Bifidobacterium capable of regulating mitochondrial dysfunction and relieving liver injury

Abstract

The invention relates to functional characteristic research of bifidobacterium, and belongs to the technical field of microorganisms. The bifidobacterium longum subsp. Longum disclosed by the invention has the effects of resisting oxidation and relieving mitochondrial dysfunction, and is specifically reflected in that the resistance of caenorhabditis elegans to oxidative stress and mitochondrial stress is remarkably improved, the activity of antioxidant enzyme in caenorhabditis elegans and the content of ATP (adenosine triphosphate) are increased, and normal mitochondrial membrane potential is maintained; the head swinging capacity and the pharyngeal pump capacity of the caenorhabditis elegans are increased, and the reproductive capacity is not influenced. The liver injury caused by acetaminophen in mice is remarkably relieved, the content of liver antioxidant enzymes and ATP is increased, normal mitochondrial membrane potential is maintained, the number of liver proinflammatory factors and macrophages is reduced, activation of cytochrome P450 2E1 is inhibited, and the liver injury caused by acetaminophen in mice is relieved through an Nrf2 signal channel.

Description

Technical Field

[0001] This invention relates to the study of the functional characteristics of Bifidobacteria, and belongs to the field of microbial technology. Background Technology

[0002] Oxidative stress refers to a state of imbalance between oxidation and antioxidation in the body, where oxidation exceeds the clearance of oxidants. This state generates a large number of free radicals, such as reactive oxygen species (ROS). ROS can directly or indirectly oxidize or damage DNA, proteins, and lipids, inducing gene mutations, protein denaturation, and lipid peroxidation, leading to physiological and pathological reactions in cells and tissues. It is considered a significant factor contributing to aging, cardiovascular disease, diabetes, and other conditions. Furthermore, the liver, as the central organ responsible for detoxification and nutrient metabolism, is particularly susceptible to oxidative stress. Some antioxidants, including enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), as well as non-enzymatic systems such as polyphenols, vitamin C, and carotenoids, can reduce redox reactions in cells and inhibit the production of free radicals. When the body is subjected to excessive oxidative stress, it is necessary to activate the body's antioxidant levels to prevent oxidative damage.

[0003] Mitochondria are the primary site of cellular oxidation and energy conversion. Most ATP in cells is produced through oxidative phosphorylation in mitochondria, accounting for over 80% of the energy required for life activities. Mitochondria are also the main site of reactive oxygen species (ROS) production in cells. Under normal conditions, intracellular ROS act as intracellular signaling molecules to regulate normal physiological functions. When tissues are damaged, ROS levels in mitochondria increase significantly, thereby damaging mitochondria and leading to mitochondrial dysfunction. Mitochondrial dysfunction can decrease respiratory chain enzyme activity, reduce mitochondrial membrane potential, decrease ATP synthesis, disrupt intracellular calcium homeostasis, alter mitochondrial permeability, and inhibit β-oxidation of fatty acids. Accumulation of fatty acids in cells increases oxidative stress, and mtDNA oxidative damage leads to decreased mitochondrial biosynthesis, further exacerbating mitochondrial dysfunction and ultimately causing apoptosis.

[0004] Probiotics, as part of the gut microbiome, play a crucial role in maintaining gut health by contributing to microbial balance, reducing inflammation, and mitigating oxidative stress. Studies have shown that probiotics, including lactobacilli and bifidobacteria, possess antioxidant properties that can reduce oxidative stress. Furthermore, bifidobacteria have been shown to have anti-aging, anti-inflammatory, and oxidative stress-regulating effects.

[0005] *C. elegans* is a non-toxic, hermaphroditic, and independent nematode that feeds on microorganisms and is a model organism in molecular and developmental biology research. In a laboratory setting at 20 °C, the average lifespan of *C. elegans* is 14-21 days, and a generation takes only about 4 days. In addition, *C. elegans* has advantages such as a simple body structure, transparent body for easy observation, small size for easy culture, strong vitality for easy preservation, high reproduction rate, and a completed genome sequence, eliminating the need for animal ethics committee approval for its use. Therefore, it is widely used in laboratory research.

[0006] Acute liver failure is a clinical syndrome characterized by massive hepatocellular necrosis and severe liver function impairment within a short period of time without underlying liver disease. Causes of acute liver failure include viral hepatitis, drug poisoning, metabolic abnormalities, and organ ischemia. For example, acetaminophen (APAP), a drug used to control pain and fever, can lead to acute liver failure when overdosed. Excessive APAP leads to the accumulation of its toxic metabolite N-acetyl-p-benzoquinone (NAPQI), resulting in severe hepatotoxicity and hepatocellular necrosis. It is generally believed that APAP activation of cytochrome P450, especially CYP2E1, is a key initiating event of NAPQI hepatotoxicity. In cases of excessive NAPQI, hepatocellular proteins and DNA deplete intracellular glutathione (GSH), triggering mitochondrial oxidative stress damage and lipid metabolism disorders, ultimately leading to apoptosis and liver damage. Summary of the Invention

[0007] Based on the above problems, the first objective of this invention is to screen for Bifidobacteria with antioxidant properties and the ability to alleviate mitochondrial dysfunction using the Caenorhabditis elegans model organism and the acetaminophen mouse model organism.

[0008] A strain of Bifidobacterium sp., named Bifidobacterium sp. CCMXJ2001 (abbreviated as 1-13), was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2025, with accession number GDMCC NO: 66359.

[0009] In one embodiment, it has one or more of the following properties: (1) Scavenging free radicals of 1,1-diphenyl-2-trinitrophenylhydrazine and 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt; (2) Enhance the oxidative stress resistance of Caenorhabditis elegans and the activity of antioxidant enzymes SOD, GSH-PX, and CAT, and reduce the content of MDA. (3) Increase the resistance of Caenorhabditis elegans to mitochondrial stress, increase ATP content, and maintain normal mitochondrial membrane potential. (4) Reduce the increase in serum alanine aminotransferase and aspartate aminotransferase levels caused by acetaminophen; (5) Relieves liver tissue damage caused by acetaminophen; (6) It reduces the decrease in the activity of antioxidant enzymes SOD, GSH-PX, and CAT caused by acetaminophen and decreases the increase in MDA content. (7) Reduces the increase in liver pro-inflammatory factors IL-6 and TNF-α caused by acetaminophen; (8) It can reduce the decrease in liver ATP content caused by acetaminophen and maintain normal mitochondrial membrane potential; (9) Reduces the increase in the number of liver macrophages caused by acetaminophen and inhibits the activation of cytochrome P450 2E1.

[0010] In one embodiment, the bacterial suspension is prepared by inoculating the above-mentioned *Bifidobacterium longum* subsp. *longum* into the culture medium at an inoculation amount of 4% of the total mass of the culture medium, culturing at 37°C for 36-48 hours to obtain a culture medium; centrifuging the culture medium at 8000 rpm for 10 minutes to obtain bacterial cells; and resuspending the bacterial cells in 0.9% sterile physiological saline to obtain a bacterial suspension.

[0011] In one embodiment, the culture medium is an optimized MRS medium supplemented with 0.5% L-cysteine ​​hydrochloride.

[0012] A second objective of this invention is to provide the use of the above-mentioned *Bifidobacterium longum* subsp. *longum* in the preparation of products that regulate mitochondrial dysfunction or alleviate liver damage.

[0013] In one embodiment, the number of viable *Bifidobacterium longum* subsp. *longum* in the product is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0014] In one embodiment, the product includes food or medicine.

[0015] In one embodiment, the pharmaceutical product contains the *Bifidobacterium longum* subsp. as described in claim 1, a drug carrier, and / or pharmaceutical excipients.

[0016] In one embodiment, the pharmaceutical excipient is one or more selected from propellants, solubilizers, emulsifiers, disintegrants, binders, fillers, lubricants, flow aids, sustained-release materials, coating materials, preservatives, antioxidants, flavoring agents, or colorants commonly used in pharmaceuticals.

[0017] In one embodiment, the food comprises a health food containing the above-mentioned Bifidobacterium longum subsp. longum; or the food comprises dairy products, soy products, meat products, or fruit and vegetable products produced using the above-mentioned fermentation agent of Bifidobacterium longum subsp. longum.

[0018] Beneficial effects The *Bifidobacterium longum* subsp. obtained by screening in this invention has great potential in anti-oxidation and alleviating mitochondrial dysfunction, especially in alleviating acute liver failure caused by acetaminophen, specifically in the following ways: (1) Scavenging free radicals of 1,1-diphenyl-2-trinitrophenylhydrazine and 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt; (2) Enhance the oxidative stress resistance and the activity of antioxidant enzymes SOD, GSH-PX, and CAT in Caenorhabditis elegans; (3) Increase the resistance of Caenorhabditis elegans to mitochondrial stress, increase ATP content, and maintain normal mitochondrial membrane potential. (4) Reduce the increase in serum alanine aminotransferase and aspartate aminotransferase levels caused by acetaminophen; (5) Relieves liver tissue damage caused by acetaminophen; (6) It reduces the decrease in the activity of antioxidant enzymes SOD, GSH-PX, and CAT caused by acetaminophen and decreases the increase in MDA content. (7) Reduces the increase in liver pro-inflammatory factors IL-6 and TNF-α caused by acetaminophen; (8) It can reduce the decrease in liver ATP content caused by acetaminophen and maintain normal mitochondrial membrane potential; (9) Reduces the increase in the number of liver macrophages caused by acetaminophen and inhibits the activation of cytochrome P450 2E1.

[0019] Therefore, Bifidobacterium longum subsp. longum has a promising future in clinical treatment.

[0020] 2. The *Bifidobacterium longum* subsp. obtained by screening in this invention is a type of probiotic and has been included in the "List of Microbial Strains that Can Be Used in Food" issued by the Ministry of Health. Therefore, it will not pose any potential safety risks to patients.

[0021] Preservation of biological materials The long subspecies of Bifidobacterium longum has been named Bifidobacterium sp. CCMXJ2001, with the taxonomic name Bifidobacterium sp. It was deposited on May 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 66359. Attached Figure Description

[0022] Figure 1 The effect of Bifidobacteria on the survival rate of nematodes under oxidative stress; Figure 2 Effects of Bifidobacterium on superoxide dismutase activity in nematodes; Figure 3 The effect of Bifidobacterium on the activity of glutathione peroxidase in nematodes; Figure 4 The effect of Bifidobacterium on catalase activity in nematodes; Figure 5 The effect of Bifidobacterium on mitochondrial stress survival in nematodes; Figure 6 The effect of Bifidobacterium on mitochondrial ATP content in nematodes; Figure 7 The effect of Bifidobacteria on the mitochondrial membrane potential of nematodes; Figure 8 The effect of Bifidobacterium on the red-green fluorescence ratio of nematode mitochondrial membrane potential; Figure 9 Effect of Bifidobacterium on serum alanine aminotransferase levels in mice; Figure 10 Effect of Bifidobacterium on serum aspartate aminotransferase levels in mice; Figure 11 The effects of Bifidobacterium on mouse liver histopathology; Figure 12 Effects of Bifidobacterium on superoxide dismutase activity in mouse liver tissue; Figure 13 Effects of Bifidobacterium on lipid peroxide content in mouse liver tissue; Figure 14 The effect of Bifidobacterium on glutathione peroxidase activity in mouse liver; Figure 15 Effects of Bifidobacterium on catalase activity in mouse liver tissue; Figure 16 Effects of Bifidobacterium on tumor necrosis factor-α levels in mouse liver tissue; Figure 17 The effect of Bifidobacterium on the interleukin-6 content in mouse liver tissue; Figure 18 The effect of Bifidobacterium on ATP content in mouse liver tissue; Figure 19 Effects of Bifidobacteria on mitochondrial membrane potential in mouse liver tissue; Figure 20 The effect of Bifidobacterium on the red-green fluorescence ratio of mitochondrial membrane potential in mouse liver tissue; Figure 21Effects of Bifidobacterium on macrophages in mouse liver tissue; Figure 22 Effect of Bifidobacterium on CYP2E1 protein expression in mouse liver tissue; Figure 23 Effects of Bifidobacterium on Nrf2 pathway genes in mouse liver tissue. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0024] Mupirocin, L-cysteine ​​hydrochloride, cholesterol, malonic acid, and 5-fluorouracil used in the following examples were purchased from Maclean's Biochemical Technology (Shanghai) Co., Ltd.; MRS and BBL culture media used in the following examples were purchased from Qingdao Haibo Biotechnology Co., Ltd.; the kits for detecting SOD, MDA, GSH-PX, CAT, ATP, mitochondrial membrane potential, ALT, and AST used in the following examples were purchased from Nanjing Jiancheng Bioengineering Institute; and the ELISA kits for detecting IL-6, IL-10, IL-1β, and TNF-α used in the following examples were purchased from Hangzhou Lianchuan Biotechnology Co., Ltd.

[0025] KPO4 solution: 1.08 g anhydrous KH2PO4, 0.36 g anhydrous K2HPO4, dissolved in 10 ml pure water, adjusted to pH 6.

[0026] 5 mg / mL cholesterol solution: 0.05 g cholesterol dissolved in 10 mL anhydrous ethanol, store at -20°C.

[0027] Preparation of NGM medium: KCl 0.94g, NaCl 1.2g, agar 7-8g, peptone 2g, add 0.4ml of 1M CaCl2, 1M MgSO4 and KPO4 solution, add pure water to 400mL, sterilize at 121℃ for 20 min, cool to 70℃, and add 0.4ml of cholesterol solution.

[0028] LB medium: 1g peptone, 1g NaCl, 0.5g yeast extract powder, dissolved in 100ml pure water, supplemented with 2g agar for solid medium, sterilized at 121℃ for 20 min.

[0029] M9 buffer: 5.9g anhydrous Na2HPO4, 3.0g anhydrous KH2PO4, 5.8g NaCl, 1ml 1M MgSO4 solution, dissolved in 1L pure water, sterilized at 121℃ for 20 min.

[0030] Nematode lysate: 1 g NaOH, 20 mL 5.25% NaOCl, 80 mL pure water.

[0031] Mupirocin solution: Dissolve 1g of mupirocin in 20mL of anhydrous ethanol, dispense into 2mL centrifuge tubes, and store at -20℃.

[0032] 1M malonic acid solution: Dissolve 5.203g of malonic acid in 50mL of pure water, filter using a 0.22μm sterile filter, and store at 4℃ for later use.

[0033] Example 1: Isolation and Identification of Bifidobacteria Stool samples with added glycerol should be stored at -80°C and thawed at room temperature before use. Take an appropriate amount of sample and serially dilute with sterile water. Take 10... -3 10 -4 Spread 100 μL onto BBL solid plates (after sterilizing the medium and cooling to below 70°C, add 1‰ mupirocin solution). Spread two plates for each gradient and incubate anaerobically at 37°C for approximately 72 h. After purifying single colonies twice by streaking, pick single colonies and inoculate them into MRS liquid medium (with 0.5% L-cysteine ​​hydrochloride) for enrichment. Incubate anaerobically at 37°C for approximately 48 h, then store in glycerol. After enrichment, perform PCR identification of the bacterial suspension. Take 1 mL of bacterial suspension, centrifuge at 8000 rpm for 6 min at 4°C, discard the supernatant, wash the bacteria twice with sterile water, and resuspend in 500 μL of sterile water as a template. The PCR reaction system is shown in Table 1, using Bifidobacterium-specific primers Bif-groEL-F (5'-TCCGATTACGAYCGYGAGAAGCT-3') and Bif-groEL-R (5'-CSGCYTCGGTSGTCAGGAACAG-3'). The amplification conditions were: 95 °C for 10 min; 95 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s, 30 cycles, followed by 72 °C for 10 min and 12 °C for 10 min. The PCR amplification products were spotted into 1.5% agarose gel wells and run at 120 V for 30 min. The amplification results were observed using a gel imaging system. Successfully amplified strains were sent to Youkang Biotechnology for sequencing, and then BLAST was performed on NCBI to determine the species of Bifidobacterium.

[0034] Table 1 PCR reaction system A total of 62 strains of Bifidobacterium were isolated and identified. In vitro antioxidant screening was performed using 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS). Bifidobacterium suspension cultured under MRS was selected as the target for antioxidant capacity determination. After MRS activation, 1 mL of the bacterial suspension was centrifuged at 8000 r / min for 10 min at 4℃ to obtain bacterial sludge. This sludge was washed twice with an equal volume of physiological saline and then resuspended in physiological saline to obtain a complete cell suspension. (For specific methods, please refer to FANG ZF, LIU N, FAN ZX, et al. Cistanche deserticola polysaccharides—Companilactobacillus crustorumLBK 1001 complex relieves aging in Caenorhabditis elegans [J]. FoodBioscience, 2025, 68: 106486. DOI: 10.1016 / j.fbio.2025.106486.) The test results are shown in the table below. Based on the combined in vitro DPPH and ABTS free radical scavenging rate experiments of Bifidobacterium, a total of 15 strains of Bifidobacterium sp. CCMXJ2001 (abbreviated as 1-13, the same below), 2-2, 3-5, 4-6, 5-11, 6-1, 10-1, 13-4, 15-4, 16-26, 17-14, 17-19, 18-7, 19-17, and 25-1 were selected for further screening.

[0035] Table 2. In vitro DPPH and ABTS free radical scavenging rates of Bifidobacteria Example 2: Screening of Bifidobacteria with antioxidant capacity based on the Caenorhabditis elegans model *Caenorhabditis elegans* was cultured in nematode growth medium (NGM) and fed with *E. coli* OP50. To ensure that the nematodes were in the same growth cycle and to eliminate the influence of individual differences on the experiment, the nematodes were synchronized. Plates with most nematodes in the oviposition stage were selected. The nematodes were washed into centrifuge tubes with M9 buffer and centrifuged at 5000 rpm for 5 min. The supernatant was aspirated, and the nematodes at the bottom were washed twice with M9 solution to remove culture medium and other impurities. An appropriate amount of nematode lysis buffer was added, and the mixture was vortexed for 1 min and allowed to stand for 5 min until the nematodes lysed and turned yellow. The mixture was then centrifuged at the same speed for 3 min, and washed twice with M9 solution to remove the nematode lysis buffer. Finally, the egg mixture was transferred to NGM culture dishes containing *E. coli* OP50 and incubated at 20°C for 72 hours. The nematodes reached the L4 stage and were used for the experiment.

[0036] Fifteen Bifidobacterium strains selected according to Example 1 were used as the experimental group. Synchronized nematodes were washed with M9 buffer, and an appropriate amount of nematode solution was transferred to NGM medium containing E. coli OP50 (to inhibit oviposition, 25 μg / mL of 5-FU was added to the NGM medium). 600 µL of Bifidobacterium was pre-spread in the experimental group, while the control group was fed only E. coli OP50. Three plates were used in each group, and the plates were incubated at 20°C. To ensure the viability of Bifidobacterium, the plates were changed after 48 hours, and the nematodes were transferred to new, identical NGM medium. After 48 hours, the plates were ready for experiments. An oxidative stress experiment was conducted after the Bifidobacterium culture of the nematodes (see reference for specific methods). At the end of the Bifidobacterium culture, the nematodes were transferred to blank plates with a final hydrogen peroxide concentration of 2 mM. The nematode survival rate was recorded after 5 hours.

[0037] The results are as follows Figure 1 As shown, the average survival rate of the control group under oxidative stress was 20.42%. Compared with the control group, the culture of Bifidobacterium strains 1-13, 2-2, 15-4, 17-14, 18-7, and 25-1 significantly improved the survival rate of nematodes (p < 0.01). The average survival rates under oxidative stress were 44.66%, 42.42%, 43.15%, 44.68%, 47.45%, and 43.62%, respectively, indicating that the above-mentioned Bifidobacterium strains have good antioxidant properties.

[0038] To determine the antioxidant indices of nematodes, nematodes cultured with Bifidobacterium groups 1-13, 2-2, 18-7, 17-14, 15-4, and 25-1 according to the above-described culture method were collected and washed with M9 buffer. Tissue homogenates were prepared using a tissue homogenizer, and protein concentrations were determined using a BCA kit. Samples were then diluted to appropriate concentrations. Activities were measured according to the procedures outlined in the SOD, CAT, and GSH-PX assay kits. Results were expressed as per milligram of protein. Figures 2-4 As shown.

[0039] Depend on Figure 2 The average superoxide dismutase (SOD) activity in the control group was 43.03 U / mgprot. Compared with the control group, the Bifidobacterium groups 1-13, 2-2, 15-4, and 25-1 significantly increased the SOD activity of nematodes (p < 0.05), with average SOD activities of 51.50 U / mgprot, 49.78 U / mgprot, 49.65 U / mgprot, and 50.70 U / mgprot, respectively.

[0040] Depend on Figure 3 It was found that the activity of glutathione peroxidase (GSH-PX) in the blank group was 86.96 U / mgprot. Compared with the blank group, the experimental groups of Bifidobacterium 1-13, 2-2, 18-7, and 25-1 significantly increased the activity of glutathione peroxidase in nematodes (p<0.05), and the GSH-PX activities were 110.3 U / mgprot, 104.4 U / mgprot, 104.4 U / mgprot, and 109.3 U / mgprot, respectively.

[0041] Depend on Figure 4 It was found that the catalase (CAT) activity in the blank group was 8.006 U / mgprot. Compared with the blank group, the experimental groups of Bifidobacterium 1-13, 2-2, 18-7, and 25-1 significantly increased the CAT enzyme activity in nematodes (p < 0.05), with CAT activities of 10.33 U / mgprot, 10.44 U / mgprot, 10.32 U / mgprot, and 10.80 U / mgprot, respectively.

[0042] The above results indicate that Bifidobacterium 1-13 can enhance the antioxidant stress resistance of nematodes by increasing the activity of enzymes such as CAT, GSH-PX, and SOD.

[0043] Example 3: Screening for Bifidobacteria that alleviate mitochondrial dysfunction based on a Caenorhabditis elegans model *Caenorhabditis elegans* was cultured in nematode growth medium (NGM) and fed with *E. coli* OP50. To ensure that the nematodes were in the same growth cycle and to eliminate the influence of individual differences on the experiment, the nematodes were synchronized. Plates with most nematodes in the oviposition stage were selected. The nematodes were washed into centrifuge tubes with M9 buffer and centrifuged at 5000 rpm for 5 min. The supernatant was aspirated, and the nematodes at the bottom were washed twice with M9 solution to remove culture medium and other impurities. An appropriate amount of nematode lysis buffer was added, and the mixture was vortexed for 1 min and allowed to stand for 5 min until the nematodes lysed and turned yellow. The mixture was then centrifuged at the same speed for 3 min, and washed twice with M9 solution to remove the nematode lysis buffer. Finally, the egg mixture was transferred to NGM culture dishes containing *E. coli* OP50 and incubated at 20°C for 72 hours. The nematodes reached the L4 stage and were used for the experiment.

[0044] Six Bifidobacterium strains (1-13, 2-2, 18-7, 17-14, 15-4, and 25-1) selected according to Example 2 were used as the experimental group. The synchronized nematodes were washed with M9 buffer, and an appropriate amount of the nematode solution was transferred to NGM medium containing E. coli OP50 (to inhibit oviposition, 25 μg / mL of 5-FU was added to the NGM medium). 600 µL of Bifidobacterium was pre-spread on the experimental group, while the control group was fed only E. coli OP50. Three plates were used for each group, and the cultures were incubated at 20°C. To ensure the viability of the Bifidobacterium, the plates were changed after 48 hours, and the nematodes were transferred to new, identical NGM medium. The medium was ready for experiments after 48 hours.

[0045] Malonic acid (MA), as an inhibitor of the mitochondrial respiratory chain, can induce mitochondrial stress. This experiment used a malonic acid survival assay in nematodes to analyze their survival rate after 16 hours, providing supplementary evidence to reflect the mitochondrial stress resistance of nematodes. After nematode culture, 300 μl of malonic acid solution was spread into NGM medium (the volume of NGM in the small dish was 10 mL, so the final concentration of MA was 30 mM). At the end of the Bifidobacterium culture, the nematodes were transferred to the medium, and their survival rate was recorded after 16 hours.

[0046] Mitochondria are the primary site of ATP production. ATP and mitochondrial membrane potential were measured according to the kit instructions, and protein concentration was determined using the BCA kit. Samples were diluted to appropriate concentrations. This was used to measure ATP levels and mitochondrial membrane potential. The relative ratio of red to green fluorescence of the JC-1 probe was used to represent mitochondrial membrane potential; a lower value indicates a higher degree of mitochondrial depolarization, i.e., a lower membrane potential.

[0047] Depend on Figure 5The mean mitochondrial stress survival rate of the control group was 19.80%. Compared with the control group, the experimental groups of Bifidobacterium 1-13, 2-2, 18-7 and 25-1 significantly improved the mitochondrial stress resistance of nematodes (p<0.05), by 30.47%, 27.78%, 29.70% and 28.4%, respectively, demonstrating that they have a certain protective effect on mitochondrial function.

[0048] Depend on Figure 6 The results showed that the ATP content in the control group was 47.55 µmol / mgprot. Compared with the control group, the experimental groups of Bifidobacterium 1-13, 18-7, and 25-1 significantly improved the decrease in ATP content in nematodes (p < 0.05), with ATP contents of 84.60 µmol / mgprot, 84.67 µmol / mgprot, and 77.63 µmol / mgprot, respectively. This demonstrates that Bifidobacterium has a certain protective effect on the function of mitochondrial respiration and ATP production.

[0049] Depend on Figure 7 and Figure 8 The results showed that the ratio of red to green fluorescence in the mitochondria of the blank group was 0.9359. Compared with the blank group, the Bifidobacterium groups 1-13, 18-7, and 25-1 could significantly improve the decrease in mitochondrial membrane potential (p<0.05), with the ratios of red to green fluorescence in the mitochondria being 1.149, 1.124, and 1.103, respectively, demonstrating that they have a positive effect on maintaining normal mitochondrial membrane potential.

[0050] Example 4: Effects of Bifidobacterium on alleviating liver injury and histopathology in acetaminophen model mice Bifidobacteria frozen at -80℃ were thawed at room temperature and an appropriate amount was added to MRS liquid medium. After anaerobic culture at 37℃ for 48 h, the culture was inoculated into fresh MRS liquid medium at an inoculum rate of 4%, and cultured anaerobically at 37℃ for 48 h. After two generations of activation, the bacterial cells were collected by centrifugation. The bacterial sludge was resuspended in physiological saline, counted, and stored at 4℃ for later use. Fresh bacterial culture was prepared every 5 days.

[0051] The experimental animals were 6-8 week old SPF-grade C57BL / 6J mice, housed in an environment with a temperature of 20±2℃, humidity of 40%, and a 12-hour light / dark cycle, with free access to food and water. After one week of acclimatization, the mice were randomly divided into three groups: a normal control group (CON), a model group (MOD), and Bifidobacterium groups (1-13, 18-7, and 25-1), with 8 mice in each group. After the acclimatization period, the CON and MOD groups were administered 200 μL of physiological saline by gavage daily for two weeks, while the Bifidobacterium groups were administered 200 μL of bacterial suspension with a CFU of 1×10⁹ (bacterial weight suspended in physiological saline) by gavage. On day 15 of the experiment, the MOD and Bifidobacterium groups were intraperitoneally injected with 300 mg / kg acetaminophen solution, while the normal control group was intraperitoneally injected with an equal volume of physiological saline. Feces were collected 8 hours after intraperitoneal injection, and the mice were sacrificed 24 hours later.

[0052] During dissection, blood samples were left to stand at room temperature for 30 min, then centrifuged at 4°C and 3000 r / min for 15 min to collect serum as the test samples. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice were measured using the microplate method, following the instructions of the ALT and AST assay kits.

[0053] During dissection, mouse livers were collected, and liver tissue of approximately 0.5 × 0.5 × 0.5 cm was cut from the same location. The tissue was immediately fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin, sectioned, and stained with H&E.

[0054] Depend on Figure 9 The results showed that the serum ALT level in the CON group was 11.20 U / L, while the serum ALT level in the MOD group was 133.0 U / L, significantly higher than that in the CON group (p < 0.001). Serum ALT levels represent hepatocyte damage, indicating that APAP treatment caused hepatocyte damage in mice. The serum ALT level in the Bifidobacterium 1-13 group was 61.32 U / L, significantly lower than that in the MOD group (p < 0.05), demonstrating that intervention with Bifidobacterium 1-13 significantly improved the increase in serum ALT levels induced by APAP and improved hepatocyte damage in mice. The serum ALT level in the Bifidobacterium 18-7 group was 80.34 U / L, and the serum ALT level in the 25-1 group was 107.90 U / L, showing a decreasing trend compared to the MOD group, but the difference was not statistically significant.

[0055] Depend on Figure 10The results showed that the serum AST level in the CON group was 14.05 U / L, while that in the MOD group was 133.0 U / L, significantly higher than that in the CON group (p < 0.001). Serum AST levels represent the degree of hepatocyte necrosis, indicating that APAP treatment caused hepatocyte necrosis in mice. The serum AST level in the Bifidobacterium 1-13 group was 50.38 U / L, significantly lower than that in the MOD group (p < 0.05), demonstrating that intervention with Bifidobacterium 1-13 significantly improved the increase in serum AST levels induced by APAP and reduced hepatocyte necrosis in mice. The serum AST level in the Bifidobacterium 18-7 group was 89.83 U / L, and that in the 25-1 group was 78.50 U / L, showing a decreasing trend compared to the MOD group, but the difference was not statistically significant.

[0056] Depend on Figure 11 It can be seen that in the HE staining of liver tissue, the hepatocytes in the CON group were tightly arranged, and no obvious dilation or compression of the hepatic sinusoids was observed, nor were any inflammatory changes observed; in the MOD group, obvious dilation and compression of the hepatic sinusoids were observed, with pyknosis, deep staining, fragmentation or dissolution of hepatocyte nuclei, accompanied by inflammatory cell infiltration; in groups 1-13, no obvious dilation or compression of liver tissue and hepatic sinusoids were observed, and mild inflammatory cell infiltration could be observed; in groups 18-7, dilation and compression of the hepatic sinusoids were observed, with pyknosis, deep staining, fragmentation or dissolution of hepatocyte nuclei, and inflammatory cell infiltration; in group 25-1, dilation and compression of the hepatic sinusoids were observed, with pyknosis and deep staining of hepatocyte nuclei, accompanied by a small amount of inflammatory cell infiltration.

[0057] Experimental groups 1-13 significantly improved the increase in serum ALT and AST levels in mice caused by APAP (p<0.05) and improved liver lesions. Groups 18-7 and 25-1 also showed a trend of protective effect on mouse liver, proving the protective effect of Bifidobacterium on liver and verifying the conclusion that Bifidobacterium with antioxidant and mitochondrial dysfunction was screened based on the Caenorhabditis elegans model.

[0058] Example 5: Effects of Bifidobacterium on antioxidant indicators and inflammatory factor levels in mice with acetaminophen-induced irritation. The bacterial culture was prepared according to the method in Example 4 and mice were fed with it. Liver tissue frozen at -80℃ was taken out, and 20mg of liver tissue was accurately weighed under ice bath conditions. Nine times the volume of physiological saline was added, i.e., 180μL, to prepare a 10% liver tissue homogenate. Grinding beads were added, and the homogenate was ground in a grinder at 60Hz with 45s intervals of 15s, for 3 times. The homogenate was then centrifuged at 3000 r / min at 4℃ for 15 min, and the supernatant was collected. The protein concentration in the liver tissue homogenate was determined using a BCA protein concentration assay kit. Then, the contents of SOD, GSH-Px, CAT, and MDA in the liver tissue were detected by the kit. The results were expressed as milligrams of protein.

[0059] Retrieve liver tissue frozen at -80℃, accurately weigh 20mg of liver tissue under ice bath conditions, then prepare 1×PBS, dilute PMSF to 1μM (dilution within half an hour before use), add 100μL of PMSF solution (five times the sample weight), add grinding beads, and grind in a grinder at 60Hz for 45s intervals with 15s intervals, repeating 3 times. Centrifuge at 12000 r / min for 10 min at 4℃, collect the supernatant in a 200μL centrifuge tube, and store at -20℃ for later use. Detect the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) according to the kit instructions.

[0060] Depend on Figure 12 The results showed that the SOD enzyme activity in the liver tissue of the CON group mice was 162.3 U / mgprot, and that in the MOD group it was 118.4 U / mgprot, which was significantly lower than that in the CON group (p < 0.01). The SOD enzyme activity in the Bifidobacterium 1-13 group was 155.6 U / mgprot, and that in the 18-7 group was 152.2 U / mgprot, which was significantly higher than that in the MOD group (p < 0.05). The SOD enzyme activity in the Bifidobacterium 25-1 group was 143.6 U / mgprot, which was higher than that in the MOD group, but the difference was not statistically significant.

[0061] Depend on Figure 13 The results showed that the MDA content in the liver tissue of the CON group mice was 8.48 mmol / mgprot, and that in the MOD group it was 24.74 mmol / mgprot, which was significantly higher than that in the CON group (p < 0.01). The MDA content in the Bifidobacterium 1-13 group was 12.99 mmol / mgprot, which was significantly lower than that in the MOD group (p < 0.05). The MDA content in the Bifidobacterium 18-7 group was 19.36 mmol / mgprot, and that in the 25-1 group was 20.68 mmol / mgprot, which was lower than that in the MOD group, but the difference was not statistically significant.

[0062] Depend on Figure 14 The results showed that the GSH-PX enzyme activity in the liver tissue of the CON group mice was 682.0 U / mgprot, and that in the MOD group it was 481.4 U / mgprot, which was significantly lower than that in the CON group (p < 0.001). The activity in the Bifidobacterium 1-13 group was 636.3 U / mgprot, which was significantly higher than that in the MOD group (p < 0.01). The enzyme activity in the Bifidobacterium 18-7 group was 619.7 U / mgprot, and that in the 25-1 group it was 605.1 U / mgprot, which was significantly higher than that in the MOD group.

[0063] Depend on Figure 15The results showed that the CAT enzyme activity in the liver tissue of the CON group mice was 37.95 U / mgprot, and that in the MOD group it was 20.92 U / mgprot, which was significantly lower than that in the CON group (p < 0.001). The activity in the Bifidobacterium 1-13 group was 33.18 U / mgprot, which was significantly higher than that in the MOD group (p < 0.05). The CAT enzyme activity in the Bifidobacterium 18-7 group was 28.76 U / mgprot, and that in the 25-1 group was 25.94 U / mgprot, which was higher than that in the MOD group, but the difference was not statistically significant.

[0064] It is evident that Bifidobacterium can significantly increase the levels of antioxidant enzymes SOD, GSH-PX, and CAT in mouse liver tissue, while decreasing the level of lipid peroxide MDA. Among these, Bifidobacterium 1-13 showed the most significant effect, demonstrating its good antioxidant activity. This is consistent with the results obtained in the *C. elegans* experiment. Since APAP is metabolized in the liver to produce toxic metabolites and consumes glutathione (GSH), and the GSH-PX kit utilizes the specific catalytic reaction of GSH to hydrogen peroxide to quantify it, the enzyme content also reflects the GSH content in liver tissue. Bifidobacterium 1-13 showed the most significant alleviating effect on GSH-PX (p < 0.01), demonstrating that Bifidobacterium 1-13 has a protective effect against APAP-induced liver oxidative stress.

[0065] Depend on Figure 16 The results showed that the TNF-α content in the liver tissue of the CON group mice was 9.08 pg / ml, and that in the MOD group it was 23.62 pg / ml, both significantly higher than that in the CON group (p < 0.001). The TNF-α content in the Bifidobacterium 1-13 group was 11.29 pg / ml, significantly lower than that in the MOD group (p < 0.01). The TNF-α content in the Bifidobacterium 18-7 group was 13.73 pg / ml, significantly lower than that in the MOD group (p < 0.05), and that in the 25-1 group was 12.05 pg / ml, significantly lower than that in the MOD group (p < 0.01).

[0066] Depend on Figure 17 The results showed that the tumor necrosis factor-α content in the liver tissue of the CON group mice was 9.08 pg / ml, and that in the MOD group it was 23.62 pg / ml, both significantly higher than that in the CON group (p < 0.001). The content in the Bifidobacterium 1-13 group was 11.29 pg / ml, significantly lower than that in the MOD group (p < 0.01). The content of TNF-α in the Bifidobacterium 18-7 group was 13.73 pg / ml, significantly lower than that in the MOD group (p < 0.05), and that in the 25-1 group was 12.05 pg / ml, significantly lower than that in the MOD group (p < 0.01).

[0067] Depend on Figure 17 The results showed that the IL-6 content in the liver tissue of the CON group mice was 51.3 pg / ml, and that in the MOD group it was 131.4 pg / ml, which was significantly higher than that in the CON group (p < 0.001). The IL-6 content in the Bifidobacterium 1-13 group was 85.0 pg / ml, which was significantly lower than that in the MOD group (p < 0.01). The TNF-α content in the Bifidobacterium 18-7 group was 96.8 pg / ml, which showed a decreasing trend compared with the MOD group but was not statistically significant. The TNF-α content in the 25-1 group was 12.05 pg / ml, which was significantly lower than that in the MOD group (p < 0.01).

[0068] It is evident that Bifidobacterium can significantly reduce the levels of liver pro-inflammatory factors TNF-α and IL-6, and the effect of Bifidobacterium 1-13 is even more significant, proving that Bifidobacterium 1-13 can alleviate liver oxidative stress caused by APAP by reducing the inflammatory response.

[0069] Example 6: Bifidobacterium's effect on alleviating mitochondrial dysfunction in acetaminophen model mice The bacterial culture was prepared according to the method in Example 4 and fed to mice. Liver tissue frozen at -80℃ was taken out, and 20mg of liver tissue was accurately weighed under ice bath conditions. Nine times the volume of physiological saline was added, i.e., 180μL, to prepare a 10% liver tissue homogenate. Grinding beads were added, and the homogenate was ground in a grinder at 60Hz with 45s intervals of 15s, for 3 times. The homogenate was then centrifuged at 3000 r / min at 4℃ for 15 min, and the supernatant was collected. The protein concentration in the liver tissue homogenate was determined using a BCA protein concentration assay kit. Then, the ATP content in the liver tissue was detected using the kit. The results were expressed as milligrams of protein, and the ratio of mitochondrial red-green fluorescence was detected using a mitochondrial membrane potential assay kit.

[0070] Depend on Figure 18 The results showed that the ATP content in the liver tissue of mice in the CON group was 44.84 µmol / mgprot, while that in the MOD group was 30.50 µmol / mgprot, which was significantly lower than that in the CON group (p < 0.05). The ATP content in the Bifidobacterium 1-13 group was 42.75 µmol / mgprot, which was significantly higher than that in the MOD group (p < 0.05). The ATP content in the Bifidobacterium 18-7 group was 37.14 µmol / mgprot, and that in the Bifidobacterium 25-1 group was 41.89 µmol / mgprot, which were higher than those in the MOD group but not statistically significant.

[0071] Depend on Figure 19 and Figure 20The results showed that the proportion of red-green fluorescence in mitochondria of the liver tissue of mice in the CON group was 0.9629, while that in the MOD group was 0.7565, both significantly lower than that in the CON group (p < 0.001), demonstrating that APAP caused a decrease in mitochondrial membrane potential in mice. The proportion of red-green fluorescence in Bifidobacterium groups 1-13 was 0.9085, and in group 18-7 it was 0.9437, both significantly higher than that in the MOD group (p < 0.01). The proportion of red-green fluorescence in Bifidobacterium group 25-1 was 0.8658, significantly higher than that in the MOD group (p < 0.05), demonstrating that Bifidobacterium plays a positive role in maintaining normal mitochondrial membrane potential.

[0072] Example 7: Effects of Bifidobacterium on Immunohistochemistry and CYP2E1 Enzyme Content of Liver Macrophages in Mice with Acetaminophen-Reduced Injury. The bacterial culture was prepared according to the method in Example 4 and fed to mice. Liver tissues of groups 1-13 of Bifidobacterium, which were frozen at -80℃, were taken out. 20mg of liver tissue was accurately weighed under ice bath conditions, washed 1-2 times with PBS, and tissue protein extraction reagent was added (protease inhibitor was added a few minutes before use). Grinding beads were added, and the mixture was ground in a grinder at 60Hz with 45s intervals of 15s for 3 times. The mixture was then centrifuged at 12000 r / min for 10 min at 4℃. The supernatant was collected in a centrifuge tube for later use. The protein concentration of the sample was adjusted using a BCA protein concentration assay kit, and SDS-PAGEA electrophoresis was performed. After the electrophoresis, the gel was transferred to a PVDE membrane, incubated with antibody, and imaged using a chemiluminescence analyzer. The gray values ​​of the bands were analyzed using IPWIN60 software.

[0073] APAP is metabolized into the toxic intermediate NAPQI, which depletes GSH and triggers oxidative stress, leading to hepatocyte necrosis. Necrotic cells release DAMPs, activating Toll-like receptors and inflammasomes, and further activating macrophages. Figure 21 It was found that F4 / 80 staining revealed macrophages to be brown. In the CON group, the number of liver macrophages was low and the color was light, with an average gray value of 5975. In the MOD group, the number of macrophages increased, and the color became darker, with an average gray value of 22938, significantly higher than the CON group (p < 0.001). In the Bifidobacterium 1-13 group, the number of liver macrophages was low and the color was light, with an average gray value of 13799, significantly lower than the model group (p < 0.05). This demonstrates that Bifidobacterium 1-13 can alleviate APAP-induced activation of liver macrophages.

[0074] Depend on Figure 22It was found that the expression level of CYP2E1 in the liver of CON group mice was low, with the ratio of its internal component to CYP2E1 gray level being 0.0985, and 0.5346 in MOD group, which was significantly lower than that in model group (p < 0.001). The expression level in Bifidobacterium 1-13 group was 0.3684, which was significantly lower than that in model group (p < 0.05). This demonstrates that the intervention of Bifidobacterium 1-13 alleviated the activation of liver CYP2E1 caused by APAP and reduced the expression level of CYP2E1.

[0075] Example 8: Bifidobacteria alleviate liver oxidative stress in acetaminophen model mice by activating the Nrf2 pathway Nrf2 is a major regulator of cellular redox homeostasis, and this signaling pathway has been shown to play a crucial role in preventing APAP-induced liver injury. To determine whether Bifidobacterium alleviates APAP-induced oxidative stress by activating the Nrf2 pathway, we performed Q-PCR experiments to verify the upstream regulator Keap1 and downstream target genes HO-1 and NQO1 of Nrf2. According to Examples 4-6, the Bifidobacterium groups 1-13 were the most effective in alleviating APAP in mouse models; therefore, mice in the Bifidobacterium groups 1-13 were selected for subsequent experiments. Bacterial culture was prepared and mice were fed according to the method in Example 4. Liver tissue frozen at -80℃ was removed, and 50 mg of liver tissue was accurately weighed under ice bath conditions. RNA was extracted from the liver tissue according to the kit instructions and reverse transcribed into cDNA. Based on the internal reference gene GAPDH, quantitative analysis was performed using SYBR_GREEN reagent on a real-time PCR system (QuantStudio™ 1 System). The primers used are shown in Table 3.

[0076] Table 3. qPCR primer sequences Depend on Figure 23 The intervention with Bifidobacterium 1-13 significantly inhibited Keap1 gene expression, thereby reducing Keap1-mediated ubiquitination and degradation of Nrf2, which in turn activated Nrf2 gene expression, as well as the expression of its downstream genes HO-1 and NQO1 (p<0.05). These results suggest that Bifidobacterium may alleviate APAP-induced hepatotoxicity through the Nrf2 signaling pathway.

Claims

1. A strain of *Bifidobacterium longum* sp., characterized in that, The long subspecies of Bifidobacterium longum was named Bifidobacterium sp. CCMXJ2001 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 19, 2025, with accession number GDMCC NO: 66359.

2. The *Bifidobacterium longum* subsp. according to claim 1, characterized in that, It possesses one or more of the following properties: (1) Scavenging free radicals of 1,1-diphenyl-2-trinitrophenylhydrazine and 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt; (2) Enhance the oxidative stress resistance of Caenorhabditis elegans and the activity of antioxidant enzymes SOD, GSH-PX, and CAT, and reduce the content of MDA. (3) Increase the resistance of Caenorhabditis elegans to mitochondrial stress, increase ATP content, and maintain normal mitochondrial membrane potential. (4) Reduce the increase in serum alanine aminotransferase and aspartate aminotransferase levels caused by acetaminophen; (5) Relieves liver tissue damage caused by acetaminophen; (6) It reduces the decrease in the activity of antioxidant enzymes SOD, GSH-PX, and CAT caused by acetaminophen and decreases the increase in MDA content. (7) Reduces the increase in liver pro-inflammatory factors IL-6 and TNF-α caused by acetaminophen; (8) It can reduce the decrease in liver ATP content caused by acetaminophen and maintain normal mitochondrial membrane potential; (9) Reduces the increase in the number of liver macrophages caused by acetaminophen and inhibits the activation of cytochrome P450 2E1.

3. The use of the *Bifidobacterium longum* subsp. according to claim 1 in the preparation of products that regulate mitochondrial dysfunction or alleviate liver damage.

4. The use according to claim 3, characterized in that, The product contains at least 1 × 10⁻⁶ live bacteria of Bifidobacterium longum subsp. longum. 9 CFU / mL or 1×10 9 CFU / g.

5. The use according to claim 3, characterized in that, The products include food or medicine.

6. The use as described in claim 3, characterized in that, The drug contains the long subspecies of Bifidobacterium longum as described in claim 1, a drug carrier, and / or pharmaceutical excipients.

7. The use as described in claim 3, characterized in that, The food product contains a health food product containing *Bifidobacterium longum* subsp. *longum* as described in claim 1; or the food product contains dairy products, soy products, meat products, or fruit and vegetable products produced using a fermentation agent of *Bifidobacterium longum* subsp. *longum* as described in claim 1.

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