Bifidobacterium longum subsp. infantis pb17 and its use in preventing and treating liver injury

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to Bifidobacterium longum subsp. infantis PB17 and application thereof in preventing and treating liver injury. The Bifidobacterium longum subsp. infantis PB17 provided by the application has a preservation number of GDMCC No: 67092. The strain of the application plays a hepatoprotective effect through a systematic mechanism of “intestinal homeostasis regulation-system inflammation inhibition-liver self-repair”, and realizes long-term, mild and effective protection of the liver while maintaining intestinal health.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bifidobacterium longum subsp. Infant PB17 and its applications. Background Technology

[0002] As the metabolic center of the human body, the liver undertakes vital physiological functions such as detoxification, synthesis, storage, and immune regulation. However, when the liver is continuously exposed to exogenous toxins (such as alcohol, drugs, and pollutants) and endogenous metabolic stress, it is prone to liver damage. The pathological process includes steatosis, inflammation, and fibrosis, which may eventually develop into cirrhosis or even liver cancer. Currently, clinical interventions mainly focus on symptomatic treatment in the middle and late stages of the disease (such as anti-inflammatory, antioxidant, and anti-fibrotic drugs). These methods often have limitations such as single target, significant side effects, or inability to fundamentally regulate the liver microenvironment.

[0003] In recent years, the gut-liver axis theory has provided a novel perspective for the prevention and treatment of liver diseases. This theory posits that the gut and liver are anatomically and functionally interconnected through the portal vein, bile ducts, and systemic circulation. Disruption of gut microbiota homeostasis leads to impaired intestinal barrier function, causing endotoxins and inflammatory mediators to translocate to the liver via the portal vein, triggering persistent inflammatory responses and oxidative stress. This is a crucial mechanism driving the occurrence and development of various liver diseases, such as non-alcoholic fatty liver disease, alcoholic liver disease, and drug-induced liver injury. Therefore, regulating the gut microbiota to repair the intestinal barrier and alleviate endotoxemia has become an important approach to the prevention and / or treatment of various liver diseases.

[0004] Probiotics, as live microorganisms, can have beneficial effects on the host's health when ingested in sufficient quantities. Among them, Bifidobacterium is one of the gut probiotics, possessing various physiological functions such as immune regulation, strengthening the intestinal barrier, and antagonizing pathogens. Bifidobacterium longum has attracted considerable attention due to its excellent intestinal colonization ability, acid-producing characteristics, and broad clinical safety profile. Preliminary studies have shown that certain probiotic strains can improve liver injury markers in animal models induced by high-fat diets or alcohol; however, their specific mechanisms of action are mostly described using general anti-inflammatory or antioxidant terms, with unstable effects and a lack of strain specificity.

[0005] The primary use of Bifidobacterium longum in liver health is for liver protection, including the prevention and / or treatment of cholestatic liver disease; including the following: The invention CN120514740A, titled "Application of Probiotics in the Treatment of Primary Biliary Cholangitis," discloses that *Bifidobacterium longum* subsp. *longum*, with the accession number CCTCC NO: M2013689, can prevent and / or treat liver function damage, cholestasis, inflammatory lesions, and / or liver fibrosis caused by cholestatic liver disease. A mouse model of primary biliary cholangitis was constructed using α-naphthyl isothiocyanate (ANIT, dissolved in olive oil).

[0006] The invention CN117305157A, entitled "A B. longum strain that promotes postoperative liver function recovery and its application," provides a B. longum strain that promotes postoperative liver function recovery, with accession number CCTCC M 20231518 and named Bifidobacterium longum JWA3. This strain can simultaneously produce high levels of 5-HT and GABA, with a 5-HT production of up to 10.18 ng / mL and a GABA production of up to 12.93 μg / mL. Verification using an animal liver injury model (DDC treatment) showed that this strain significantly improves liver injury and promotes liver function recovery after hepatectomy. As is common knowledge, the DDC (3,5-diethoxycarbonyl-1,4-dihydrocortisone) treatment model often simulates primary sclerosing cholangitis-like liver injury.

[0007] "An Investigation into the Inhibitory Mechanism of Probiotic VSL#3 on Carbon Tetrachloride-Induced Liver Fibrosis in Mice": This literature mentions that the probiotic VSL#3 contains various beneficial bacteria, including Bifidobacterium (including related strains of Bifidobacterium longum). Experiments were conducted with groups such as VSL#3 + CCl4. HE staining, serum AST and ALT measurements revealed that VSL#3 significantly alleviated CCl4-induced liver fibrosis and liver damage in mice, reduced hepatocyte necrosis foci and inflammatory cell infiltration, and significantly inhibited the activation of the Stat3 signaling pathway, thereby alleviating liver fibrosis. Probiotic VSL#3 contains multiple strains, including Lactobacillus acidophilus and Bifidobacterium. However, Bifidobacterium alone cannot exert the above effects.

[0008] It should be noted that the CCl4 (carbon tetrachloride)-induced mouse liver model is mainly used to simulate human alcoholic liver disease and cirrhosis caused by chemical liver damage. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a strain of Bifidobacterium longum infantis PB17 and its applications.

[0010] To solve the above-mentioned technical problems, the present invention provides a *Bifidobacterium longum* subspecies *PB17*, the taxonomic name of which is... Bifidobacterium longumsubsp.infantis, accession number GDMCC No: 67092.

[0011] The present invention also provides the application of the above-mentioned Bifidobacterium longum infant subspecies PB17 in the preparation of drugs for the prevention and treatment of liver injury.

[0012] An improvement to the application of this invention is to reduce the initial degree of liver damage.

[0013] As a further improvement to the application of the present invention: the reduction of the initial degree of liver damage includes reducing the area of ​​liver necrosis and reducing the degree of inflammatory cell infiltration.

[0014] As a further improvement to the application of the present invention: the reduction of the initial degree of liver damage includes intervening in the abnormal activation of hepatic stellate cells and reducing cirrhosis.

[0015] As a further improvement to the application of the present invention: the reduction of the initial degree of liver damage includes reducing the levels of AST and ALT in serum (thereby reducing the toxic effects on the liver caused by drugs, etc.).

[0016] As a further improvement to the application of the present invention: the reduction of the initial degree of liver damage includes reducing serum inflammatory factors, thereby reducing the degree of inflammation. Serum inflammatory factors include IL-6, IL-1β, and TNF-α.

[0017] This invention employs a classic CCl4 (carbon tetrachloride)-induced mouse liver model, which can be used to simulate human alcoholic liver disease and cirrhosis caused by chemical liver damage.

[0018] The preservation information of this invention is as follows: Collection Name: Bifidobacterium longum subsp.infantis, deposited at: Guangdong Provincial Center for Microbial Culture Collection, deposited at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposited on: October 13, 2025, deposit number: GDMCC No: 67092.

[0019] The beneficial effects of this invention are mainly reflected in the following: The strain of this invention does not work through a single pathway, but exerts its liver-protective effect through a multi-linked systemic mechanism of "intestinal homeostasis regulation - systemic inflammation suppression - liver self-repair". It maintains intestinal health while achieving long-term, gentle and effective protection of the liver, thereby improving the feasibility and market persuasiveness of subsequent product development. Attached Figure Description

[0020] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Figure 1This is a colony diagram of Bifidobacterium longum subsp. infantis PB17; Figure 2 The growth curve of Bifidobacterium longum subsp. infantis PB17; Figure 3 Serum AST and ALT levels for blank control, model, and PB17; Figure 4 Serum inflammatory factor levels of blank, model, and PB17; Figure 5 HE staining of liver cells (blank, model, and PB17); Figure 6 This is a comparison chart of the necrotic area of ​​the model and PB17. Note: In the above figure, blank represents the blank group, model represents the CCl4 model group, and PB17 represents the Bifidobacterium longum infantis subsp. PB17-CCl4 treatment group. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Example 1: Isolation, culture and identification of Bifidobacterium longum subsp. infantis PB17 Human intestinal isolates from Hangzhou were used as samples. After serial dilution with physiological saline, they were plated on MRS medium and anaerobic cultured at 37°C. Single colonies were picked and expanded. Sequencing and homology comparison were performed to obtain *Bifidobacterium longum* subsp. infantis PB17. Its 16S rDNA sequence identification results are shown in SEQ ID NO:1.

[0023] Morphological identification of *Bifidobacterium longum* subsp. infantis PB17: Gram-positive, non-spore-forming, long rod-shaped, curved, forked, colonies smooth, convex, with intact edges, creamy to white, shiny, and soft in texture. Figure 1 As shown.

[0024] The preservation information for Bifidobacterium longum subsp. infantis PB17 of this invention is as follows: Collection Name: Bifidobacterium longum subsp.infantis, deposited at: Guangdong Provincial Center for Microbial Culture Collection, deposited at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposited on: October 13, 2025, deposit number: GDMCC No: 67092.

[0025] Growth curve determination of the strain: Bifidobacterium longum subsp. infantis PB17 was inoculated into MRS liquid medium and cultured at 37°C under anaerobic conditions. The absorbance of the cultured bacteria at 600 nm was measured every 2 hours. The results are as follows: Figure 2As shown in the growth curve of Bifidobacterium longum subsp. infantis PB17, it can be seen that 0h~2h is the lag phase, 2h~10h is the logarithmic growth phase, 10h~22h is the plateau phase, and decline begins after 22h.

[0026] Antibiotic susceptibility testing of strains: After activation, strain PB17 was cultured in MRS medium to the late logarithmic growth stage. A suspension (normalized to 0.5 McFarland units) was then evenly spread onto agar plates using a glass spreader. Subsequently, standard antibiotic susceptibility testing discs containing five commonly used clinical antibiotics were placed on the plates. After anaerobic incubation at 37°C for 24–48 hours, the diameter of the inhibition zone around each antibiotic disc was precisely measured and recorded. The interpretation of the results strictly followed the relevant guidelines of the Clinical and Laboratory Standards Institute (CLSI) or the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to determine the susceptibility of the strain to each antibiotic. The experimental results are shown in Table 1.

[0027] Table 1. Antibiotic susceptibility results (inhibition zone diameter / mm)

[0028] Note: S indicates sensitive; I indicates moderately sensitive; R indicates insensitive.

[0029] Experimental results showed that *Bifidobacterium longum* subsp. infantis PB17 exhibited high sensitivity to five commonly used clinical antibiotics, providing important evidence for its combination therapy and safe clinical application. This characteristic not only ensures that the strain can be effectively controlled by conventional antibiotics during use, reducing potential application risks, but also lays the foundation for developing probiotic preparations that can be used in synergy with antibiotics, further broadening its application prospects in comprehensive interventions such as liver protection, prevention of liver damage, and liver cirrhosis caused by liver damage.

[0030] Example 2: Antibacterial test of Bifidobacterium longum subsp. infantis PB17 The inhibition zone diameter was measured using the Oxford cup agar diffusion method to assess the inhibitory effect of lactic acid bacteria on indicator bacteria. *Escherichia coli* ATCC8739 and *Staphylococcus aureus* ATCC6538 were used as indicator bacteria. The supernatant (unadjusted / pH adjusted to 7.0) filtered after 24 hours of culture of *Bifidobacterium longum* subsp. infantis PB17 was used to evaluate the inhibitory effect on the two pathogenic bacteria. Penicillin-streptomycin was used as a positive control, and MRS medium was used as a control group. The results are shown in Table 2. The metabolites of *Bifidobacterium longum* showed inhibitory effects on the pathogenic bacteria, and the inhibitory effect was good.

[0031] Note: The specific preparation process of the supernatant after 24 hours of culturing Bifidobacterium longum subsp. infantis PB17 is as follows: One loopful of Bifidobacterium longum subsp. infantis PB17 is inoculated into 5 ml of MRS liquid medium and cultured at 37°C under anaerobic conditions for 24 hours. Then, the mixture is filtered through a 0.22 μm filter, and the filtrate is collected to obtain the supernatant after 24 hours of culturing Bifidobacterium longum subsp. infantis PB17.

[0032] Table 2. Results of antibacterial test (diameter of inhibition zone / mm)

[0033] Example 3: Determination of acid and bile salt tolerance of Bifidobacterium longum subsp. infantis PB17 I. Acid resistance test: The activated and expanded bacterial suspension was inoculated into MRS liquid medium at a rate of 1% (v / v). After anaerobic culture at 37°C to the logarithmic growth phase, the cells were collected by centrifugation for 5 min, washed twice with phosphate buffer (pH 7.0), and then resuspended in MRS liquid medium at pH 3.0 and pH 1.0. The initial viable count was corrected to approximately 10⁻⁶ cells / mL. 8 CFU / mL, anaerobic incubation at 37℃ for 3 h. Viable bacteria in the 0 h and 3 h samples were counted using the plating method to determine their survival rate. The survival rate was calculated using the following formula:

[0034] N0 represents the viable cell count (CFU / mL) of the test strain after 0 hours of anaerobic culture; N t To test the viable cell count (CFU / mL) of the strain after anaerobic culture for 3 hours.

[0035] The results are shown in Table 3 below.

[0036] Table 3 Results of acid tolerance of the strains

[0037] II. Determination of tolerance to bile salts (sodium taurocholate): The activated and expanded bacterial suspension was inoculated into MRS liquid medium at a rate of 1%, and cultured anaerobically at 37°C until the logarithmic growth phase. After vortexing, the initial viable count was corrected to approximately 10⁻⁶. 8 CFU / mL. 10% of the samples were inoculated into MRS liquid medium containing 0.5% and 0.3% (m / v) ox bile salts, with MRS liquid medium without ox bile salts used as a control. The samples were anaerobically incubated at 37°C for 3 hours. The viable bacterial count was then determined using the spread plate method. The survival rate was calculated using the following formula:

[0038] N0 represents the viable cell count (CFU / mL) of the test strain after 0 hours of anaerobic culture; N t To test the viable cell count (CFU / mL) of the strain after anaerobic culture for 3 hours.

[0039] The results are shown in Table 4 below.

[0040] Table 4 Results of strain tolerance to bile salts

[0041] Example 4: Bifidobacterium longum subsp. infantis PB17 can alleviate the CCl4-induced liver injury phenotype in mice, according to the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)", as follows: (1) Preparation of WT mice Fifteen 7-week-old wild-type C57 / BL6 mice were purchased from Yaokang Jicui and housed in an SPF barrier system. After one week of acclimatization, the mice were randomly divided into three groups: ① blank group, ② CCl4 model group, and ③ Bifidobacterium longum infant subsp. PB17-CCl4 treatment group.

[0042] (2) Preparation of Bifidobacterium longum subsp. infantis PB17 bacterial culture Bifidobacterium longum subsp. infantis PB17 was inoculated into liquid MRS medium and cultured anaerobicly at 37°C for 20–22 h. The cells were then collected by centrifugation at 3000 rpm for 5 min, washed twice with PBS buffer (pH=7), and resuspended in PBS buffer to obtain a final concentration of approximately 2 × 10⁻⁶. 9 CFU / mL Bifidobacterium longum subsp. infantis PB17 suspension.

[0043] (3) Establish a mouse model of CCl4-induced liver injury CCl4 was dissolved in edible vegetable oil to obtain edible vegetable oil containing 1% CCl4 by mass.

[0044] Bifidobacterium longum subsp. infantis PB17-CCl4 treatment group: Each mouse was administered 200 μL of Bifidobacterium longum subsp. infantis PB17 suspension by gavage daily for 10 days; on the 10th day of gavage, the mice were fasted overnight for 16 hours, and then administered edible vegetable oil containing CCl4 by gavage at a dose of 5 ml / kg (mouse body weight, BW); and then each mouse was administered 200 μL of Bifidobacterium longum subsp. infantis PB17 suspension by gavage once more (at least 4 hours after the CCl4 gavage).

[0045] CCl4 treatment group: The oral administration of Bifidobacterium longum subsp. infantis PB17 bacterial solution was replaced with an equal volume of PBS buffer, and the rest was the same as the Bifidobacterium longum subsp. infantis PB17-CCl4 treatment group. Control group: The oral administration of Bifidobacterium longum subsp. infantis PB17 bacterial solution was replaced with an equal volume of PBS buffer, and the oral administration of edible vegetable oil containing CCl4 was replaced with edible vegetable oil (same volume); the rest were the same as the Bifidobacterium longum subsp. infantis PB17-CCl4 treatment group.

[0046] Throughout the experiment, the mice were observed. Depending on the situation, animals were sacrificed 24-48 hours after CCl4 administration for relevant testing.

[0047] Based on experimental results, after 30 hours of CCl4 induction, mouse plasma was collected via cardiac sampling into heparin sodium anticoagulant tubes. After incubation at room temperature for 30 minutes, the tubes were centrifuged at 3000 r / min for 10 minutes at 4°C. The supernatant serum sample was collected, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were measured. ELISA kits were then used to measure the levels of inflammatory factors IL-6, IL-1β, and TNF-α. Subsequently, the mouse liver was removed, fixed in formalin, and stained with hematoxylin and eosin (HE).

[0048] ALT and AST are commonly used biochemical indicators to assess liver function and liver damage, and are typically used for the diagnosis and monitoring of liver health. Figure 3 As shown, Bifidobacterium longum infantis subspecies PB17 significantly reduced serum AST and ALT levels in mice, alleviating the drug's hepatotoxic effects.

[0049] IL-6, IL-1β, and TNF-α are classic inflammatory factors; their elevation in a liver injury model may reflect the activation of inflammatory and immune responses. Figure 4 The results showed that Bifidobacterium longum infantis subspecies PB17 significantly reduced serum inflammatory factors and decreased the degree of inflammation in mice.

[0050] Results of HE staining of mouse liver as follows Figure 5 As shown, obvious necrotic areas are visible in the liver tissue of the model group, and the area of ​​liver necrosis ( Figure 6 ) and the degree of inflammatory cell infiltration ( Figure 5 The levels of PB17 were significantly reduced in the PB17 group, indicating that PB17 significantly alleviated liver damage and linking its effects to cirrhosis prevention from a pathophysiological perspective. The essence of cirrhosis is chronic injury leading to excessive repair and fibrotic scar formation. *Bifidobacterium longum* subsp. infantis directly reduced the intensity and duration of pro-fibrotic signals by mitigating the initial degree of damage (i.e., reducing the area of ​​liver necrosis and decreasing the degree of inflammatory cell infiltration), thereby intervening in the abnormal activation of hepatic stellate cells—a central link in liver fibrosis. Therefore, the application of *Bifidobacterium longum* subsp. infantis provides an innovative preventative solution targeting the pre-cirrhotic stage (i.e., the stage of liver injury and fibrosis).

[0051] Note: In this invention, the existing BL-G301, JWA3, and probiotic VSL#3 were also tested for Bifidobacterium using the method described in Example 4. The Bifidobacterium in BL-G301, JWA3, and probiotic VSL#3 significantly reduced the level of inflammatory factors in mouse serum less than that of Bifidobacterium PB17 (which is close to the results in the model group), and the area of ​​liver necrosis was significantly higher than that of PB17 (approximately 2 to 4 times the necrosis area corresponding to PB17). This indicates that PB17 of this invention can play a better role in alleviating liver damage, and that Bifidobacterium PB17 alone can play a better role.

[0052] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Bifidobacterium longum subsp. infantis, which helps prevent liver damage ( Bifidobacterium longum subsp.infantis)PB17, characterized by: The taxonomic name of strain PB17 is Bifidobacterium longum The subsp. infantis, with accession number GDMCC No: 67092, has a liver injury classified as chemical liver injury.

2. The application of Bifidobacterium longum subsp. infantis PB17 as described in claim 1 in the preparation of a drug for preventing and treating chemically induced liver injury, characterized in that: Prevention and treatment of chemically induced liver injury includes intervening in the abnormal activation of hepatic stellate cells to reduce the initial degree of liver damage. This reduction in the initial degree of liver damage includes reducing the area of ​​liver necrosis and decreasing the degree of inflammatory cell infiltration.

3. The application according to claim 2, characterized in that: The reduction of initial liver damage includes lowering serum AST and ALT levels.

4. The application according to claim 3, characterized in that: The reduction of initial liver damage includes lowering serum inflammatory factors, thereby reducing the degree of inflammation.

5. The application according to claim 4, characterized in that: Serum inflammatory factors include IL-6, IL-1β, and TNF-α.

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