Bifidobacterium animalis subsp. Lactis, product and application of bifidobacterium animalis subsp. Lactis in liver protection

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bifidobacterium animalis subsp. Lactis, a product and application of the bifidobacterium animalis subsp. Lactis in liver protection. The preservation number of the bifidobacterium animalis subsp. Lactis BB-35 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No.27836, and the preservation number of the bifidobacterium animalis subsp. Lactis BB-35 provided by the invention is CGMCC No.27836. The strain can be used for reducing the expression condition of a senescence-related marker p21 in liver cells of mice, and reducing the level of senescence-related secretory phenotype factors, the level of cystatin C and liver function-related biochemical indexes, so that the liver senescence condition of the mice is improved, and the influence on other organs is reduced; in addition, the strain can also improve oxidative stress index malonaldehyde and superoxide dismutase content, reduce inflammatory factor TNF-alpha and IL-6 levels, and provide technical support for preparation of products for preventing, relieving and / or treating liver aging and liver injury.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of animal Bifidobacterium lactis subspecies, a product and an application thereof in protecting the liver. Background Art

[0002] The liver is one of the most important metabolic organs in the human body, and it undertakes many physiological functions, including the metabolism of nutrients, detoxification, protein synthesis, and immune regulation. During the life process, the liver will gradually show aging-related changes with age. For example, the regenerative capacity of liver cells gradually decreases, the accumulation of extracellular matrix in liver tissue increases, and the metabolic functions of the liver, such as the metabolic efficiency of lipids, carbohydrates, and drugs, decrease. At the same time, the liver's antioxidant defense system function declines, leading to the accumulation of free radicals, further damaging the structure and function of liver cells and organelles. These changes make the liver more susceptible to various endogenous and exogenous damage factors, increasing the risk of liver diseases such as fatty liver and cirrhosis, which seriously affect overall health and quality of life.

[0003] In the field of anti-aging research, a variety of 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 targets. For example, some chemically synthesized antioxidants may produce harmful intermediates during metabolism in the body or interfere with normal physiological functions. In terms of non-drug interventions, such as dietary adjustments and exercise, although beneficial to health, the targeted improvement effect on liver aging is not significant enough and difficult to accurately quantify and standardize. In addition, some emerging technologies such as gene therapy and stem cell therapy are still in the early stages of research and face challenges in many aspects such as technical complexity, safety and ethics, making it difficult to be widely used in clinical practice.

[0004] As a kind of active microorganisms that are beneficial to the host, probiotics have been increasingly studied in the field of health in recent years. Studies have shown that probiotics play an important role in maintaining intestinal health, and can regulate the balance of intestinal flora, enhance intestinal barrier function, and regulate immune response. With the deepening of research, people have gradually discovered that there is a close "intestinal-liver axis" connection between the intestine and the liver. Intestinal flora and its metabolites can enter the liver through the portal vein system and affect the physiological function and pathological state of the liver. Some studies have preliminarily suggested that specific probiotic strains may indirectly affect the lipid metabolism, inflammatory response, and antioxidant status of the liver by regulating the composition and metabolism of intestinal flora. For example, some probiotics can reduce the level of endotoxins produced by harmful bacteria in the intestine and reduce the inflammatory response caused by endotoxins entering the liver through the portal vein; other studies have shown that probiotics can promote the expression of antioxidant enzymes in liver cells and enhance the antioxidant capacity of the liver. However, most of these studies are relatively scattered, lacking systematic and in-depth exploration of the comprehensive mechanism of probiotics on liver aging and patented technologies specifically used to improve liver aging.

[0005] In summary, as liver aging and damage are receiving increasing attention and existing methods have many shortcomings, in-depth research on the mechanism of action of probiotics in improving liver aging and damage and the development of related patented 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 damage. Summary of the invention

[0006] In view of the above shortcomings, the present invention provides a strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ) BB-35, with the deposit number of CGMCC No.27836, was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 7, 2023. The strain is cultured, fermented, and freeze-dried to obtain bacterial powder and postbiotic products (including BB-35 inactivated bacteria and / or metabolites), which can effectively intervene in liver aging and damage.

[0007] The technical solution of the present invention is: In one aspect, the present invention provides a strain of Bifidobacterium animalis subsp. lactis, whose deposit number is CGMCC No.27836.

[0008] In another aspect, the present invention provides a culture, wherein the culture is obtained by inoculating the aforementioned Bifidobacterium animalis subsp. lactis into a culture medium and culturing the culture medium.

[0009] More specifically, the inoculation method includes, but is not limited to, any one or more of: plate streak method, slant inoculation method, pour culture method, puncture inoculation method, and liquid inoculation method.

[0010] More specifically, the inoculation amount of the inoculation can be 0.1%-20%, and in some cases it can also be a higher or lower inoculation amount. Specifically, the inoculation amount can 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%.

[0011] Further specifically, the culture medium can be a solid culture medium, a semi-solid culture medium or a liquid culture medium, more specifically, it can be any suitable culture medium type disclosed in the prior art, it can also be a culture medium that is further improved on the culture medium type disclosed in the prior art to improve the strain performance, or it can be a culture medium that is not disclosed in the prior art but can be used for the cultivation of the aforementioned Lactococcus lactis subspecies cremoris.

[0012] Preferably, the components of the culture medium may include, by weight: 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 parts of MgSO4·7H2O, 0.1-0.5 parts of MnSO4·5H2O, 0.5-1.5 parts of Tween-80 and 0.1-0.5 parts of L-cysteine ​​hydrochloride.

[0013] Further preferably, the components of the culture medium may include, by weight: 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 parts of MgSO4·7H2O, 0.3 parts of MnSO4·5H2O, 1.0 parts of Tween-80 and 0.3 parts of L-cysteine ​​hydrochloride.

[0014] In another aspect, the present invention provides the use of the aforementioned animal Bifidobacterium lactis subspecies or culture, wherein the use comprises at least one of the following: (1) Application in the preparation of products for preventing, alleviating and / or treating liver aging; (2) Use in the preparation of products for preventing, alleviating and / or treating liver damage.

[0015] Specifically, the products described in (1) and (2) include bacterial preparations, health products, medicines or pharmaceutical raw materials.

[0016] Specifically, (2) the liver injury includes but is not limited to traumatic liver injury or disease-induced liver injury.

[0017] In another aspect, the present invention provides a bacterial agent, comprising the aforementioned Bifidobacterium animalis subsp. lactis.

[0018] Specifically, the bacterial agent includes one or more of the fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, freeze-dried powder and cell lysate of Bifidobacterium animalis subsp. lactis.

[0019] More specifically, the fermentation liquid refers to the liquid obtained by inoculating the bacteria into the culture medium and culturing it for a period of time.

[0020] To be more specific, the fermentation broth supernatant refers to the clarified liquid on the upper layer of the fermentation broth after centrifugation; it contains abundant metabolites of bacterial growth and reproduction and some bacterial fragments. The acidic substances and bacteriocins secreted by bacteria have antagonistic and killing effects on harmful bacteria; the amino acids and synthesized vitamins after bacteria decompose food are all in the culture medium, and also include 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.

[0021] More specifically, the fermentation liquid precipitate refers to the liquid precipitate obtained by centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix, mainly protein and intracellular matrix.

[0022] More specifically, the live bacteria, also called active flora, can colonize and multiply in the intestine, which is beneficial to increase the number of beneficial bacteria.

[0023] More specifically, the dead bacteria are microorganisms that have lost their vitality and are unable to grow and reproduce. The probiotics lose their vitality due to the production process, such as high temperature treatment or excessive drying.

[0024] More specifically, the lyophilized powder is obtained by freeze-drying the aforementioned culture solution. The lyophilized powder generally also includes a lyophilization protectant. The lyophilization protectant includes, but is not limited to, pH buffer, filler, sugar, nonionic 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, and bovine serum albumin. The sugar may be a disaccharide, such as any one or more of sucrose or trehalose. The nonionic surfactant includes, but is not limited to, Tween, and the Tween may be Tween-20, Tween-60, Tween-80, etc. The lyophilization protectant may also include antioxidants, etc. The lyophilization protectant may also specifically include albumin, polyethylene glycol, etc.

[0025] More specifically, the cell lysis solution can be obtained by lysing the bacteria obtained by 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 hydrolysis, and the enzymatic hydrolysis can be hydrolase or oxidase. The lysis can also be performed by increasing the intracellular pressure to cause the cells to rupture by themselves.

[0026] Specifically, the bacterial count of the animal Bifidobacterium lactis subspecies is not less than 5.0×10 8 CFU / mL or 5.0×10 8 CFU / g.

[0027] More specifically, the bacterial count of the animal Bifidobacterium lactis subspecies 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.

[0028] Specifically, the bacterial agent can be a solid preparation or a liquid preparation.

[0029] In another aspect, the present invention provides a medicine, comprising the aforementioned Bifidobacterium animalis subspecies lactis or culture.

[0030] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0031] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, and antioxidants.

[0032] Specifically, the dosage form of the drug includes, but is not limited to, tablets, liquids, capsules, powders, suppositories or granules.

[0033] In another aspect, the present invention provides a pharmaceutical raw material, which comprises the aforementioned Bifidobacterium animalis subspecies lactis or culture.

[0034] Specifically, the pharmaceutical raw material may be in solid form, liquid form or semi-solid form.

[0035] The beneficial effects of the present invention are: The BB-35 provided by the present invention can be used to reduce the expression of the aging-related marker p21 in mouse liver cells, reduce the levels of aging-related secretory phenotypic factors, cystatin C levels and biochemical indicators related to liver function, thereby improving the liver aging of mice and reducing the impact on other organs. In addition, the strain can also improve the oxidative stress indicators malondialdehyde and superoxide dismutase content, and reduce the levels of inflammatory factors TNF-α and IL-6. The strain can be used to prepare products for preventing, alleviating and / or treating liver aging and liver damage.

[0036] Collection information: Biomaterial: BB-35; Classification name: Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalum subsp. milk ); Deposit number: CGMCC No.27836; Deposit date: July 7, 2023; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Abbreviation of depository unit: CGMCC; Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0037] In the present invention, the following terms are used: p21: p21 is a cyclin-dependent kinase inhibitor (CKIs), which can inhibit cell cycle progression, causing hepatocytes to enter a senescent state and exhibit aging-related characteristics, such as cell cycle arrest and the appearance of the senescence-associated secretory phenotype (SASP). In a variety of tissues and cell types, such as liver, kidney, brain, and lung, the expression level of p21 is significantly increased in aging models.

[0038] SASP: The SASP factors secreted by aging hepatocytes can act on the surrounding normal hepatocytes, inducing their aging. They can also enter the blood circulation, reach distant organs and tissues, affect the cell aging process of other organs, and cause multiple organ dysfunction.

[0039] TGFβ1: TGFβ1 is a key profibrotic factor that is involved in regulating the aging of liver cells. On the one hand, it can induce liver cell aging by activating intracellular signaling pathways. For example, TGFβ1 can upregulate the expression of CKIs such as p15 and p21, which can inhibit the activity of cyclin-cyclin-dependent kinase (cyclin-CDK) complexes, causing cell cycle arrest, leading to liver cell aging. On the other hand, TGFβ1 also plays a role in SASP. TGFβ1 secreted by aging 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 impact of aging cells.

[0040] TGFβ2: TGFβ2 is similar to TGFβ1 and is involved in the induction of liver cell senescence, activating intracellular signaling pathways, leading to upregulation of CKIs expression, arresting the cell cycle, and thus inducing liver cells to enter a senescent state.

[0041] CCL2: C-C motif chemokine ligand 2 (CCL2) is an important mediator of inflammatory response. Aging liver cells release CCL2, which can attract immune cells such as monocytes and lymphocytes to gather in liver tissue. CCL2 can also transmit aging signals between cells. CCL2 secreted by aging liver cells can act on the surrounding normal liver cells and induce them to enter a senescent state.

[0042] Cystatin C: Cystatin C is an endogenous marker that reflects glomerular filtration rate (GFR). Changes in its level in the blood can sensitively indicate the state of renal function. Changes in liver function may cause systemic effects, including renal function.

[0043] ALT: ALT is mainly present in the cytoplasm of liver cells. When liver cells are damaged, the permeability of the cell membrane changes, and the ALT in the cells will be released outside the cells and then enter the blood circulation, leading to an increase in the ALT level in the serum. Bilirubin plays an important role in evaluating the bilirubin metabolism function of the liver. When the liver is damaged, such as hepatitis, cirrhosis, intrahepatic bile duct obstruction, etc., the uptake, binding or excretion process of bilirubin will be affected, resulting in an increase in the bilirubin concentration in the blood and the appearance of jaundice.

[0044] ALP: In the hepatobiliary system, ALP is mainly present on the microvilli of the bile capillary surface of hepatocytes and is involved in the formation and excretion of bile. When the liver or bile duct system is diseased, the synthesis and release of ALP by hepatocytes increases, leading to an increase in serum ALP. Therefore, they are sensitive indicators commonly used in clinical practice and research to reflect the degree of hepatocyte damage.

[0045] MDA: MDA is one of the end products of lipid peroxidation. Its content can usually indirectly reflect the degree of lipid peroxidation in the body and the severity of free radical attack on cells. During liver damage in mice, if affected by drugs, chemicals, ischemia-reperfusion and other factors, a large number of free radicals will be produced in liver cells, triggering lipid peroxidation and leading to increased MDA content. Therefore, the determination of MDA content is often used as one of the important indicators for evaluating the level of oxidative stress in liver damage.

[0046] SOD: SOD is an important antioxidant enzyme, which is widely present in the cytoplasm, mitochondria and other parts 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. However, when liver damage occurs, due to the massive production of free radicals, the activity of SOD may be affected, and its ability to scavenge free radicals will also change accordingly.

[0047] TNF-α: TNF-α is mainly secreted by activated macrophages, Kupffer cells, etc. It can activate multiple 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 and IL-6, thereby initiating and amplifying the inflammatory response, attracting more immune cells to infiltrate the liver tissue, and aggravating inflammatory damage.

[0048] IL-6: IL-6 is a pleiotropic cytokine that is produced by a variety of cells such as macrophages, lymphocytes, and hepatocytes during liver damage. It can act on a variety of cell types, promote the activation, proliferation, and migration of inflammatory cells, enhance the phagocytic and killing abilities 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 synergize with TNF-α to form a complex inflammatory network, jointly participating in the pathological process of liver damage.

[0049] The present invention will be further described in detail below by way of examples. The following examples are only a part of the present invention and are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0050] Example 1 Preparation of a bacterial powder Bacterial species: Bifidobacterium animalis subspecies lactis BB-35, with the deposit number of CGMCC No.27836; Preparation method of bacterial powder: (1) Activation of bacterial strains The strain stored in -40°C was inoculated into a modified MRS liquid culture medium sterilized at 121°C for 15 min, cultured anaerobically at 37°C for 24 h, and subcultured twice to obtain an activated strain.

[0051] (2) Optimizing the preparation of culture medium The optimized culture medium composition is as follows: sucrose 23.5kg, lactose 12.0kg, soy peptone 15.0kg, yeast powder 5.0kg, yeast peptone 12kg, Na2HPO321.0kg, citric acid 2.0kg, MgSO4·7H2O 0.6kg, MnSO4·5H2O 0.3kg, Tween-80 1.0kg, L-cysteine ​​hydrochloride 0.3kg, and distilled water 1000L.

[0052] The components of the optimized culture medium were prepared in proportion, mixed evenly, adjusted to pH 6.5, and sterilized at 121°C for 15 min.

[0053] (3) Preparation of seed solution The activated bacteria were inoculated into the optimized culture medium and cultured anaerobically at 37°C until the pH value reached 4.6 to obtain the seed solution.

[0054] (4) Inoculation and fermentation Fermentation was carried out in a BLBIO-10GJ fermenter produced by Bailun Biotechnology. 100 L of optimized culture medium was placed in the fermenter. After sterilization, the seed liquid was inoculated into the optimized culture medium at a volume fraction of 1‰ and fermented for 18 h under controlled fermentation conditions.

[0055] The fermentation conditions were controlled as follows: constant temperature culture at 30°C in the early stage of fermentation, and natural fermentation until the pH reached 5.0; then the fermentation temperature was adjusted to constant temperature culture at 37°C, and the pH was controlled to remain at 6.0 to maintain anaerobic fermentation.

[0056] During the fermentation process, the pH was controlled by adding a neutralizer, wherein the neutralizer was a 25% NaOH solution; and the anaerobic condition was achieved by passing nitrogen gas once every two hours.

[0057] (5) Termination of fermentation: When the bacteria stop producing acid, the fermentation is terminated to obtain a high-density fermentation liquid of each strain, and the number of live bacteria in the fermentation liquid reaches 5×10 10 cfu / mL or more. The cessation of acid production can be judged by the fact that the pH no longer decreases and the addition of the neutralizer stops.

[0058] (6) Freeze drying: a. Bacterial cell concentration: The high-density fermentation broth is concentrated by centrifugation at 12000g.

[0059] b. Add protective agent: Add 5 times the protective agent solution to the bacterial concentrate; the protective agent solution composition is as follows: 15kg skim milk, 12kg lactose, 1kg vitamin C, 1kg sodium glutamate, 1000L distilled water.

[0060] c. Drying: The bacterial suspension after adding the protective agent is freeze-dried (model: Sihuan LGJ-12C) to obtain freeze-dried bacterial powder, and the total number of viable bacteria in the bacterial powder reaches 5.0×10 11 CFU / g and above.

[0061] Example 2 Preparation of a probiotic postbiotic powder On the basis of Example 1, after terminating the fermentation, the following steps are performed: Inactivation: Mix maltodextrin according to production requirements (to provide 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 sterilize and inactivate. The sterilization and inactivation parameters are 75°C and 15 minutes. Spray drying: The inactivated bacterial suspension is spray dried (inlet air temperature 160°C, outlet air temperature 45°C) to obtain probiotic postbiotic powder.

[0062] Example 3 Preparation of a bacterial agent Weigh 0.1 g of the bacterial powder in Example 1, add 0.9 g of maltodextrin as an auxiliary material, mix well, and the number of viable bacteria is not less than 5.0 × 10 10 CFU / g.

[0063] Example 4 Preparation of a postbiotic Weigh 0.1 g of the probiotic postbiotic powder in Example 2, add 0.9 g of maltodextrin as an auxiliary material, mix well, and the bacterial count is not less than 5.0 × 10 10 Cells / g.

[0064] Effect Example 1: Probiotics Improve Liver Aging 1.1 Preparation of inoculants According to Example 3 and Example 4, the bacterial agent and postbiotics of Bifidobacterium animalis subsp. lactis BB-35 were prepared respectively.

[0065] 1.2 Mouse background and modeling C57Bl6 mice aged 8-12 weeks (weight 20-25g, purchased from Speffort), half male and half female, were selected. The mice were housed in an animal room at a temperature of 22 ± 2℃ and a humidity of 50-60%, with free access to food and water. The experiment was started after acclimatization for 1 week. Mice carrying the Mdm2tm2.1Glo allele (reference: Grier JD, 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 TA, Sunkin SM, et al. A robust and high-throughput Cre reporting and characterization systemfor the whole mouse brain[J]. Nature Neuroscience, 2010, 13(1):133-140. DOI:10.1038 / nn.2467.) were injected intravenously with the hepatocyte-specific AAV8-TBG-Cre vector, causing genetic recombination in liver cells (Δ Mdm2 Hep ), resulting in the removal of the p53 binding domain of MDM2, weakening the inhibitory effect of MDM2 on p53, and the accumulation of p53 in hepatocytes, thereby inducing liver senescence and achieving the establishment of a liver aging model.

[0066] 1.3 Experimental Grouping Model group (20 mice): 2×10 AAV8-TBG-Cre vectors were injected every day 11 GC / pc; Probiotic group (20 mice): AAV8-TBG-Cre vector injection and intragastric administration of bacteria were performed every day. AAV8-TBG-Cre vector 2×10 11 GC / head, bacterial agent (Example 3) gavage 5.0×10 8 CFU / piece; Postbiotic group (20 mice): AAV8-TBG-Cre vector injection and postbiotics gavage were performed every day. AAV8-TBG-Cre vector 2×10 11GC / head, postbiotics (Example 4) gavage 5.0×10 8 CFU / piece; Blank group (20 rats): 100 μL of normal saline was injected per rat every day.

[0067] 1.4 Detection Method At the beginning of the intervention and 30 days after the intervention, blood samples were collected from mice by cardiac puncture to measure the levels of aging-related secretory phenotypic factors, cystatin C levels, and biochemical indices related to liver function in the mice. The mice were killed 30 days later, and liver tissue samples were collected to analyze the expression of the aging-related marker p21 in hepatocytes.

[0068] p21 detection: Immunohistochemistry was used to quantitatively analyze the expression level of p21 in mouse liver tissue. The specific method is referenced in 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. Cystatin C assay: The experiment was performed using the 'mouse / rat cystatin C' immunoassay kit (R&D Technologies, MSCTC0) according to the manufacturer's instructions.

[0069] Levels of senescence-related secretory phenotype SASP factors (TGFβ1, TGFβ2, and C-C motif chemokine ligand 2 (CCL2)): Hengyuan Biotechnology mouse transforming growth factor β (TGF-β) ELISA kit and Shanghai Ailenda Biotechnology Co., Ltd. ELISA kit were used to detect the levels of senescence-related secretory phenotype factors in mouse plasma (TGFβ1, TGFβ2, and CCL2 catalog numbers are B163862, HY-30006K, and ALD26231, respectively). Reference: Zhou M, Liu C, Li B, et al. Cell surface patching via CXCR4-targeted nanothreads for cancer metastasisinhibition[J]. Nature Communications[2025-01-14]. DOI:10.1038 / s41467-024-47111-z. Biochemical indicators related to liver function (alanine aminotransferase (ALT), bilirubin, alkaline phosphatase (ALP)): Alanine aminotransferase (ALT) was measured using the Shanghai Allianda Mouse ALT Detection Kit (Cat. No.: ALD27658), and alkaline phosphatase (ALP) content was measured using the Xibao kit (Cat. No.: EDM0210A), and bilirubin content was measured using the Shanghai Caiyou kit (Cat. No.: E-26023). The specific method can be operated according to the instructions of the kit.

[0070] 1.5 Experimental Results 1.5.1 p21 expression level As shown in Table 1, the p21 expression level of mice was analyzed and it was found that during the entire intervention period, the p21 levels of the probiotic group and the postbiotic group were slightly higher than those of the blank group, but significantly lower than those of the model group ( P <0.05), indicating that BB-35 and its inactivated bacteria intervention can significantly reduce the level of p21, thereby achieving the effect of improving liver aging.

[0071] Table 1 Percentage of p21+ hepatocytes in four groups of mice before and after intervention

[0072] Note: The same letters in the same row of data indicate no significant difference ( P >0.05, different letters indicate significant differences ( P <0.05).

[0073] 1.5.2 SASP factor levels 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 with the blank group (P<0.05), indicating that BB-35 and its inactivated bacteria intervention can significantly reduce the level of SASP factors, thereby achieving the effect of improving liver aging.

[0074] Table 2 SASP factor levels in the four groups of mice before and after intervention

[0075] Note: The same letters in the same column indicate no significant difference ( P >0.05, different letters indicate significant differences ( P <0.05).

[0076] 1.5.3 Cystatin C Levels As shown in Table 3, by measuring the level of cystatin C in mouse plasma, the cystatin C levels in the probiotic group and the postbiotic group were significantly lower than those in the model group, and there was no significant difference with the blank group (P<0.05). The concentration of cystatin C in the blood increased in the liver aging model, which is an early sign of renal dysfunction, indicating that changes in liver function may affect changes in renal function, and the intervention of probiotics and postbiotics can improve and prevent this change.

[0077] Table 3 Cystatin C levels in four groups of mice before and after intervention

[0078] Note: The same letters in the same row of data indicate no significant difference ( P >0.05, different letters indicate significant differences ( P <0.05).

[0079] 1.5.4 Changes in biochemical indicators related to liver function Alanine aminotransferase (ALT), bilirubin, and alkaline phosphatase (ALP) are often used together to evaluate liver function.

[0080] As shown in Table 4, through comparative analysis of biochemical indices related to liver function in mice (ALT, ALP and bilirubin), it was found that the ALT, ALP and bilirubin levels in the probiotic group and the postbiotic group were significantly lower than those in the model group, and there was no significant difference with the blank group ( P <0.05). This indicates that BB-35 and its inactivated bacteria intervention can significantly improve the liver function of mice.

[0081] Table 4 Biochemical index levels of the four groups of mice before and after intervention

[0082] Note: The same letters in the same column indicate no significant difference ( P >0.05, different letters indicate significant differences ( P <0.05).

[0083] 1.6 Conclusion The bacterial preparation prepared from the probiotic animal Bifidobacterium lactis subsp. BB-35 can prevent, alleviate and / or treat liver aging by reducing the expression of the aging-related marker p21 in mouse hepatocytes, reducing the levels of aging-related secretory phenotypic factors, cystatin C levels and biochemical indicators related to liver function, thereby improving the liver aging of mice and reducing the impact on other organs.

[0084] Effect Example 2: Probiotics Improve Liver Damage 2.1 Preparation of inoculants According to Example 3 and Example 4, the bacterial agent and postbiotics of Bifidobacterium animalis subsp. lactis BB-35 were prepared respectively.

[0085] 2.2 Mouse background and modeling Healthy male C57BL / 6 mice (weight 20-25g, purchased from Spefoc) were selected and randomly divided into model group, probiotic group, postbiotic group and control group, with 10 mice in each group. Liver injury was induced by intraperitoneal injection of carbon tetrachloride (CCl4). The specific operation was as follows: CCl4 was diluted with olive oil to a solution with a volume fraction of 10%, and intraperitoneally injected twice a week at a dose of 0.2mL / 100g body weight for 4 consecutive weeks.

[0086] 2.3 Experimental Grouping Model group (10 rats): injected with CCl4 0.2 mL / 100 g body weight; Probiotic group (10 rats): injection of CCl4 0.2 mL / 100 g body weight and intragastric administration of bacterial agent (Example 3), intragastric administration of bacterial powder 5.0×10 8 CFU / piece; Postbiotic group (10 rats): injected with CCl4 0.2 mL / 100 g body weight and gavaged with postbiotics (Example 4), postbiotics 5.0×10 8 Cells / cell; Control group (10 rats): injected with 0.2 mL / 100 g body weight of normal saline.

[0087] 2.4 Detection Method At the beginning of the intervention and 7 weeks after the intervention, all mice were anesthetized and killed, 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 expression levels of oxidative stress indicators (such as malondialdehyde (MDA) content and superoxide dismutase (SOD) activity) and inflammatory factors (such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)) in liver tissue.

[0088] ALT and TBIL determination: ALT and TBIL levels were determined using Shanghai Ailenda Mouse Alanine Aminotransferase (ALT) ELISA Kit and Shanghai Caiyou Mouse Total Bilirubin Kit (Cat. No.: ALD27658 and E-26023 respectively). The specific method can be operated according to the instructions of the kit.

[0089] MDA and SOD determination: The content of MDA and SOD was determined using the Bioson malondialdehyde (MDA) content detection kit and the Bioson superoxide dismutase (SOD) activity detection kit (catalog numbers: AK289 and AK060, respectively).

[0090] TNF-α and IL-6 determination: ELISA kits (enzyme-linked immunosorbent assay kit, Shanghai ELISA Biological, catalog numbers: ml002095 and ml098430 respectively) were used to detect the levels of inflammatory factors TNF-α and IL-6.

[0091] 2.5 Experimental Results 2.5.1 ALT and TBIL levels As shown in Table 5, through comparative analysis of the liver function indicators ALT and TBIL in mice, it was found that the ALP and TBIL levels in the probiotic group and the postbiotic group were significantly lower than those in the model group ( P <0.05), and there was no significant difference with the blank group. This showed that BB-35 and its inactivated bacteria intervention could significantly improve the liver injury of mice.

[0092] Table 5 Changes of ALT and TBIL levels in the four groups of mice before and after intervention

[0093] Note: The same letters in the same column indicate no significant difference ( P >0.05, different letters indicate significant differences ( P <0.05).

[0094] 2.5.2 Oxidative stress indicators MDA and SOD 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), and there was no significant difference with the blank group. This showed that BB-35 and its inactivated bacteria intervention could significantly improve the liver injury of mice.

[0095] Table 6 Changes of MDA and SOD levels in four groups of mice before and after intervention

[0096] Note: The same letters in the same column indicate no significant difference ( P >0.05, different letters indicate significant differences ( P <0.05).

[0097] 2.5.3 Levels of inflammatory factors TNF-α and IL-6 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 postbiotic group remained almost unchanged and had no significant difference from the blank group. This indicates that BB-35 and its inactivated bacteria intervention can significantly improve the liver damage in mice.

[0098] Table 7 Changes in TNF-α and IL-6 levels in the four groups of mice before and after intervention

[0099] Note: The same letters in the same column indicate no significant difference ( P >0.05, different letters indicate significant differences ( P <0.05).

[0100] 2.6 Conclusion The bacterial agent prepared with the probiotic animal Bifidobacterium lactis subspecies BB-35 can prevent, alleviate and / or treat liver damage. It improves the liver damage in mice by reducing the levels of alanine aminotransferase and total bilirubin, the liver function indicators of mice, reducing the content of malondialdehyde and superoxide dismutase, the indicators of oxidative stress, and reducing the levels of inflammatory factors tumor necrosis factor α and interleukin 6, thereby providing new directions and ideas for the prevention and treatment of liver damage.

[0101] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

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

2. A culture, characterized in that The culture is obtained by inoculating the animal Bifidobacterium lactis subsp. lactis described in claim 1 into a culture medium for cultivation.

3. The culture according to claim 2, characterized in that The components of the culture medium include, by weight: 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 parts of MgSO4·7H2O, 0.1-0.5 parts of MnSO4·5H2O, 0.5-1.5 parts of Tween-80 and 0.1-0.5 parts of L-cysteine ​​hydrochloride.

4. The culture according to claim 3, characterized in that The components of the culture medium include, by weight: 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 parts of MgSO4·7H2O, 0.3 parts of MnSO4·5H2O, 1.0 parts of Tween-80 and 0.3 parts of L-cysteine ​​hydrochloride.

5. The use of the Bifidobacterium animalis subsp. lactis according to claim 1 or the culture according to any one of claims 2 to 4, characterized in that: The application includes at least one of the following: (1) Application in the preparation of products for preventing, alleviating and / or treating liver aging; (2) Use in the preparation of products for preventing, alleviating and / or treating liver damage.

6. The use according to claim 5, characterized in that: The products mentioned in (1) and (2) include bacterial preparations, health products, medicines or pharmaceutical raw materials.

7. A bacterial agent, characterized in that The bacterial agent comprises the Bifidobacterium animalis subsp. lactis according to claim 1.

8. The bacterial agent according to claim 7, characterized in that The bacterial agent comprises one or more of the fermentation liquid, fermentation liquid supernatant, fermentation liquid precipitation, live bacteria, dead bacteria, freeze-dried powder and cell lysate of Bifidobacterium animalis lactis subspecies.

9. The bacterial agent according to claim 8, characterized in that The bacterial count of the animal Bifidobacterium lactis subspecies is not less than 5.0×10 8 CFU / mL or 5.0×10 8 CFU / g.

10. The bacterial agent according to any one of claims 7 to 9, characterized in that: The bacterial agent is a solid preparation or a liquid preparation.

11. A drug, characterized in that The medicament comprises the Bifidobacterium animalis subsp. lactis of claim 1 or the culture of any one of claims 2-4.

12. The drug according to claim 11, characterized in that The drug also includes pharmaceutically acceptable excipients.

13. The drug according to claim 12, characterized in that The pharmaceutically acceptable excipients include any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, and antioxidants.

14. The drug according to any one of claims 11 to 13, characterized in that The dosage form of the drug is tablet, liquid, capsule, powder, suppository or granule.

15. A pharmaceutical raw material, characterized in that: The pharmaceutical raw material comprises the Bifidobacterium animalis subspecies lactis according to claim 1 or the culture according to any one of claims 2-4.

16. The pharmaceutical raw material according to claim 15, characterized in that: The pharmaceutical raw material is in solid form, liquid form or semi-solid form.

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