subsp. lactis BA-C10 bacterial cells. Add 1 mL of normal saline to every 50 mg of bacterial cells for dissolution to obtain an inactivated Bifidobacterium animalis subsp. lactis BA-C10 bacterial suspension.

[0065] Example 4: Promotion of alcohol metabolism by inactivated Bifidobacterium animalis subsp. lactis BA-C10 bacterial cells

[0066] 3.1. Laboratory animals

[0067] 48 male C57BL / 6 mice, purchased from Shanghai SLAC Laboratory Animal Co., Ltd., with the company license number: SCXK (Shanghai) 2013-0016, were housed in the SPF-grade animal feeding room of the Laboratory Animal Center of Zhejiang Chinese Medical University. Feeding environment: room temperature 20-25°C, 12-hour light-dark cycle, relative humidity of about 50%, uniformly matched feed, drinking water and other supplies, good indoor ventilation, daily cleaning to maintain a good hygienic environment in the cages. All experiments complied with the animal ethics code and relevant regulations of the Laboratory Animal Ethics Committee, and were specifically carried out in accordance with the IAC category and other relevant standard operating procedures (SOP). After the experiment, blood was collected via abdominal aorta under anesthesia, and the carcasses were subjected to unified harmless treatment.

[0068] 3.2. Reagents: absolute ethanol (CAS-NO: 64-17-5, lichrosolv), ALT assay kit (Cat. No.: C009-2, Nanjing Jiancheng Bioengineering Institute), AST assay kit (Cat. No.: C010-2, Nanjing Jiancheng Bioengineering Institute), glutathione peroxidase detection kit (Cat. No.: S0057S, Beyotime Biotechnology), alcohol dehydrogenase detection kit (Cat. No.: BC1085, Solarbio).

[0069] 3.3. Methods

[0070] After one week of adaptive feeding in the SPF-grade animal laboratory, 8-week-old C57BL / 6 mice were randomly divided into 2 groups, with 24 mice in each group. Intervention method: The BA-C10 group was gavaged with 0.2 mL of BA-C10 heat-killed bacterial suspension (BA-C10 heat-killed) every day, and the CON group was gavaged with 0.2 mL of normal saline every day. After 4 weeks of continuous intervention, the mice were anesthetized by intraperitoneal injection of 1% pentobarbital, and blood was collected from the inferior vena cava to determine indicators such as plasma ethanol.

[0071] 3.1 Gas chromatography (GC) is used to detect plasma ethanol content via headspace vials;

[0072] Dilute the sample (mouse plasma) with ultrapure water (1:1). Gently shake; if the sample contains many impurities, centrifuge lightly (4℃, 3000×g, 5 minutes) and collect the supernatant. Mix the supernatant with chromatographic grade tert-butanol (1:5), filter through a 0.45μm disposable filter, and inject into a chromatographic vial. The gas chromatograph includes an AOC-20S autosampler, a flame ionization detector (FID), a GC-2010, and an Rtx-BAC1 column (30m×0.32mm×1.8μm). Injection method: split injection, split ratio 20:1; carrier gas control: constant linear velocity, carrier gas is nitrogen (2ml / min), injection volume 0.2μL. The column temperature program can be adjusted as follows: initial temperature 40℃, hold for 2 minutes, increase to 240℃ at a rate of 10℃ / min, hold for 1 minute, total time 23min.

[0073] 3.2 Plasma ALT detection

[0074] Mouse plasma samples were directly collected for testing. The matrix solution was preheated to 37°C. 20 μL of matrix solution and 5 μL of the test sample were added to the sample wells and mixed thoroughly. 20 μL of matrix solution was added to the control wells, and the mixture was incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to both the test and control wells, and 5 μL of the test sample was added to the control wells. The mixture was then incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed thoroughly, and incubated at room temperature for 15 min. The OD value of each well was measured using a microplate reader at 510 nm. The corresponding ALT / GPT activity units were determined by referring to the standard curve.

[0075] 3.3 Plasma AST detection

[0076] Mouse plasma samples were directly collected for testing. The matrix solution was preheated to 37°C. 20 μL of matrix solution and 5 μL of the test sample were added to the sample wells and mixed thoroughly. 20 μL of matrix solution was added to the control wells, and the mixture was incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to both the test and control wells, and 5 μL of the test sample was added to the control wells. The mixture was then incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed thoroughly, and incubated at room temperature for 15 min. The OD values ​​of each well were measured using a microplate reader at 510 nm. The corresponding AST / GPT activity units were determined by referring to the standard curve.

[0077] 3.4 Determination of Liver Alcohol Dehydrogenase (ADH) Content

[0078] To detect alcohol dehydrogenase (ADH) activity, the tissue mass (g) and extraction liquid volume (mL) were first homogenized under ice bath conditions at a ratio of 1:5-10 (approximately 0.1g of tissue was recommended to be added to 1mL of extraction liquid). The homogenate was then centrifuged at 16,000×g for 20 minutes at 4°C, and the supernatant was placed on ice for analysis. ADH catalyzes the reduction of acetaldehyde to ethanol by NADH. NADH exhibits an absorption peak at 340nm, while NAD+ shows no absorption. ADH activity was calculated by measuring the rate of decrease in absorbance at 340nm. A microplate reader was preheated for 30 minutes, set to a wavelength of 340nm, and zeroed with distilled water. Reagent 1 was preheated in a 25°C water bath for at least 30 minutes. Blank tube: Add 20 μL of distilled water, 8 μL of reagent II, 152 μL of reagent I, and 20 μL of reagent III to a 96-well plate. Mix well and measure the absorbance values ​​A1 and A2 at 340 nm after 15 seconds and 75 seconds, respectively. Calculate ΔAblank tube = A1 - A2. Test tube: Add 20 μL of supernatant instead of distilled water and repeat the above operation. Record the absorbance values ​​A3 and A4 at 15 seconds and 75 seconds, respectively. Calculate ΔAtest tube = A3 - A4. Calculate ADH activity based on the difference between ΔAtest tube and ΔAblank tube.

[0079] 3.5. Determination of liver glutathione reductase (GR) content;

[0080] To detect glutathione reductase (GR) activity, animals were first perfused with physiological saline containing 0.16 mg / mL heparin to remove blood. Subsequently, tissue samples were collected and homogenized at a ratio of 200 μL lysis buffer per 20 mg of tissue. Homogenization was performed using TissueMaster. TM The tissue homogenizer was used at 4°C. After homogenization, the sample was centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was used to detect GR activity. During the assay, the sample was reacted with oxidized glutathione (GSSG) and the chromogenic substrate DTNB. The generated yellow TNB showed a linear positive correlation with GR activity. The amount of TNB generated was measured at an A412 wavelength using a microplate reader to calculate GR activity. Since glutathione reductase is the rate-limiting factor in the reaction system, the amount of TNB generated reflects the activity of GR.

[0081] 3.4. Index Measurement:

[0082] Figure 5 Effects of inactivated BA-C10 on plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice. Figure 5 The results showed that the CON group mice were administered 0.2 mL of physiological saline by gavage daily, while the BA-C10 group mice received 0.2 mL of 10% saline solution. 9The BA-C10 bacterial suspension at CFU / mL was administered to the group receiving the inactivated BA-C10 suspension via gavage, while the inactivated BA-C10 group received an equal volume of inactivated bacterial suspension. After gavage administration of ethanol (5 g / kg), plasma ALT and AST levels were measured (p < 0.05), showing that serum ALT and AST levels in both the BA-C10 and inactivated BA-C10 groups were significantly lower than those in the CON group. This indicates that inactivation of the BA-C10 bacterial suspension can effectively alleviate ethanol-induced liver injury.

[0083] Figure 6 Effects of inactivated BA-C10 on the rate of alcohol metabolism in mice. Figure 6 The results showed that the ethanol levels in mouse plasma were measured at 1 h, 2 h, 4 h, and 8 h after oral administration of ethanol (5 g / kg). The results indicated that the plasma ethanol levels in the BA-C10 group and the inactivated BA-C10 group were significantly lower than those in the CON group (p < 0.05). This suggests that both live and inactivated BA-C10 bacterial solutions can effectively promote ethanol metabolism and reduce plasma ethanol levels.

[0084] Figure 7 Effects of inactivated BA-C10 on the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in mice. Figure 7 The results showed that after oral administration of ethanol (5 g / kg), the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in mice in the BA-C10 group and the inactivated BA-C10 group were significantly higher than those in the CON group (p<0.05). This indicates that both live BA-C10 bacteria and the inactivated bacterial solution can significantly enhance the ethanol metabolism and antioxidant capacity of mice.

[0085] Example 5: Using Bifidobacterium lactis subsp. BA-C10 as the research object, a gastrointestinal tolerance experiment was simulated, and Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019 were used as comparative examples.

[0086] Tolerance test in gastrointestinal fluid: Simulated human gastrointestinal fluid was prepared, and Bifidobacterium animalis subsp. lactis BA-C10, Bifidobacterium lactis BL-99, and Bifidobacterium lactis HN019 were treated in the simulated gastrointestinal fluid for different time periods. The viable counts of Bifidobacterium animalis subsp. lactis BA-C10, Bifidobacterium lactis BL-99, and Bifidobacterium lactis HN019 after treatment in the simulated gastrointestinal fluid were determined by plate count method.

[0087] Preparation of simulated gastric juice: Prepare a pepsin solution with a concentration of 3 mg / mL using PBS at pH 2.5. This is the artificial gastric juice. After sterilization by filtration through a 0.22 μm microporous membrane, it is ready for use.

[0088] Preparation of simulated intestinal fluid: Prepare a 1 mg / mL trypsin solution with pH 8.0 PBS, add bovine bile salts to make the final concentration 1.8%, and the resulting solution is the artificial intestinal fluid. After filtration through a 0.22 μm microporous membrane for sterilization, it is ready for use.

[0089] Bifidobacterium animalis subsp. lactis BA-C10, Bifidobacterium lactis BL-99, and Bifidobacterium lactis HN019 bacterial powders were added to sterile PBS (pH 7.3), shaken to resuspend, and 500 μL of the resuspended solution was added to 4.5 mL of simulated gastric fluid. After incubation at 37°C for 3 hours, bacterial counts were performed using the pour plate method. Then, another 500 μL of the bacterial suspension after incubation in simulated gastric fluid for 3 hours was added to 4.5 mL of simulated intestinal fluid, and incubated at 37°C for 8 hours, followed by counts using the pour plate method. The survival rate was calculated using the following formula.

[0090] Survival rate = Number of colonies after treatment (CFU / mL) / Initial number of colonies (CFU / mL) × 100%. The gastrointestinal tolerance of *Bifidobacterium animalis* subsp. *lactamase* BA-C10, *Bifidobacterium lactis* BL-99, and *Bifidobacterium lactis* HN019 is shown in Table 1 below.

[0091]

[0092] Table 1

[0093] The table shows that after 3 hours in gastric fluid, the viable count of *Bifidobacterium animalis* subsp. *lactamase* BA-C10 remained at 36.33%, while after 8 hours in intestinal fluid, the viable count decreased to 80.7% of the number of viable bacteria treated with gastric fluid. This indicates that *Bifidobacterium animalis* subsp. *lactamase* BA-C10 has high gastrointestinal tolerance and, compared with *Bifidobacterium lactis* BL-99 and *Bifidobacterium lactis* HN019, exhibits higher tolerance to simulated gastrointestinal fluid.

[0094] In summary, the *Bifidobacterium lactis* subsp. BA-C10 of this invention promotes alcohol metabolism by reducing plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and increasing hepatic alcohol dehydrogenase and glutathione peroxidase (GLP) activities, thereby reducing alcohol-induced liver damage. Experiments have demonstrated that both the live and inactivated bacterial solutions of *Bifidobacterium lactis* subsp. BA-C10 of this invention possess excellent alcohol metabolism-promoting capabilities and are safe, effective, and have no significant side effects. The development of the live and inactivated bacterial solutions of *Bifidobacterium lactis* subsp. BA-C10 of this invention opens up new directions for the research and improvement of products that promote alcohol metabolism.

[0095] The above description is merely a selection of preferred embodiments of this disclosure and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in the embodiments of this disclosure is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in the embodiments of this disclosure.

Claims

1. The application of *Bifidobacterium animalis* subsp. *lactamase* BA-C10 in the preparation of products that improve alcoholic liver injury, enhance ethanol metabolism, reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and increase alcohol dehydrogenase and glutathione peroxidase (GLP) activities, wherein the products are bacterial agents or drugs; characterized in that: The Bifidobacterium animalis subsp. lactis BA-C10 was deposited at the China Center for Type Culture Collection on March 11, 2024, with accession number: CCTCC NO: M 2024462; The drug comprises a live and / or inactivated solution of Bifidobacterium lactis subsp. BA-C10.

2. The application according to claim 1, characterized in that: The drug comprises a live and / or inactivated solution of Bifidobacterium lactis subsp. BA-C10, and / or other drugs compatible with the live and / or inactivated solution of Bifidobacterium lactis subsp. BA-C10, as well as pharmaceutically acceptable carriers and / or excipients.

3. The application according to claim 1, characterized in that: The preparation of the live bacterial culture of Bifidobacterium lactis subsp. BA-C10 is as follows: The strain BA-C10, preserved in glycerol tubes, was first streaked 2-3 times on LB agar plates for activation. Then, single colonies were picked and cultured in LB liquid medium at 37°C for 18-24 hours until the bacterial concentration reached 10⁻⁶. 9 ~10 10 CFU / mL was used as a live bacterial solution.

4. The application according to claim 3, characterized in that: The inactivated bacterial solution of Bifidobacterium lactis subsp. BA-C10 was prepared as follows: The live bacterial solution was inactivated by high temperature and high pressure at 121℃ for 30 minutes to obtain the inactivated bacterial solution.

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