A strain of Bifidobacterium animalis subsp. lactis, a product and its application in protecting the liver

Abstract

The present invention belongs to the field of microbial technology, and particularly relates to a strain of Bifidobacterium animalis subsp. lactis, a product and its application in protecting the liver. The Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) BB-35 provided by the present invention has a preservation number of CGMCC No. 27836. This strain can be used to reduce the expression of the senescence-related marker p21 in mouse hepatocytes, reduce the levels of senescence-related secretory phenotype factors, cystatin C levels and liver function-related biochemical indicators, thereby improving the liver senescence condition of mice and reducing the impact on other organs. In addition, this strain can also improve the oxidative stress indicators malondialdehyde and superoxide dismutase contents, and reduce the levels of inflammatory factors TNF-α and IL-6, providing technical support for the preparation of products for preventing, alleviating and / or treating liver senescence and liver injury.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a strain of Bifidobacterium animalis subsp. lactis, a product and its application in protecting the liver. Background Art

[0002] The liver is one of the most important metabolic organs in the human body, undertaking numerous physiological functions, including metabolism of nutrients, detoxification, protein synthesis, immune regulation, etc. During the life process, the liver gradually shows aging-related changes with the increase of age. For example, the regenerative ability of hepatocytes gradually decreases, the accumulation of extracellular matrix in liver tissue increases, the metabolic functions of the liver such as the metabolic efficiency of lipids, sugars and drugs decrease, and at the same time, the function of the antioxidant defense system of the liver declines, resulting in the accumulation of free radicals, further damaging the structure and function of hepatocytes and cell organelles. These changes make the liver more vulnerable to various internal and external injury factors, increasing the risk of liver diseases such as fatty liver and cirrhosis, seriously affecting the overall health status and quality of life.

[0003] In the field of anti-aging research, various approaches and methods have been explored. In terms of drugs, although some antioxidants and drugs that regulate cell metabolism have been studied for anti-aging, they often have problems such as large side effects, limited effects or single action targets. For example, some chemically synthesized antioxidants may produce harmful intermediate products during the metabolic process in the body or interfere with normal physiological functions. In terms of non-drug interventions, such as diet adjustment and exercise, although they are beneficial to health, the targeted improvement effects on liver aging are not significant enough and are difficult to accurately quantify and standardize. In addition, some emerging technologies such as gene therapy and stem cell therapy are still in the initial stage of research, facing challenges in terms of technical complexity, safety and ethics, and are difficult to be widely applied in clinical practice.

[0004] Probiotics, as a type of active microorganisms beneficial to the host, have been increasingly widely studied in the field of health in recent years. Existing research has shown that probiotics play an important role in maintaining intestinal health, capable of regulating the balance of intestinal flora, enhancing intestinal barrier function, regulating immune responses, etc. With the in-depth research, it has gradually been found that there is a close "gut-liver axis" connection between the intestine and the liver. The intestinal flora and their metabolites can enter the liver through the portal vein system, affecting the physiological functions and pathological states of the liver. Some preliminary studies suggest that specific probiotic strains may indirectly affect liver lipid metabolism, inflammatory responses, and antioxidant status, etc., by regulating the composition and metabolism of the intestinal flora. For example, certain probiotics can reduce the level of endotoxins produced by harmful bacteria in the intestine, reducing the inflammatory responses triggered after endotoxins enter the liver through the portal vein; there are also studies showing that probiotics can promote the expression of antioxidant enzymes in liver cells, enhancing the antioxidant capacity of the liver. However, most of these studies are relatively scattered, lacking systematic in-depth exploration of the comprehensive mechanism of action of probiotics on liver aging and targeted patent technologies for improving liver aging.

[0005] In summary, against the background that the problems of liver aging and injury are attracting increasing attention and there are many deficiencies in existing means, in-depth research on the mechanism of action of probiotics in improving liver aging and liver injury and the development of related patent technologies have important scientific significance and potential clinical application value, and are expected to provide a new, safer and more effective strategy for the intervention of liver aging and liver injury. Summary of the Invention

[0006] In view of the above deficiencies, the present invention provides a strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) BB-35, with the deposit number CGMCC No. 27836, which was deposited at the China General Microbiological Culture Collection Center on July 7, 2023. After culturing, fermenting, and freeze-drying this strain, bacterial powder and postbiotics products (including inactivated BB-35 cells and / or metabolites) are obtained, which can effectively intervene in liver aging and injury.

[0007] The technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides a strain of Bifidobacterium animalis subsp. lactis, with the deposit number CGMCC No. 27836.

[0009] On the other hand, the present invention provides a culture, which is obtained by inoculating the aforementioned Bifidobacterium animalis subsp. lactis on a culture medium.

[0010] More specifically, the inoculation methods include, but are not limited to: any one or more of streak plate method, slant inoculation method, pour plate method, stab inoculation method, liquid inoculation method.

[0011] More specifically, the inoculation amount of the inoculation may be 0.1%-20%, and in some cases, it may also be a higher or lower inoculation amount. Specifically, the inoculation amount may be 1%-20%, 2%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-8%, 5%-15%, 5%-10%, 5%-8%, 8%-10%, 8%-15%, 5%-12%, 2%-7%.

[0012] Further specifically, the culture medium may be a solid culture medium, a semi-solid culture medium or a liquid culture medium. More specifically, it may be any suitable culture medium type already disclosed in the prior art, or a culture medium obtained by further improving the culture medium type disclosed in the prior art to improve the performance of the strain, or a culture medium not disclosed in the prior art but capable of being used for culturing the foregoing Lactococcus lactis subsp. cremoris.

[0013] Preferably, the components of the culture medium may include, by weight parts: 20-30 parts of sucrose, 10-15 parts of lactose, 10-20 parts of soy peptone, 3-8 parts of yeast powder, 10-15 parts of yeast peptone, 20-25 parts of Na2HPO3, 1-3 parts of citric acid, 0.1-1.0 part of MgSO4·7H2O, 0.1-0.5 part of MnSO4·5H2O, 0.5-1.5 parts of Tween-80, and 0.1-0.5 part of L-cysteine hydrochloride.

[0014] More preferably, the components of the culture medium may include, by weight parts: 23.5 parts of sucrose, 12.0 parts of lactose, 15.0 parts of soy peptone, 5.0 parts of yeast powder, 12.0 parts of yeast peptone, 21.0 parts of Na2HPO3, 2 parts of citric acid, 0.6 part of MgSO4·7H2O, 0.3 part of MnSO4·5H2O, 1.0 part of Tween-80, and 0.3 part of L-cysteine hydrochloride.

[0015] On the other hand, the present invention provides the use of the foregoing Bifidobacterium animalis subsp. lactis or culture, and the use includes at least one of the following:

[0016] (1) Use in the preparation of products for preventing, alleviating and / or treating liver aging;

[0017] (2) Use in the preparation of products for preventing, alleviating and / or treating liver injury.

[0018] Specifically, the products in (1) and (2) include bacterial agents, drugs or pharmaceutical raw materials.

[0019] Specifically, the liver injury in (2) includes, but is not limited to, traumatic liver injury or disease-induced liver injury.

[0020] In another aspect, the present invention provides a bacterial agent, which comprises the aforementioned Bifidobacterium animalis subsp. lactis.

[0021] Specifically, the bacterial agent comprises one or more of the fermentation broth, supernatant of the fermentation broth, precipitate of the fermentation broth, live bacteria, dead bacteria, freeze-dried powder and cell lysate of Bifidobacterium animalis subsp. lactis.

[0022] More specifically, the fermentation broth refers to the liquid obtained by inoculating the bacterial strain into a culture medium and culturing for a period of time.

[0023] More specifically, the supernatant of the fermentation broth refers to the clear liquid in the upper layer after centrifugation of the fermentation broth; it contains rich metabolites and some bacterial debris during the process of bacterial growth and reproduction, and the acidic substances and bacteriocins secreted by bacteria have antagonistic and killing effects on harmful bacteria; the amino acids after the bacteria decompose food, as well as the synthesized vitamins are all in the culture solution, and it also includes the enzymes secreted by bacteria that are useful to the human body; and some bacterial components also have an immune-promoting effect on the human body.

[0024] More specifically, the precipitate of the fermentation broth refers to the liquid precipitate obtained by centrifugation, including free proteins, residual bacteria, broken cells, and residues of the culture medium matrix, mainly proteins and intracellular matrix.

[0025] More specifically, the live bacteria, also known as active bacterial flora, can colonize and multiply in the intestine, which is beneficial to increasing the number of beneficial bacteria.

[0026] More specifically, the dead bacteria are microorganisms that have lost their vital activity and cannot grow and reproduce, and the loss of vitality of probiotics is caused by the production process, such as high-temperature treatment or excessive drying.

[0027] More specifically, the freeze-dried powder is obtained by freeze-drying the aforementioned culture solution. The freeze-dried powder generally also includes a freeze-drying protectant. The freeze-drying protectant includes but is not limited to: pH buffer, filler, sugar, non-ionic surfactant, ligand, etc. The pH buffer includes but is not limited to any one or more of Tris, amino acids or their salts, citric acid or its salts, acetic acid or its salts. The filler includes but is not limited to any one or more of mannitol, glycine, bovine serum albumin. The sugar can be a disaccharide, such as any one or more of sucrose or trehalose. The non-ionic surfactant includes but is not limited to Tween, and the Tween can be Tween-20, Tween-60, Tween-80, etc. The freeze-drying protectant can also include antioxidants, etc. The freeze-drying protectant can specifically also include albumin, polyethylene glycol, etc.

[0028] More specifically, the cell lysate can be obtained by lysing the bacterial cells obtained from the aforementioned culture. The lysis can be physical lysis or chemical lysis. The physical lysis includes, but is not limited to, grinding, ultrasonic disruption, etc. The chemical lysis includes, but is not limited to, chemical reagent lysis and enzymatic lysis. The enzymatic lysis can be hydrolase or oxidase. The lysis can also be achieved by increasing the intracellular pressure to cause the cells to rupture spontaneously.

[0029] Specifically, the number of Bifidobacterium animalis subsp. lactis is not less than 5.0×10 8 CFU / mL or 5.0×10 8 CFU / g.

[0030] More specifically, the number of Bifidobacterium animalis subsp. lactis is not less than 5.0×10 8 CFU / mL - 5.0×10 12 CFU / mL or 5.0×10 8 CFU / g - 5.0×10 12 CFU / g.

[0031] Specifically, the microbial agent can be a solid preparation or a liquid preparation.

[0032] In another aspect, the present invention provides a drug, which comprises the aforementioned Bifidobacterium animalis subsp. lactis or culture.

[0033] Specifically, the drug further comprises pharmaceutically acceptable excipients.

[0034] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, antioxidants.

[0035] Specifically, the dosage forms of the drug include, but are not limited to, tablets, liquids, capsules, powders, suppositories or granules.

[0036] In another aspect, the present invention provides a pharmaceutical raw material, which comprises the aforementioned Bifidobacterium animalis subsp. lactis or culture.

[0037] Specifically, the pharmaceutical raw material can be in solid form, liquid form or semi-solid form.

[0038] The beneficial effects of the present invention are as follows:

[0039] BB-35 provided by the present invention can be used to reduce the expression of the senescence-related marker p21 in mouse hepatocytes, reduce the levels of senescence-related secretory phenotype factors, cystatin C levels, and liver function-related biochemical indicators, thereby improving the liver senescence condition of mice and reducing the impact on other organs. In addition, this strain can also improve the oxidative stress indicators malondialdehyde and superoxide dismutase contents, and reduce the levels of inflammatory factors TNF-α and IL-6. This strain can be used to prepare products for preventing, alleviating, and / or treating liver senescence and liver injury.

[0040] Preservation information:

[0041] Biological material: BB-35;

[0042] Taxonomic name: Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis )

[0043] Preservation number: CGMCC No. 27836;

[0044] Preservation date: July 7, 2023;

[0045] Preservation unit: China General Microbiological Culture Collection Center;

[0046] Abbreviation of the preservation unit: CGMCC;

[0047] Preservation address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed implementation manners

[0048] Terms of the present invention:

[0049] p21: p21 is a cyclin-dependent kinase inhibitor (CKIs), which can inhibit the cell cycle process, cause hepatocytes to enter the senescent state, and exhibit senescence-related characteristics, such as cell cycle arrest and the appearance of senescence-related secretory phenotype (SASP). In various tissues and cell types, such as the liver, kidney, brain, and lung, the expression level of p21 is significantly increased in senescence models.

[0050] SASP: The SASP factors secreted by senescent hepatocytes can act on surrounding normal hepatocytes, induce their senescence, and can also enter the blood circulation, reach distant organs and tissues, and affect the cell senescence process of other organs, leading to multi-organ dysfunction.

[0051] TGFβ1: TGFβ1 is a key profibrotic factor that participates in regulating the senescence of liver cells. On the one hand, it can induce hepatocyte senescence by activating intracellular signaling pathways. For example, TGFβ1 can upregulate the expression of CKIs such as p15 and p21, and these CKIs can inhibit the activity of cyclin - cyclin - dependent kinase (cyclin - CDK) complexes, arresting the cell cycle and thus leading to hepatocyte senescence. On the other hand, TGFβ1 also plays a role in SASP. TGFβ1 secreted by senescent hepatocytes can act on surrounding or distant cells in a paracrine or endocrine manner, inducing them to enter a senescent state or regulating their functions, thereby expanding the scope and influence of senescent cells.

[0052] TGFβ2: Similar to TGFβ1, TGFβ2 participates in the induction process of hepatocyte senescence, activates intracellular signaling pathways, leads to upregulation of the expression of CKIs, arrests the cell cycle, and thus induces hepatocytes to enter a senescent state.

[0053] CCL2: C - C motif chemokine ligand 2 (CCL2) is an important mediator of the inflammatory response. Senescent liver cells release CCL2, which can chemotactically attract immune cells such as monocytes and lymphocytes to aggregate in the liver tissue. Moreover, CCL2 can transmit senescence signals between cells. CCL2 secreted by senescent hepatocytes can act on surrounding normal hepatocytes, inducing them to enter a senescent state.

[0054] Cystatin C: Cystatin C is an endogenous marker reflecting glomerular filtration rate (GFR), and changes in its blood level can sensitively indicate the status of renal function. Changes in liver function may cause systemic effects, including renal function.

[0055] ALT: ALT is mainly present in the cytoplasm of hepatocytes. When hepatocytes are damaged, the permeability of the cell membrane changes, and intracellular ALT is released into the extracellular space and then enters the blood circulation, resulting in an increase in the serum ALT level; bilirubin plays an important role in evaluating the bilirubin metabolism function of the liver. When the liver is damaged, such as in hepatitis, cirrhosis, intrahepatic bile duct obstruction, etc., the processes of bilirubin uptake, conjugation, or excretion are affected, leading to an increase in the blood bilirubin concentration and the appearance of jaundice.

[0056] ALP: In the hepatobiliary system, ALP is mainly present on the microvilli of the canalicular surface of hepatocytes and participates in the formation and excretion of bile. When there are lesions in the liver or biliary system, the synthesis and release of ALP by hepatocytes increase, resulting in an increase in serum ALP. Therefore, they are sensitive indicators commonly used in clinical and research to reflect the degree of hepatocyte damage.

[0057] MDA: MDA is one of the end products of lipid peroxidation. The level of its content can usually indirectly reflect the degree of lipid peroxidation in the body and the severity of cells being attacked by free radicals. During the process of liver injury in mice, when affected by factors such as drugs, chemicals, ischemia-reperfusion, etc., a large amount of free radicals will be generated in liver cells, triggering lipid peroxidation reactions and leading to an increase in MDA content. Therefore, the determination of MDA content is often used as one of the important indicators for evaluating the oxidative stress level in liver injury.

[0058] SOD: SOD is an important antioxidant enzyme, widely present in the cytoplasm, mitochondria, etc. of liver cells. Its main function is to catalyze the dismutation reaction of superoxide anion free radicals to generate hydrogen peroxide and oxygen, thereby reducing the damage of superoxide anion free radicals to liver cells. Under normal physiological conditions, SOD can maintain the dynamic balance of free radicals in cells and protect liver cells from oxidative stress damage. However, when liver injury occurs, due to the large production of free radicals, the activity of SOD may be affected, and its ability to scavenge free radicals will also change accordingly.

[0059] TNF-α: TNF-α is mainly secreted by activated macrophages, Kupffer cells, etc. It can activate various intracellular signaling pathways, such as the NF-κB pathway, promote the expression of inflammation-related genes, and further induce the production and release of other inflammatory factors such as IL-1, IL-6, etc., thereby initiating and amplifying the inflammatory response, attracting more immune cells to infiltrate into the liver tissue, and aggravating the inflammatory damage.

[0060] IL-6: IL-6 is a pleiotropic cytokine, which is produced by various cells such as macrophages, lymphocytes, hepatocytes, etc. during the process of liver injury. It can act on various cell types, promote the activation, proliferation and migration of inflammatory cells, enhance the phagocytosis and killing ability of inflammatory cells, and further aggravate the inflammatory response of the liver. At the same time, IL-6 can also regulate the expression and release of other inflammatory factors, and cooperate with TNF-α, etc. to form a complex inflammatory network, jointly participating in the pathological process of liver injury.

[0061] The present invention will be further clearly and completely elaborated through the following examples. The following examples are only a part of the examples of the present invention, not used to limit the present invention, only used to illustrate the present invention. The experimental methods used in the following examples are all conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0062] Example 1 Preparation of a bacterial powder

[0063] Strain: Bifidobacterium animalis subsp. lactis BB-35, with the preservation number of CGMCC No. 27836;

[0064] Method for preparing bacterial powder:

[0065] (1) Activation of the strain

[0066] Inoculate the strain stored at -40°C into the improved MRS liquid medium sterilized at 121°C for 15 min, and anaerobically culture at 37°C for 24 h. Passage culture is carried out 2 times to obtain the activated strain.

[0067] (2) Preparation of the optimized medium

[0068] The composition of the optimized medium is as follows: sucrose 23.5 kg, lactose 12.0 kg, soy peptone 15.0 kg, yeast powder 5.0 kg, yeast peptone 12 kg, Na2HPO3 21.0 kg, citric acid 2.0 kg, MgSO4·7H2O 0.6 kg, MnSO4·5H2O 0.3 kg, Tween-80 1.0 kg, L-cysteine hydrochloride 0.3 kg, distilled water 1000 L.

[0069] Prepare each component of the optimized medium according to the ratio, mix evenly, adjust the pH value to 6.5, and sterilize at 121°C for 15 min.

[0070] (3) Preparation of the seed liquid

[0071] Inoculate the activated strain into the optimized medium and stop anaerobic culture at 37°C when the pH value reaches 4.6. The seed liquid is obtained.

[0072] (4) Inoculation and fermentation

[0073] Use the Bailun Biology BLBIO-10GJ fermenter for fermentation. Load 100 L of the optimized medium into the fermenter. After sterilization, inoculate the seed liquid into the optimized medium at a volume fraction of 1‰ and ferment for 18 h under the controlled fermentation conditions.

[0074] The fermentation conditions are controlled as follows: keep a constant temperature of 30°C for the early stage of fermentation and ferment naturally until the pH reaches 5.0; then adjust the fermentation temperature to 37°C for constant temperature culture and control the pH to remain at 6.0, maintaining anaerobic fermentation.

[0075] During the fermentation process, control the pH by adding a neutralizing agent dropwise. The neutralizing agent is a 25% NaOH solution; the anaerobic condition is achieved by purging nitrogen every two hours.

[0076] (5) Terminate fermentation: Terminate fermentation when the acid production of the bacteria stops, and obtain the high-density fermentation broth of each strain. The viable count of the fermentation broth reaches 5×10 10above cfu / mL. The cessation of acid production can be judged by the fact that the pH no longer decreases and the addition of the neutralizing agent stops.

[0077] (6)Freeze-drying:

[0078] a. Bacterial cell concentration: The high-density fermentation broth is centrifuged at 12,000 g to concentrate the bacterial cells.

[0079] b. Addition of protective agent: 5 times the protective agent solution is added to the concentrated bacterial cell solution; the composition of the protective agent solution is as follows: 15 kg of skim milk, 12 kg of lactose, 1 kg of vitamin C, 1 kg of sodium glutamate, and 1000 L of distilled water.

[0080] c. Drying: The above-mentioned bacterial suspension after adding the protective agent is freeze-dried (model: Sihuan LGJ-12C) to obtain freeze-dried bacterial powder, and the total viable count in the bacterial powder reaches 5.0×10 11 CFU / g or more.

[0081] Example 2 Preparation of a probiotic postbiotic bacterial powder

[0082] On the basis of Example 1, after terminating the fermentation, the following steps are carried out:

[0083] Inactivation: Mix maltodextrin according to production requirements (to provide an ideal liquid dispersion for spray drying), preheat to 58 °C, homogenize at a primary pressure of 19.0 Mpa and a secondary pressure of 5.0 Mpa, and then carry out sterilization inactivation. The parameter conditions for sterilization inactivation are 75 °C and 15 min;

[0084] Spray drying: The inactivated bacterial suspension is spray-dried (inlet air temperature 160 °C, outlet air temperature 45 °C) to obtain probiotic postbiotic powder.

[0085] Example 3 Preparation of a bacterial agent

[0086] Weigh 0.1 g of the bacterial powder in Example 1, add 0.9 g of the auxiliary material maltodextrin, mix evenly, and the viable count is not less than 5.0×10 10 CFU / g.

[0087] Example 4 Preparation of a postbiotic

[0088] Weigh 0.1 g of the probiotic postbiotic bacterial powder in Example 2, add 0.9 g of the auxiliary material maltodextrin, mix evenly, and the cell count is not less than 5.0×10 10 Cells / g.

[0089] Effect Example 1 Probiotics improve liver aging

[0090] 1.1 Preparation of bacterial agent

[0091] Prepare the bacterial agent and postbiotics of Bifidobacterium animalis subsp. lactis BB-35 according to Example 3 and Example 4 respectively.

[0092] 1.2 Mouse background and modeling

[0093] Select C57Bl6 mice aged 8 - 12 weeks (weighing 20 - 25 g, purchased from SPF Biotechnology Co., Ltd.), with an equal number of males and females. The mice are housed in an animal room at a temperature of 22 ± 2°C and a humidity of 50 - 60%, with free access to food and water. The experiment starts after 1 week of environmental adaptation. Use mice with the Mdm2tm2.1Glo allele (Reference: Grier J D, Yan W, Lozano G. Conditional allele of mdm2 which encodes a p53 inhibitor[J]. Genesis, 2010, 32(2):145 - 147. DOI:10.1002 / gene.10066.) and the Gt(ROSA)26Sortm14(CAG - tdTomato)Hze allele (Reference: Madisen L, Zwingman T A, Sunkin S M, et al. A robust and high - throughput Cre reporting and characterization system for the whole mouse brain[J]. Nature Neuroscience, 2010, 13(1):133 - 140. DOI:10.1038 / nn.2467.). Through intravenous injection of the hepatocyte - specific AAV8 - TBG – Cre vector, gene recombination occurs in hepatocytes (Δ Mdm2 Hep ) resulting in the excision of the p53 - binding domain of MDM2, weakening the inhibitory effect of MDM2 on p53, the accumulation of p53 in hepatocytes, and thus inducing liver aging to establish a liver aging model.

[0094] 1.3 Experimental grouping

[0095] Model group (20 mice): Inject 2×10 11 GC / mouse of the AAV8 - TBG – Cre vector every day;

[0096] Probiotic group (20 mice): Inject the AAV8 - TBG – Cre vector and intragastrically administer the bacterial agent every day. The AAV8 - TBG – Cre vector is 2×10 11GC / animal, gavaged with the bacterial agent (Example 3) at 5.0×10 8 CFU / animal;

[0097] Postbiotics group (20 animals): Injected with AAV8 - TBG – Cre vector and gavaged with postbiotics daily, AAV8 - TBG – Cre vector at 2×10 11 GC / animal, gavaged with postbiotics (Example 4) at 5.0×10 8 CFU / animal;

[0098] Blank group (20 animals): Injected with 100 μL of normal saline per animal daily.

[0099] 1.4 Detection methods

[0100] At the start of the intervention and 30 days after the intervention, mouse blood samples were collected by cardiac puncture to measure the levels of senescence - associated secretory phenotype factors, cystatin C levels, and liver - function - related biochemical indicators in mice. The mice were sacrificed 30 days later, and liver tissue samples were collected to analyze the expression of the senescence - related marker p21 in hepatocytes.

[0101] p21 detection: Immunohistochemistry was used to quantitatively analyze the cellular expression level of p21 in the liver tissue of mice. For the specific method, refer to the literature: Li, Xu, Han, et al. Expression of p53, p21(CIP1 / WAF1) and eIF4E in the adjacent tissues of oral squamous cell carcinoma: establishing the molecular boundary and a cancer progression model. [J]. International journal of oral science, 2015. DOI: 10.1038 / ijos.2015.5.

[0102] Cystatin C determination: The experiment was carried out using the'mouse / Rat cystatin C' immunoassay kit (R&D Technologies, MSCTC0) according to the manufacturer's instructions.

[0103] Levels of senescence-associated secretory phenotype (SASP) factors (transforming growth factor-β1 (TGFβ1), TGFβ2, and C-C motif chemokine ligand 2 (CCL2)): The levels of senescence-associated secretory phenotype factors (TGFβ1, TGFβ2, and CCL2, catalog numbers: B163862, HY-30006K, and ALD26231, respectively) in mouse plasma were detected using a TGF-β ELISA kit from Hengyuan Biotech and an ELISA kit from Shanghai Ailand Biotechnology Co., Ltd. Reference: Zhou M, Liu C, Li B, et al. Cell surface patching via CXCR4-targeted nanothreads for cancer metastasis inhibition[J]. Nature Communications[2025-01-14]. DOI: 10.1038 / s41467-024-47111-z.

[0104] Biochemical indicators related to liver function (alanine aminotransferase (ALT), bilirubin, alkaline phosphatase (ALP)): The alanine aminotransferase (ALT) was measured using a mouse ALT detection kit from Shanghai Ailand (catalog number: ALD27658), the alkaline phosphatase (ALP) content was measured using a kit from Sigma (catalog number: EDM0210A), and the bilirubin content was measured using a kit from Shanghai Caiyou (catalog number: E-26023). The specific methods were operated according to the kit instructions.

[0105] 1.5 Experimental results

[0106] 1.5.1 p21 expression level

[0107] As shown in Table 1, through the analysis of the p21 expression level in mice, it was found that during the entire intervention period, the p21 levels in the probiotic group and the postbiotic group were slightly higher than those in the blank group but significantly lower than those in the model group ( P <0.05), indicating that the intervention with BB-35 and its inactivated bacteria can significantly reduce the p21 level, thus achieving the effect of improving liver senescence.

[0108] Table 1 Percentage of p21+ hepatocytes in four groups of mice before and after intervention

[0109]

[0110] Note: Data with the same superscript letter in the same row indicate no significant difference ( P >0.05), and different letters indicate significant difference ( P <0.05).

[0111] 1.5.2 SASP factor levels

[0112] As shown in Table 2, through the analysis of the levels of SASP factors (TGFβ1, TGFβ2, and CCL2), it was found that the levels of TGFβ1, TGFβ2, and CCL2 in the probiotic group and the postbiotic group were significantly lower than those in the model group, and there was no significant difference from the blank group (P < 0.05), indicating that the intervention of BB-35 and its inactivated bacteria could significantly reduce the levels of SASP factors, thus achieving the effect of improving liver aging.

[0113] Table 2 SASP factor levels in four groups of mice before and after intervention

[0114]

[0115] Note: Data with the same superscript letter in the same column indicate no significant difference ( P > 0.05), and different letters indicate significant difference ( P < 0.05).

[0116] 1.5.3 Cystatin C levels

[0117] As shown in Table 3, through the determination of the levels of cystatin C in the plasma of mice, it was found that the levels of cystatin C in the probiotic group and the postbiotic group were significantly lower than those in the model group, and there was no significant difference from the blank group (P < 0.05). In the liver aging model, the concentration of cystatin C in the blood increased, which is an early signal of kidney dysfunction, indicating that the changes in liver function may affect the changes in kidney function, and the intervention of probiotics and postbiotics can improve and prevent this change.

[0118] Table 3 Cystatin C levels in four groups of mice before and after intervention

[0119]

[0120] Note: Data with the same superscript letter in the same row indicate no significant difference ( P > 0.05), and different letters indicate significant difference ( P < 0.05).

[0121] 1.5.4 Changes in biochemical indicators related to liver function

[0122] Alanine aminotransferase (ALT), bilirubin, and alkaline phosphatase (ALP) are usually used together to evaluate the liver function.

[0123] As shown in Table 4, through the comparative analysis of the biochemical indicators related to liver function in mice (ALT, ALP, and bilirubin), it was found that the contents of ALT, ALP, and bilirubin in the probiotic group and the postbiotic group were significantly lower than those in the model group, and there was no significant difference from the blank group ( P(<0.05). It indicates that the intervention of BB-35 and its inactivated bacteria can significantly improve the liver function of mice.

[0124] Table 4 Biochemical index levels of four groups of mice before and after intervention

[0125]

[0126] Note: Data in the same column with the same superscript letter indicate no significant difference ( P (>0.05), and different letters indicate significant difference ( P (<0.05).

[0127] 1.6 Conclusion

[0128] The bacterial agent prepared from the probiotic Bifidobacterium animalis subsp. lactis BB-35 can prevent, alleviate and / or treat liver aging, by reducing the expression of the senescence-related marker p21 in mouse hepatocytes, decreasing the levels of senescence-related secretory phenotype factors, cystatin C, and liver function-related biochemical indexes, thereby improving the liver aging condition of mice and reducing the impact on other organs.

[0129] Effect Example 2 Probiotics improve liver injury

[0130] 2.1 Preparation of bacterial agent

[0131] Prepare the bacterial agent and postbiotic of Bifidobacterium animalis subsp. lactis BB-35 according to Example 3 and Example 4 respectively.

[0132] 2.2 Mouse background and modeling

[0133] Select healthy male C57BL / 6 mice (weight 20 - 25 g, purchased from Speywood), and randomly divide them into a model group, a probiotic group, a postbiotic group, and a control group, with 10 mice in each group. Carbon tetrachloride (CCl4) was intraperitoneally injected to induce liver injury. The specific operation is as follows: Dilute CCl4 with olive oil to a solution with a volume fraction of 10%, and intraperitoneally inject it twice a week at a dose of 0.2 mL / 100 g body weight for 4 consecutive weeks.

[0134] 2.3 Experimental grouping

[0135] Model group (10 mice): Inject 0.2 mL / 100 g body weight of CCl4;

[0136] Probiotic group (10 mice): Inject 0.2 mL / 100 g body weight of CCl4 and intragastric administration of the bacterial agent (Example 3), and intragastric administration of the bacterial powder at 5.0×10 8 CFU / mouse;

[0137] Postbiotics group (10 mice): Injected with 0.2 mL / 100 g body weight of CCl4 and gavaged with postbiotics (Example 4), 5.0×10 8 Cells / mouse;

[0138] Control group (10 mice): Injected with 0.2 mL / 100 g body weight of normal saline.

[0139] 2.4 Detection method

[0140] At the beginning and 7 weeks after the intervention, all mice were anesthetized and sacrificed, and blood and liver tissue samples were collected. Blood samples were used to detect liver function indicators such as alanine aminotransferase (ALT) and total bilirubin (TBIL); liver tissue samples were used to determine the oxidative stress indicators in liver tissue (such as malondialdehyde (MDA) content and superoxide dismutase (SOD) activity) and the expression levels of inflammatory factors (such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)).

[0141] Determination of ALT and TBIL: The contents of ALT and TBIL were determined using the Shanghai Ailianda Mouse Alanine Aminotransferase (ALT) ELISA Detection Kit and the Shanghai Caiyou Mouse Total Bilirubin Kit (product numbers: ALD27658 and E-26023 respectively). The specific method can be operated according to the kit instructions.

[0142] Determination of MDA and SOD: The contents of MDA and SOD were determined using the Bioray Malondialdehyde (MDA) Content Detection Kit and the Bioray Superoxide Dismutase (SOD) Activity Detection Kit (product numbers: AK289 and AK060 respectively).

[0143] Determination of TNF-α and IL-6: ELISA kits (enzyme-linked immunosorbent assay kits, Shanghai Enzyme-linked Biology, product numbers: ml002095 and ml098430 respectively) were used to detect the levels of inflammatory factors TNF-α and IL-6.

[0144] 2.5 Experimental results

[0145] 2.5.1 ALT and TBIL levels

[0146] As shown in Table 5, through the comparative analysis of the liver function indicators ALT and TBIL in mice, it was found that the contents of ALP and TBIL in the probiotic group and the postbiotics group were significantly lower than those in the model group ( P <0.05), and there was no significant difference from the blank group. It indicated that the intervention with BB-35 and its inactivated bacteria could significantly improve the liver injury in mice.

[0147] Table 5 Changes in ALT and TBIL levels in four groups of mice before and after intervention

[0148]

[0149] Note: The same superscript letter for the data in the same column indicates no significant difference ( P >0.05), and different letters indicate significant difference ( P <0.05).

[0150] 2.5.2 Oxidative stress indices MDA and SOD

[0151] As shown in Table 6, the SOD content in the probiotic group and the postbiotic group was significantly higher than that in the model group ( P <0.05), and the MDA content was significantly lower than that in the model group ( P <0.05), showing no significant difference from the blank group. It indicates that the intervention with BB-35 and its heat-inactivated bacteria can significantly improve the liver injury in mice.

[0152] Table 6 Changes in MDA and SOD levels in four groups of mice before and after intervention

[0153]

[0154] Note: The same superscript letter for the data in the same column indicates no significant difference ( P >0.05), and different letters indicate significant difference ( P <0.05).

[0155] 2.5.3 Levels of inflammatory factors TNF-α and IL-6

[0156] As shown in Table 7, after intervention, the inflammatory factors TNF-α and IL-6 in the model group increased significantly (P<0.05), while the levels of inflammatory factors in the probiotic group and the postbiotic group of mice remained almost unchanged, showing no significant difference from the blank group. It indicates that the intervention with BB-35 and its heat-inactivated bacteria can significantly improve the liver injury in mice.

[0157] Table 7 Changes in TNF-α and IL-6 levels in four groups of mice before and after intervention

[0158]

[0159] Note: The same superscript letter for the data in the same column indicates no significant difference ( P >0.05), and different letters indicate significant difference ( P <0.05).

[0160] 2.6 Conclusion

[0161] The bacterial agent prepared from Bifidobacterium animalis subsp. lactis BB-35 can prevent, alleviate and / or treat liver injury by reducing the levels of alanine aminotransferase and total bilirubin, which are liver function indicators in mice, decreasing the contents of malondialdehyde and superoxide dismutase, which are oxidative stress indicators, and lowering the levels of tumor necrosis factor-α and interleukin-6, which are inflammatory factors, thereby improving the liver injury condition in mice and providing a new direction and idea for the prevention and treatment of liver injury.

[0162] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ), characterized in that Its preservation number is CGMCC No. 27836.

2. Use of the Bifidobacterium animalis subsp. lactis according to claim 1, characterized in that, The applications include at least one of the following: (1) Application in the preparation of drugs for preventing, alleviating and / or treating liver aging; (2) Application in the preparation of drugs for preventing, alleviating and / or treating liver injury.

3. A bacterial agent, characterized in that, The bacterial agent includes the Bifidobacterium animalis subsp. lactis described in claim 1.

4. The bacterial agent according to claim 3, characterized in that, The bacterial agent includes one or more of the fermentation broth, live bacteria, dead bacteria, freeze-dried powder and cell lysate of Bifidobacterium animalis subsp. lactis.

5. The microbial agent according to claim 4, characterized in that, The number of bacteria of Bifidobacterium animalis subsp. lactis is not less than 5.0×10 8 CFU / mL or 5.0×10 8 CFU / g.

6. The microbial agent according to any one of claims 3-5, characterized in that, The bacterial agent is a solid preparation or a liquid preparation.

7. A drug, characterized in that, The drug includes the Bifidobacterium animalis subsp. lactis described in claim 1.

8. The medicament according to claim 7, characterized in that, The drug further includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that, The pharmaceutically acceptable excipients include excipients.

10. The drug according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more of stabilizers, diluents, binders, preservatives, lubricants, antioxidants.

11. The drug according to any one of claims 7-10, characterized in that, The dosage form of the drug is tablets, liquids, capsules, powders, suppositories or granules.

12. A pharmaceutical raw material, characterized in that, The pharmaceutical raw material includes the Bifidobacterium animalis subsp. lactis described in claim 1.

13. The pharmaceutical raw material according to claim 12, wherein, The pharmaceutical raw material is in solid form, liquid form or semi-solid form.

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