Bifidobacterium animalis subsp. Lactis BA-C10 for improving alcohol metabolism or liver injury and application thereof

Abstract

The invention discloses an animal bifidobacterium subsp. Lactis BA-C10 for improving alcohol metabolism or liver injury and application of the animal bifidobacterium subsp. Lactis BA-C10, the animal bifidobacterium subsp. Lactis BA-C10 is preserved in 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 subsp. Lactis BA-C10 has the effect of promoting alcohol metabolism, and can reduce the damage of alcohol to the liver.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly relates to a Bifidobacterium animalis subsp. lactis BA-C10 for improving alcohol metabolism or liver injury and its application. Background Art

[0002] With the improvement of social living standards and the increase of pressure, the frequency of drinking behavior in daily social activities has increased significantly, resulting in an increasing trend of liver injury caused by alcohol year by year. 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 effect of alcohol on the liver, the abnormal activation of the immune system, and the damage mechanism of oxidative stress.

[0003] In the process of alcohol metabolism, ethanol is converted into acetaldehyde by Alcohol Dehydrogenase (ADH), and the latter is then converted into acetate by Aldehyde Dehydrogenase (ALDH). As a highly reactive toxic metabolite, acetaldehyde can trigger a significant oxidative stress response, causing oxidative damage to intracellular lipids, proteins, and DNA, thereby inducing apoptosis and necrosis of hepatocytes. In addition, alcohol causes a large amount of intestinal toxins and harmful metabolites (such as ammonia, indole, phenolic compounds) to be unable to be effectively cleared, leading to intestinal microecological imbalance, promoting the overgrowth of harmful bacteria, increasing the absorption of endotoxin, and then triggering liver inflammation and injury. Therefore, it has important scientific significance to develop effective intervention strategies and treatment means.

[0004] In recent years, probiotics, as beneficial microorganisms that regulate the intestinal microecology, have received extensive attention. Studies have found that probiotics have a protective effect on liver injury caused by alcohol by regulating the intestinal flora, enhancing the intestinal barrier function, and reducing the absorption of endotoxin. These effects of probiotics can effectively reduce the toxicity of alcohol to the liver and alleviate the progression of alcoholic liver disease. Therefore, probiotics, as an adjuvant treatment means, have potential application value in the prevention and treatment of alcoholic liver disease. Summary of the Invention

[0005] The object of the present invention is to provide a Bifidobacterium animalis subsp. lactis BA-C10 for improving alcohol metabolism or liver injury and its application. The product prepared from the Bifidobacterium animalis subsp. lactis BA-C10 of the present invention has the effect of promoting alcohol metabolism and can reduce the damage of alcohol to the liver.

[0006] Technical solution of the present invention: A Bifidobacterium animalis subsp. lactis BA-C10, which was deposited at the China Center for Type Culture Collection on March 11, 2024, with the deposit number: CCTCC NO: M2024462.

[0007] Application of the above-mentioned Bifidobacterium animalis subsp. lactis BA-C10 in the preparation of a product for promoting alcohol metabolism.

[0008] For the aforementioned application, the product is a bacterial agent, food, and / or drug.

[0009] For the aforementioned application, the food is one or more of fermented milk, cheese, milk-containing beverage, solid beverage, milk powder; the food contains the live bacterial liquid and / or inactivated bacterial liquid of Bifidobacterium animalis subsp. lactis BA-C10.

[0010] For the aforementioned application, the drug contains the live bacterial liquid and / or inactivated bacterial liquid of Bifidobacterium animalis subsp. lactis BA-C10, and / or other drug classes compatible with the live bacterial liquid and / or inactivated bacterial liquid of Bifidobacterium animalis subsp. lactis BA-C10, as well as pharmaceutically acceptable carriers and / or excipients.

[0011] For the aforementioned application, the Bifidobacterium animalis subsp. lactis BA-C10 is used for the preparation of a bacterial agent, food, and / or drug for increasing the activities of alcohol dehydrogenase and glutathione peroxidase.

[0012] For the aforementioned application, the Bifidobacterium animalis subsp. lactis BA-C10 is used for the preparation of a bacterial agent, food, and / or drug for reducing the levels of alanine aminotransferase and aspartate aminotransferase.

[0013] For the aforementioned application, the Bifidobacterium animalis subsp. lactis BA-C10 is used for the preparation of a bacterial agent, food, and / or drug for improving alcoholic liver injury.

[0014] For the aforementioned application, the preparation of the live bacterial liquid of Bifidobacterium animalis subsp. lactis BA-C10 is as follows:

[0015] The strain BA-C10 stored in a glycerol tube is first streaked and activated on an LB agar plate 2-3 times, and then a single colony is picked and cultured in an LB liquid medium at 37°C for 18-24 h until the concentration of the bacterial liquid reaches 10 9 ~10 10 CFU / mL or so, as the live bacterial liquid.

[0016] The aforementioned application, the preparation of the inactivated bacterial solution of Bifidobacterium animalis subsp. lactis BA-C10 is as follows:

[0017] The live bacterial solution is inactivated at high temperature and high pressure of 121 °C for 30 min to obtain the inactivated bacterial solution.

[0018] Compared with the prior art, Bifidobacterium animalis subsp. lactis BA-C10 of the present invention can promote alcohol metabolism by reducing the levels of plasma alanine aminotransferase and aspartate aminotransferase and increasing the activities of liver alcohol dehydrogenase and glutathione peroxidase, and can reduce the damage of alcohol to the liver. Experiments have proved that both the live bacterial solution and the inactivated bacterial solution of Bifidobacterium animalis subsp. lactis BA-C10 of the present invention have good ability to promote alcohol metabolism, and have the advantages of safety, effectiveness and no obvious side effects. In addition, Bifidobacterium animalis subsp. lactis BA-C9 of the present invention has good gastrointestinal tolerance, so it is more likely to survive through the stomach and small intestine after the product is orally taken or consumed. The research and development of the live bacterial solution and the inactivated bacterial solution of Bifidobacterium animalis subsp. lactis BA-C10 of the present invention have opened up a new direction for the research and improvement of products for promoting alcohol metabolism. Description of the Drawings

[0019] Figure 1 It is the morphological diagram of the bacteria body of BA-C10 by Gram staining;

[0020] Figure 2 It is the influence of BA-C10 on plasma alanine aminotransferase and aspartate aminotransferase (ALT and AST) of mice;

[0021] Figure 3 It is the influence of BA-C10 on the alcohol metabolism rate of mice;

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

[0023] Figure 5 It is the influence of inactivated BA-C10 on plasma alanine aminotransferase and aspartate aminotransferase (ALT and AST) of mice;

[0024] Figure 6 It is the influence of inactivated BA-C10 on the alcohol metabolism rate of mice;

[0025] Figure 7 It is the influence of inactivated BA-C10 on the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) of mice. Detailed Embodiments

[0026] The present invention will be further described below in conjunction with the drawings and embodiments, but it shall not be used as the basis for limiting the present invention.

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

[0028] 1.1 Source of samples

[0029] The strains used in this invention were isolated from the feces of six-year-old children.

[0030] 1.2 Isolation and purification of strains

[0031] Take out the preserved strains from the -80°C refrigerator and inoculate them onto a fresh LB medium with a pH of 6 by streaking. Incubate it in a 37°C constant temperature incubator for 2 - 3 days. When monoclonal colonies grow out, pick the monoclonal colonies with an inoculation loop and inoculate them onto a fresh LB medium with a pH of 6 by sectional streaking. Preserve them in a 4°C refrigerator for later use after monoclonal colonies grow out.

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

[0033] 2.1 Colony characteristics

[0034] After culturing Bifidobacterium animalis subsp. lactis BA-C10 obtained by isolation and purification on an LB solid medium for 48 h, the colonies are nearly circular, white, of uniform size, with smooth and regular edges without wrinkles, and the surface is moist.

[0035] 2.2 Morphology under the microscope

[0036] Smear of colonies of Bifidobacterium animalis subsp. lactis BA-C10: Gram staining is negative, non-spore-forming, short bacilli with round ends, single or in short chains, as shown Figure 1 .

[0037] 2.3 16S rDNA identification

[0038] Extract the genomic DNA of the target strain using an Ezup column bacterial genomic DNA extraction kit. Use the extracted genomic DNA of the target strain as the template for PCR amplification. Perform a 16S rDNA PCR experiment using the bacterial universal primers 27F and 1492R. After the PCR reaction amplification is completed, take the PCR product for agarose gel detection and photography. The amplified fragment length is about 1.2 kbp. Send the PCR product 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 a homology of over 99% with the 16S rDNA sequence for the single-strain identification of Bifidobacterium animalis subsp. lactis. By combining the sequence alignment results and the physiological and biochemical results of the strain Bifidobacterium animalis subsp. lactis BA-C10, it was determined that the screened Bifidobacterium animalis subsp. lactis BA-C10 was Bifidobacterium animalis subsp. lactis, and thus it was named Bifidobacterium animalis subsp. lactis BA-C10. The preservation name of the strain of the present invention is Bifidobacterium animalis subsp. lactis BA-C10, the preservation unit: China Center for Type Culture Collection, the preservation address: Wuhan University, Wuhan, China, the preservation number: CCTCC NO: M2024462, the preservation time: March 11, 2024, and the taxonomic naming of the present invention is Bifidobacterium animalis subsp. lactis BA-C10.

[0041] In the present invention:

[0042] The preparation method of the BA-C10 bacterial liquid is as follows: The strain BA-C10 preserved in a glycerol tube is first streaked and activated on an LB agar plate 2 - 3 times, and then a single colony is picked and cultured in an LB liquid medium at 37°C for 18 - 24 h until the concentration of the bacterial liquid reaches 10 9 ~10 10 CFU / mL or so, serving as a bacterial suspension (a live bacterial liquid). When actually used, the bacterial liquid can be adjusted to the required concentration with physiological saline, for example, adjusted to 5×10 9 CFU / mL.

[0043] 2. The above-mentioned bacterial suspension of BA-C10 is inactivated at 121°C under high temperature and high pressure for 30 min to serve as an inactivated BA-C10 bacterial liquid.

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

[0045] 1. Experimental animals: 48 male C57BL / 6 mice were housed in the SPF environment of the Experimental Animal Center of Zhejiang Chinese Medical University. Breeding environment: Room temperature 20 - 25°C, 12-hour light-dark alternation every day, relative humidity about 50%, the feed, drinking water, etc. were all uniformly matched, the indoor environment was kept well ventilated, cleaned every day, and the good sanitary environment in the cage was maintained. All experiments complied with the animal ethics code and the relevant regulations of the Experimental Animal Ethics Committee, and were specifically carried out according to the IAC category and other relevant standard operating procedures (SOP). After the experiment, blood was taken by abdominal aortic anesthesia, and the corpses were uniformly treated harmlessly.

[0046] 2. Reagents: anhydrous ethanol (CAS-NO: 64-17-5, lichrosolv), ALT kit (Cat. No.: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (Cat. No.: C010-2 Nanjing Jiancheng Bioengineering Institute), glutathione peroxidase detection kit (Cat. No.: S0057S Biyuntian Biotechnology), alcohol dehydrogenase detection kit (Cat. No.: BC1085 Solebao).

[0047] 3. Methods:

[0048] After one week of adaptation feeding in an SPF animal laboratory, 8-week-old C57BL / 6 mice were randomly divided into 2 groups, with 24 mice in each group. Intervention method: BA-C10 group was gavaged with 0.2 ml BA-C10 bacterial solution (concentration of bacterial solution 109 CFU / mL) every day, and CON group was gavaged with 0.2 ml normal saline every day. After 4 weeks of continuous intervention, mice were anesthetized with 1% pentobarbital intraperitoneal injection, and blood was collected from the inferior vena cava to measure plasma ethanol and other indicators.

[0049] 3.1. Gas chromatography (GC) was used to detect plasma ethanol content through headspace vials;

[0050] The sample to be tested (mouse plasma) was diluted with ultrapure water (1:1). Shake gently. If the sample contains more impurities, centrifuge gently (4°C, 3000×g, 5 minutes) and take the supernatant. After mixing the supernatant with chromatography-grade tert-butyl alcohol (1:5), filter it with a 0.45μm disposable filter and inject it into a chromatographic bottle. The gas chromatograph includes an automatic sampler AOC-20S, a flame ionization detector (FID), a GC-2010 and an Rtx-BAC1 chromatographic column (specifications are 30m×0.32mm×1.8μm). Injection mode: split injection, split ratio 20:1, carrier gas control mode: constant linear velocity, carrier gas is nitrogen (2ml / min), injection volume 0.2μL. The column temperature program can be adjusted as follows: the initial temperature is 40°C for 2 minutes, the temperature is increased to 240°C at a rate of 10°C / min, and it is maintained for 1 minute, and the total time is 23min.

[0051] 3.2. Plasma ALT detection;

[0052] Take the mouse plasma sample for direct sampling and determination. Preheat the matrix solution at 37°C in advance. Add 20 μL of the matrix solution and 5 μL of the sample to be tested into the sample well, mix well. Add 20 μL of the matrix solution to the control well, and incubate at 37°C for 30 min. Add 20 μL of 2,4-dinitrophenylhydrazine solution to both the measurement well and the control well, and add 5 μL of the sample to be tested to the control well, mix well, and incubate at 37°C for 20 min. Add 200 μL of 0.4 mol / L sodium hydroxide solution to each well, mix well, let stand at room temperature for 15 min, measure the OD value of each well at a wavelength of 510 nm with an enzyme-linked immunosorbent assay (ELISA) reader, check the standard curve, and obtain the corresponding ALT / GPT activity unit.

[0053] 3.3 Detection of plasma AST;

[0054] Take the mouse plasma sample for direct sampling and determination. Preheat the matrix solution at 37°C in advance. Add 20 μL of the matrix solution and 5 μL of the sample to be tested into the sample well, mix well. Add 20 μL of the matrix solution to the control well, and incubate at 37°C for 30 min. Add 20 μL of 2,4-dinitrophenylhydrazine solution to both the measurement well and the control well, and add 5 μL of the sample to be tested to the control well, mix well, and incubate at 37°C for 20 min. Add 200 μL of 0.4 mol / L sodium hydroxide solution to each well, mix well, let stand at room temperature for 15 min, measure the OD value of each well at a wavelength of 510 nm with an ELISA reader, check the standard curve, and obtain the corresponding AST / GPT activity unit.

[0055] 3.4 Determination of the content of liver alcohol dehydrogenase ADH

[0056] To detect the activity of alcohol dehydrogenase (ADH), first homogenize the tissue under ice bath conditions according to the ratio of tissue mass (g): extraction solution volume (mL) of 1:5 - 10 (it is recommended to weigh about 0.1 g of tissue and add 1 mL of extraction solution). Then, centrifuge the homogenate at 16,000×g for 20 minutes at 4°C, and take the supernatant and place it on ice for further measurement. ADH catalyzes NADH to reduce acetaldehyde to ethanol. At the same time, NADH has an absorption peak at 340 nm, while NAD+ has no absorption. Calculate the ADH activity by measuring the rate of decrease in absorbance at 340 nm. Use an ELISA reader, preheat it for 30 minutes in advance, set the wavelength to 340 nm, and zero with distilled water. Reagent 1 is preheated in a 25°C water bath for more than 30 minutes. Blank tube: Add 20 μL of distilled water, 8 μL of Reagent 2, 152 μL of Reagent 1, and 20 μL of Reagent 3 to a 96-well plate, mix well, and measure the absorbance values A1 and A2 at 15 seconds and 75 seconds at 340 nm, and calculate ΔA blank tube = A1 - A2. Measurement tube: Add 20 μL of the supernatant instead of distilled water, repeat the above operation, record the absorbance values A3 and A4 at 15 seconds and 75 seconds, and calculate ΔA measurement tube = A3 - A4. Calculate the ADH activity based on the difference between ΔA measurement tube and ΔA blank tube.

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

[0058] To detect the activity of glutathione reductase (GR), animals were first perfused with physiological saline containing 0.16 mg / mL heparin to remove blood. Then, tissue samples were collected and homogenized at a ratio of 200 μL of lysis buffer per 20 mg of tissue. The homogenization process can be carried out using a TissueMaster TM tissue grinder at 4 °C. After homogenization, the samples were centrifuged at 12,000×g for 10 minutes at 4 °C, and the supernatant was taken for detecting GR activity. During detection, the samples were reacted with oxidized glutathione (GSSG) and the chromogenic substrate DTNB, and the generated yellow TNB was linearly positively correlated with GR activity. The amount of TNB generated was measured at a wavelength of A412 using an enzyme-linked immunosorbent assay (ELISA) reader to calculate the activity of GR. Since glutathione reductase becomes the rate-limiting factor in the reaction system, the amount of TNB generated can reflect the activity of GR.

[0059] 4 Index determination;

[0060] Figure 2 Effect of BA-C10 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in mouse plasma. Figure 2 The results showed that mice in the CON group were gavaged with 0.2 mL of physiological saline daily, and mice in the BA-C10 group were gavaged with 0.2 mL of BA-C10 bacterial solution at a concentration of 109 CFU / mL daily. After gavaging with ethanol (5 g / kg), the plasma ALT and AST levels of the mice were detected (p < 0.05). The results indicated that compared with the CON group, the serum ALT and AST levels of the mice in the BA-C10 group were significantly decreased.

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

[0062] Effect of BA-C10 on the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in mice. Figure 4 The results showed that after gavaging with ethanol (5 g / kg), the activities of alcohol dehydrogenase (ADH) and glutathione peroxidase (GSH-Px) in the mice in the BA-C10 group were significantly higher than those in the CON group (p < 0.05). This indicated that the BA-C10 bacterial solution could enhance the alcohol metabolism ability and antioxidant ability.

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

[0064] Dissolve 1 g of BA-C10 bacterial powder in 500 mL of LB liquid medium. After culturing at 37 °C for 24 h, 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 away the residual medium, centrifuge again, and discard the supernatant to obtain inactivated Bifidobacterium animalis subsp. lactis BA-C10 cells. Dissolve 1 mL of physiological saline in every 50 mg of cells to obtain inactivated Bifidobacterium animalis subsp. lactis BA-C10 bacterial solution.

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

[0066] 3.1 Experimental animals

[0067] Forty-eight male C57BL / 6 mice were purchased from Shanghai Slac Laboratory Animal Center (Company License No.: SCXK (Shanghai) 2013-0016) and were housed in the SPF-level animal breeding room of the Experimental Animal Center of Zhejiang Chinese Medical University. Breeding environment: room temperature 20-25 °C, 12-hour light-dark cycle, relative humidity about 50%, feed, drinking water, etc. were uniformly matched, the indoor environment was kept well ventilated, cleaned every day, and the cage was maintained in good sanitary conditions. All experiments complied with the animal ethics code and the relevant regulations of the Experimental Animal Ethics Committee, and were specifically carried out according to Class IAC and other relevant standard operating procedures (SOP). After the experiment, blood was collected by abdominal aortic anesthesia, and the corpses were uniformly treated harmlessly.

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

[0069] 3.3 Methods

[0070] After one week of adaptive feeding of C57BL / 6 mice in the SPF-level 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 intragastrically administered 0.2 ml of BA-C10 heat-killed bacterial solution every day, and the CON group was intragastrically administered 0.2 ml of physiological 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 measure plasma ethanol and other indicators.

[0071] 3.1. Gas chromatography (GC) was used to detect plasma ethanol content through headspace vials;

[0072] The sample to be tested (mouse plasma) was diluted with ultrapure water (1:1). Shake gently. If the sample contains more impurities, centrifuge gently (4°C, 3000×g, 5 minutes) and take the supernatant. After mixing the supernatant with chromatography-grade tert-butyl alcohol (1:5), filter it with a 0.45μm disposable filter and inject it into a chromatographic bottle. The gas chromatograph includes an automatic sampler AOC-20S, a flame ionization detector (FID), a GC-2010 and an Rtx-BAC1 chromatographic column (specifications are 30m×0.32mm×1.8μm). Injection mode: split injection, split ratio 20:1, carrier gas control mode: constant linear velocity, carrier gas is nitrogen (2ml / min), injection volume 0.2μL. The column temperature program can be adjusted as follows: the initial temperature is 40°C for 2 minutes, the temperature is increased to 240°C at a rate of 10°C / min, and it is maintained for 1 minute, and the total time is 23min.

[0073] 3.2 Plasma ALT detection

[0074] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding ALT / GPT activity unit.

[0075] 3.3 Plasma AST detection

[0076] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding AST / GPT activity unit.

[0077] 3.4 Determination of liver alcohol dehydrogenase ADH content

[0078] To detect the activity of alcohol dehydrogenase (ADH), first, homogenize the tissue under ice bath conditions according to the ratio of tissue mass (g): extraction solution volume (mL) of 1:5 - 10 (it is recommended to weigh about 0.1 g of tissue and add 1 mL of extraction solution). Then, centrifuge the homogenate at 16,000×g for 20 minutes at 4°C, and take the supernatant and place it on ice for further measurement. ADH catalyzes NADH to reduce acetaldehyde to ethanol. At the same time, NADH has an absorption peak at 340 nm, while NAD+ has no absorption. Calculate the ADH activity by measuring the rate of decrease in absorbance at 340 nm. Use a microplate reader, preheat it for 30 minutes in advance, set the wavelength to 340 nm, and zero it with distilled water. Reagent 1 is preheated in a 25°C water bath for more than 30 minutes. Blank tube: Add 20 μL of distilled water, 8 μL of Reagent 2, 152 μL of Reagent 1, and 20 μL of Reagent 3 to a 96-well plate. After mixing, measure the absorbance values A1 and A2 at 15 seconds and 75 seconds at 340 nm, and calculate ΔA blank tube = A1 - A2. Measurement tube: Add 20 μL of supernatant instead of distilled water, repeat the above operation, record the absorbance values A3 and A4 at 15 seconds and 75 seconds, and calculate ΔA measurement tube = A3 - A4. Calculate the ADH activity based on the difference between ΔA measurement tube and ΔA blank tube.

[0079] 3.5 Determination of the content of glutathione reductase GR in the liver;

[0080] To detect the activity of glutathione reductase (GR), first, perfuse the animal with physiological saline containing 0.16 mg / mL heparin to remove the blood. Then, collect the tissue samples and homogenize them according to the ratio of adding 200 μL of lysis solution per 20 mg of tissue. The homogenization process can be completed using a TissueMaster TM tissue grinder at 4°C. After homogenization, centrifuge the sample at 12,000×g for 10 minutes at 4°C, and take the supernatant for detecting the GR activity. During the detection, react the sample with oxidized glutathione (GSSG) and the chromogenic substrate DTNB. The generated yellow TNB has a linear positive correlation with the GR activity. Measure the amount of TNB generated at the A412 wavelength using a microplate reader and calculate the activity of GR. Since glutathione reductase becomes the rate-limiting factor in the reaction system, the amount of TNB generated can reflect the GR activity.

[0081] 3.4 Index determination:

[0082] Figure 5 The effects of inactivated BA-C10 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in mouse plasma. Figure 5 The results showed that mice in the CON group were gavaged with 0.2 mL of physiological saline every day, while mice in the BA-C10 group received 0.2 mL of a concentration of 10 9BA-C10 bacterial solution at CFU / mL, and the inactivated BA-C10 group was gavaged with an equal volume of inactivated bacterial solution. After gavaging with ethanol (5 g / kg), plasma ALT and AST levels were detected (p < 0.05), indicating that the serum ALT and AST levels of mice in the BA-C10 group and the inactivated BA-C10 group were significantly lower than those in the CON group. This indicates that after inactivation, the BA-C10 bacterial solution can effectively reduce ethanol-induced liver injury.

[0083] Figure 6 To investigate the effect of inactivated BA-C10 on the alcohol metabolism rate in mice. Figure 6 The results showed that after gavaging with ethanol (5 g / kg), the ethanol levels in the plasma of mice were detected at 1 h, 2 h, 4 h, and 8 h respectively. The results indicated that the ethanol content in the plasma of mice in the BA-C10 group and the inactivated BA-C10 group was significantly lower than that in the CON group (p < 0.05). This indicates that both live bacteria and inactivated bacteria of BA-C10 can effectively promote ethanol metabolism and reduce the ethanol level in the plasma.

[0084] Figure 7 To investigate the effect 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 gavaging with 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 ability and antioxidant ability of mice.

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

[0086] Tolerance experiment 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 successively treated in the simulated gastrointestinal fluid for different times; the viable bacteria counts of Bifidobacterium animalis subsp. lactis BA-C10, Bifidobacterium lactis BL-99, and Bifidobacterium lactis HN019 after treatment in the simulated gastrointestinal fluid were measured by the plate counting method.

[0087] Preparation of simulated gastric juice: A pepsin solution with a concentration of 3 mg / mL was prepared with PBS at pH 2.5, which was the artificial gastric juice. After filtration and sterilization through a 0.22 μm microporous membrane, it was reserved for use.

[0088] Preparation of simulated intestinal fluid: Prepare a trypsin solution with a concentration of 1 mg / mL using PBS at pH 8.0, and add bovine bile salt to make its final concentration 1.8%. The resulting solution is the artificial intestinal fluid, which is filtered and sterilized through a 0.22 μm microporous membrane and reserved for use.

[0089] Add the bacterial powders of Bifidobacterium animalis subsp. lactis BA-C10, Bifidobacterium lactis BL-99, and Bifidobacterium lactis HN019 to sterile PBS (pH 7.3), shake to resuspend them. Take 500 μL of the resuspended solution and add it to 4.5 mL of artificial gastric juice, incubate in a 37 °C incubator for 3 h, and then count using the pour plate method. Then take another 500 μL of the bacterial suspension after incubating in artificial gastric juice for 3 h and add it to 4.5 mL of artificial intestinal fluid, incubate in a 37 °C incubator for 8 h, and then count using the pour plate method. Calculate the survival rate according to the following formula.

[0090] Survival rate = number of colonies after treatment (CFU / mL) / initial number of colonies (CFU / mL) × 100%. The gastrointestinal tolerances of Bifidobacterium animalis subsp. lactis BA-C10, Bifidobacterium lactis BL-99, and Bifidobacterium lactis HN019 are shown in Table 1 as follows:

[0091]

[0092] Table 1

[0093] It can be seen from the table that after 3 h in gastric juice, the survival rate of viable bacteria of Bifidobacterium animalis subsp. lactis BA-C10 can still remain at 36.33%, while after culturing in intestinal fluid for 8 h, the survival rate of viable bacteria decreases to 80.7% of the viable bacteria after gastric juice treatment. It shows that Bifidobacterium animalis subsp. lactis BA-C10 has a relatively high gastrointestinal tolerance and has a relatively high tolerance to simulated gastrointestinal fluid compared with Bifidobacterium lactis BL-99 and Bifidobacterium lactis HN019.

[0094] In summary, Bifidobacterium animalis subsp. lactis BA-C10 of the present invention can promote alcohol metabolism by reducing the levels of plasma alanine aminotransferase and aspartate aminotransferase and increasing the activities of liver alcohol dehydrogenase and glutathione peroxidase, and can reduce the damage of alcohol to the liver. Experiments have proved that both the viable bacteria solution and the inactivated bacteria solution of Bifidobacterium animalis subsp. lactis BA-C10 of the present invention have good abilities to promote alcohol metabolism, and have the advantages of being safe, effective, and having no obvious side effects. The research and development of the viable bacteria solution and the inactivated bacteria solution of Bifidobacterium animalis subsp. lactis BA-C10 of the present invention have opened up a new direction for the research and improvement of products for promoting alcohol metabolism.

[0095] The above description is only some preferred embodiments of the present disclosure and an explanation of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by the specific combination of the above technical features, and should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above inventive concept. For example, the technical solutions formed by mutually replacing the above features with the technical features (but not limited to) disclosed in the embodiments of the present disclosure that have similar functions.

Claims

1. An animal Bifidobacterium lactis subspecies BA-C10 for improving alcohol metabolism or liver damage, characterized in that: The animal Bifidobacterium lactis subspecies BA-C10 was deposited in the China Center for Type Culture Collection on March 11, 2024, with the deposit number: CCTCC NO: M 2024462.

2. Use of the animal Bifidobacterium lactis subspecies BA-C10 according to claim 1 in the preparation of a product that promotes alcohol metabolism.

3. The use according to claim 2, characterized in that: The product is a bacterial agent, food and / or medicine.

4. The use according to claim 3, characterized in that: The food is one or more of fermented milk, cheese, milk-containing beverage, solid beverage and milk powder; the food contains live bacterial liquid and / or inactivated bacterial liquid of Bifidobacterium animalis subspecies lactis BA-C10.

5. The use according to claim 3, characterized in that: The medicine comprises live bacterial liquid and / or inactivated bacterial liquid of animal Bifidobacterium lactis subspecies BA-C10, and / or other medicines compatible with the live bacterial liquid and / or inactivated bacterial liquid of animal Bifidobacterium lactis subspecies BA-C10, as well as pharmaceutically acceptable carriers and / or excipients.

6. The use according to claim 2, characterized in that: The animal Bifidobacterium lactis subspecies BA-C10 is used for preparing bacterial agents, foods and / or medicines for improving the activities of alcohol dehydrogenase and glutathione peroxidase.

7. The use according to claim 2, characterized in that: The animal Bifidobacterium lactis subspecies BA-C10 is used for preparing bacterial agents, foods and / or medicines for reducing the levels of alanine aminotransferase and aspartate aminotransferase.

8. The use according to claim 2, characterized in that: The animal Bifidobacterium lactis subspecies BA-C10 is used for preparing bacterial agents, foods and / or medicines for improving alcoholic liver damage.

9. The use according to claim 4, characterized in that: The preparation of the live bacterial liquid of the animal Bifidobacterium lactis subspecies BA-C10 is as follows: The strain BA-C10 stored in the glycerol tube was first activated by streaking on LB agar plates 2-3 times, and then a single colony was picked and expanded in LB liquid medium at 37°C for 18-24 hours until the concentration of the bacterial solution reached 10 9 ~10 10 CFU / mL, as live bacterial solution.

10. The use according to claim 8, characterized in that: The preparation of the inactivated bacterial solution of the animal Bifidobacterium lactis subspecies BA-C10 is as follows: The live bacterial liquid was inactivated at 121°C under high temperature and high pressure for 30 min to serve as the inactivated bacterial liquid.

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