Bifidobacterium longum CCFM1508 capable of activating TFEB-HKDC1 autophagy pathway and application of bifidobacterium longum CCFM1508 in colon aging resistance

Abstract

The invention discloses bifidobacterium longum CCFM1508 capable of activating a TFEB-HKDC1 autophagy pathway and application of the bifidobacterium longum CCFM1508 in the aspect of colon aging resistance, and belongs to the field of microorganisms. The bifidobacterium longum CCFM1508 provided by the invention has an autophagy regulation capability and can be used for effectively improving colon aging. Through intervention of the bifidobacterium longum CCFM1508, damage to colon tissue of an aged mouse can be remarkably relieved, activity of aging-related beta-galactosidase is inhibited, cell apoptosis is regulated, the level of proinflammatory cytokines is inhibited, oxidative damage degree in the colon tissue is slowed down, transcriptional levels of aging markers p21 and p53 in the colon tissue are remarkably reduced, an autophagy pathway of TFEB-HKDC1 can be activated, and the anti-aging effect is achieved. The autophagy level of the colon tissue is adjusted, and the colon aging resisting effect is achieved. Therefore, the application range of the bifidobacterium longum as probiotics is expanded, and the bifidobacterium longum has a huge application prospect in relieving colon aging.

Description

Technical Field

[0001] This invention relates to a strain of Bifidobacterium longum CCFM1508 that activates the TFEB-HKDC1 autophagy pathway and its application in anti-colon aging, belonging to the field of microbiology. Background Technology

[0002] With the increasing aging of the global population, aging and related diseases have become a major public health issue. The colon, as a key organ for digestion, absorption, immune defense, and maintenance of gut microbiota homeostasis, is experiencing increasing attention due to the age-related decline in its structure and function. Colonic aging is not only manifested in the accumulation of intestinal damage, decline in intestinal barrier function, weakened intestinal immunity, and dysbiosis of the gut microbiota, but is also closely related to various systemic age-related diseases, such as neurodegenerative diseases and metabolic syndrome. Therefore, finding safe and effective intervention strategies to actively regulate the healthy lifespan of the colon in the elderly is of great significance.

[0003] Current research indicates that one of the core mechanisms of colonic aging involves impaired autophagy. Autophagy is a highly conserved intracellular process used to degrade and recycle damaged organelles, misfolded proteins, and pathogens, and is crucial for maintaining cellular homeostasis. During aging, autophagy flux generally declines, leading to the accumulation of damaged organelles and proteins, triggering oxidative stress, mitochondrial dysfunction, and chronic inflammation, thereby accelerating cellular senescence and tissue functional decline. Therefore, targeting and activating the autophagy pathway is considered a highly promising strategy for delaying colonic aging. Recent studies have found that hexokinase domain-containing protein 1 (HKDC1), as a key protein, works synergistically with TFEB and plays an important role in mitochondrial quality control and autophagy regulation. However, how to safely and effectively target and activate the autophagy pathway locally in the intestine to combat colonic aging remains a challenge for current research.

[0004] In terms of intervention methods, existing technologies mainly include drug intervention (such as autophagy inducers like rapamycin) and lifestyle intervention. However, long-term use of chemical drugs may have side effects and safety issues, and since the drugs are mainly absorbed by the small intestine, they cannot target and regulate aging in the colon. Therefore, it is urgent to develop a natural, safe, and effective intervention method. Probiotics have become a research hotspot in the field of anti-colonic aging due to their recognized safety, broad health-promoting effects, and colonic colonization characteristics. Numerous studies have confirmed that specific probiotic strains can indirectly exert the benefits of delaying aging by regulating the balance of intestinal flora, enhancing intestinal barrier function, and inhibiting inflammatory responses. Among them, Bifidobacterium longum has received widespread attention due to its properties of regulating host immunity and anti-inflammation (Journal of immunology research, 2021, 2021(1):8030297). In existing technologies, patent CN113215035A discloses that *Bifidobacterium longum* and *Bifidobacterium animalis* can alleviate memory impairment in D-galactose-aged mice, reduce the concentration of malondialdehyde (MDA), a lipid peroxide, in serum, decrease the rate of cellular oxidation, and effectively improve the lifespan of individual cells. However, current research on the anti-aging effects of probiotics mainly focuses on macroscopic improvements. Whether they can directly target core intracellular aging mechanisms, particularly whether they can regulate autophagy pathways such as TFEB-HKDC1, remains unclear, and there is a lack of definitive evidence and highly effective strains.

[0005] In summary, there is an urgent need in this field to screen and obtain a new probiotic strain with anti-colonic aging properties. This strain can efficiently activate the autophagy pathway in colon cells, fundamentally improve cell aging caused by autophagy dysfunction, and thus provide a novel and effective microbial solution for the prevention and relief of colonic aging and related diseases. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a strain of Bifidobacterium longum CCFM1508 that activates the TFEB-HKDC1 autophagy pathway and its application in anti-colonic aging. Currently, it is unknown whether Bifidobacterium longum can directly target the core intracellular aging mechanism, especially whether it can regulate autophagy pathways such as TFEB-HKDC1. A new probiotic agent for colonic aging needs to be developed.

[0007] This invention provides a strain of Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1508, the Bifidobacterium longum CCFM1508, was deposited on July 24, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66744, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The Bifidobacterium longum CCFM1508 has the following characteristics: (1) Bacterial characteristics: Gram-positive, pleomorphic, showing “V” or “Y” shaped branching.

[0009] (2) Colony characteristics: They appear as round bulges on MRS solid medium and are milky white or opaque white.

[0010] The present invention also provides a probiotic preparation containing the aforementioned Bifidobacterium longum CCFM1508.

[0011] In one embodiment, the microbial preparation is a liquid or solid microbial agent.

[0012] In one embodiment, the viable count of Bifidobacterium longum CCFM1508 in the microbial preparation is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0013] In one embodiment, the preparation method of the microbial preparation is as follows: the seed culture of Bifidobacterium longum CCFM1508 is inoculated into MRS medium supplemented with cysteine ​​at an inoculation rate of 2% to 4%, and anaerobically cultured at 37°C for 24 h. The culture medium is centrifuged to collect the bacterial cells, washed 2 to 4 times with physiological saline, and resuspended with a protective agent to achieve a concentration of 10. 10 The bacterial agent was obtained by freeze-drying at CFU / mL.

[0014] In one embodiment, the protective agent contains 100 g / L skim milk, 30 mL / L glycerol, 100 g / L maltodextrin, 150 g / L trehalose, and 10 g / L L-monosodium glutamate.

[0015] In one embodiment, the fermentation medium contains: 10 g / L peptone, 5 g / L yeast extract, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 2 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, 1 mL / L Tween 80, 1 g / L L-cysteine ​​hydrochloride, and a pH of 6.5-6.8.

[0016] The present invention also provides the application of the aforementioned Bifidobacterium longum CCFM1508 in the preparation of products for anti-colonic aging.

[0017] In one embodiment, the product includes, but is not limited to, food, health products, or medicines.

[0018] In one embodiment, the medicament further includes a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field.

[0019] In one embodiment, the viable count of Bifidobacterium longum CCFM1508 in the functional product is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0020] In one embodiment, the food includes, but is not limited to, fermented food or fermented beverage containing the aforementioned Bifidobacterium longum CCFM1508; or fermented food produced using a microbial preparation containing the aforementioned Bifidobacterium longum CCFM1508.

[0021] In one embodiment, the improvement of colonic aging symptoms includes, but is not limited to, the following aspects: (1) Reduce the risk of age-related damage to the colonic barrier function and increased permeability; (2) Alleviates the phenomenon of shortened colon length; (3) Reduces the activity of β-galactosidase in the colon due to aging; (4) Improves the level of cell apoptosis in the colon caused by aging; (5) Reduce the content of pro-inflammatory cytokines in colon tissue, including TNF-α, IL-1β, IL-6 and IL-8; (6) Reduces oxidative stress levels in colon tissue; (7) Reduces inflammatory cell infiltration in colon tissue; (8) To alleviate the decrease in the number of goblet cells caused by aging; (9) Reduce the transcriptional levels of aging markers in the colon, including p21 and p53; (10) Regulate the transcriptional levels of colonic tight junction proteins, including Claudin-1 and ZO-1; (11) Improve the changes in autophagy levels caused by aging.

[0022] This invention provides the use of the aforementioned Bifidobacterium longum CCFM1508 or the aforementioned microbial preparation in the preparation of a drug for anti-colonic aging.

[0023] This invention provides the application of the aforementioned Bifidobacterium longum CCFM1508 or the aforementioned microbial preparation in the preparation of anti-aging health products.

[0024] The present invention has the following beneficial effects: This invention provides a strain of *Bifidobacterium longum* CCFM1508 that can alleviate age-related colonic barrier damage, colonic inflammation, and colonic tissue aging, and improve age-related autophagy dysregulation. When *Bifidobacterium longum* CCFM1508 provided by this invention is applied to aging mice, it can significantly alleviate colonic barrier damage, colonic inflammation, and colonic tissue aging, and improve age-related autophagy dysregulation. Specifically, compared with the model group: (1) The impaired colonic barrier function and increased permeability in aging mice were improved; (2) The phenomenon of shortened colon length in aging mice was improved; (3) The activity of β-galactosidase in the colon of aging mice was reduced by 27.19% compared with the model group; (4) The level of apoptosis in the colon of aging mice was improved; (5) The concentrations of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 in the colon of aged mice were reduced by 36.13%, 29.04%, 22.12% and 18.64% respectively compared with the model group; (6) The activities of SOD and CAT in the colon of aging mice increased by 17.91% and 61.23% respectively compared with the model group; (7) The GSH content in the colon of aging mice was 45.95% higher than that in the model group; (8) The concentration of MDA in the colon of aged mice was reduced by 45.81% compared with that in the model group; (9) Decreased inflammatory infiltration in the colon tissue of aged mice; (10) The number of goblet cells in the colon tissue of aging mice increased; (11) The transcriptional levels of aging markers p21 and p53 in the colon of aging mice decreased by 43.57% and 39.80% respectively compared with the model group; (12) The transcriptional levels of tight junction proteins Claudin-1 and ZO-1 are increased in the colon of aging mice; (13) Regulates the level of autophagy in colonic tissue caused by aging and increases the number of autophagosomes; (14) The transcriptional levels of autophagy-related proteins Atg3 and Atg5 in the colon of aging mice were increased by 63.56% and 91.78% respectively compared with the model group; (15) The transcriptional levels of TFEB and HKDC1 in the colon of aged mice increased by 31.04% and 154.14% respectively compared with the model group.

[0025] Therefore, Bifidobacterium longum CCFM1508 has great application potential in the preparation of products that alleviate colonic aging.

[0026] Preservation of biological materials Bifidobacterium longum CCFM1508, taxonomically named Bifidobacterium longum ( Bifidobacterium longum subsp. longum The sample was deposited on July 24, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66744. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0027] Figure 1 The levels of FITC-glucan in the serum of mice in different groups of experiments; Figure 2 Endotoxin levels in the serum of mice in different groups; Figure 3 Colon length of mice in different experimental groups; Figure 4 The activity of β-galactosidase in the colon tissue of mice in different groups of experiments; Figure 5 Tunel staining of colon tissue from different groups of experimental mice; Figure 6 Figure showing the levels of pro-inflammatory cytokines in the colon tissue of mice from different experimental groups; Figure 7 A graph showing the level of oxidative stress in the colon tissue of mice from different groups. Figure 8 H&E staining of colon tissue from different groups of experimental mice; Figure 9 AB-PAS staining of colon tissue from different groups of experimental mice; Figure 10 Plots showing the transcriptional levels of p21 and p53, markers of colonic aging, in mice from different groups. Figure 11 A graph showing the transcriptional levels of tight junction proteins in the colon of experimental mice from different groups; Figure 12 Transmission electron micrographs of colon tissue from different groups of experimental mice; Figure 13 Transcriptional levels of autophagy-related proteins Atg3 and Atg5 in the colon tissue of mice from different groups; Figure 14 Transcriptional levels of TFEB and HKDC1 in colon tissues of mice from different groups; Detailed Implementation The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments should not be construed as limiting the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0028] The BALB / c male mice used in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. TNF-α, IL-1β, IL-6, and IL-8 ELISA assay kits were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. SOD, CAT, GSH, and MDA assay kits were purchased from Nanjing Jiancheng.

[0029] The strain information involved in the following examples is as follows: Bifidobacterium longum CCFM1508, taxonomically named Bifidobacterium longum ( Bifidobacterium longum subsp. longum The sample was deposited on July 24, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66744. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology.

[0030] The culture medium preparations involved in the following examples are as follows: MRS fermentation medium: peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.1 g / L, manganese sulfate monohydrate 0.05 g / L, Tween 80 1 mL / L, L-cysteine ​​hydrochloride 1 g / L, pH 6.5~6.8.

[0031] MRS solid medium: 15 g / L agar powder is added to the fermentation medium.

[0032] Example 1: Screening, identification and preservation of Bifidobacterium longum CCFM1508 1. Screening A certain amount of fecal samples from healthy human subjects in Bo'ai County, Henan Province were serially diluted with PBS, spread on MRS solid medium, and cultured at 37°C for 48 h in an anaerobic environment. The colony morphology was observed and recorded. Single colonies were picked and inoculated into MRS liquid medium. After culturing at 37°C for 24 h in an anaerobic environment, strain CCFM1508 was obtained.

[0033] 2. Identification The whole genome DNA of strain CCFM1508 was extracted for 16S rDNA amplification. The amplified DNA fragments were collected for sequencing (performed by Shanghai Sangon Biotech Co., Ltd.). The sequenced results were aligned using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the strain was *Bifidobacterium longum*, and it was named *Bifidobacterium longum*. Bifidobacterium longum subsp. longum (CCFM1508)

[0034] 3. Save Extraction of Bifidobacterium longum ( Bifidobacterium longum subsp. longum A single colony of CCFM1508 was inoculated into MRS liquid medium and cultured anaerobically at 37°C for 24 h to obtain a bacterial suspension. 1 mL of the bacterial suspension was placed in a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, and the upper culture medium was discarded. The resulting bacterial sludge was resuspended in 1 mL of 30% (v / v) glycerol and stored in a -80°C freezer.

[0035] Example 2: Preparation of Bifidobacterium longum CCFM1508 bacterial suspension Bifidobacterium longum CCFM1508 was streaked onto solid culture medium from cryopreservation tubes and activated by anaerobic incubation at 37°C for 48 h. Single colonies were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h. After at least three subcultures, the activated bacterial culture was obtained. The activated bacterial culture was inoculated into MRS liquid medium at a 2% (v / v) ratio and incubated at 37°C for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 g for 3 min at 4°C. The obtained wet bacterial sludge was washed three times with PBS buffer (pH 7.4) and resuspended in sterile physiological saline to obtain the bacterial suspension.

[0036] Example 3: Effects of Bifidobacterium longum CCFM1508 on colonic permeability in D-galactose-induced aging mice Twenty-four 8-week-old SPF-grade male BALB / c mice were randomly divided into four groups: a control group, a model group, a Bifidobacterium longum CCFM1508 intervention group, and a Bifidobacterium longum FJSWXJ14M1 (self-screened strain) intervention group, with six mice in each group. This animal model was constructed using D-galactose. Excessive D-galactose accumulates in the mice, and under the action of galactose oxidase, produces aldose and hydrogen peroxide, promoting the production of oxygen free radicals and superoxide anions, damaging macromolecules and cellular function, and inducing aging, thus creating a relatively stable aging mouse model. The grouping and treatment methods of the experimental animals are shown in Table 1.

[0037] Table 1 Grouping of experimental animals

[0038] (1) The amount of FITC-glucan entering the bloodstream from the intestinal lumen reflects colonic permeability. FITC-glucan at a concentration of 40 mg / mL was prepared using PBS (0.1 M, pH=6.8) in the dark. After the experiment, mice were fasted for 4 hours and then administered FITC-glucan by gavage at a dose of 0.4 mg / g. Food and water were withheld after gavage. After 90 minutes, 100 μL of blood was collected from the tip of the mouse tail vein. The blood sample was allowed to stand in the dark for 30 minutes, then centrifuged at 3000 g at 25°C for 10 minutes. The supernatant serum sample was carefully aspirated. 20 μL of serum was transferred to a 96-well plate and diluted to 200 μL with PBS (0.1 M, pH=6.8). Each sample was tested in triplicate. Fluorescence intensity was detected using a microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0039] (2) Serum endotoxin levels After the experiment, in accordance with ethical requirements, the mice were enucleated to collect blood and dissected. The mouse blood was allowed to stand for 2 hours and then centrifuged at 3000 r / min for 15 min. The mouse serum was collected and the level of endotoxin in the mouse serum was determined using a kit (product number C0276S, purchased from Shanghai Beyotime Biotechnology Co., Ltd.).

[0040] like Figure 1 and Figure 2 As shown, after modeling, compared with the control group, the serum FITC-glucan and endotoxin levels in the model group mice were significantly increased. However, intervention with *Bifidobacterium longum* CCFM1508 and FJSWXJ14M1 significantly reduced the serum FITC-glucan and endotoxin levels in mice, alleviating the increased colonic permeability. *Bifidobacterium longum* CCFM1508 showed a better alleviating effect than *Bifidobacterium longum* FJSWXJ14M1, reducing serum FITC-glucan and endotoxin levels in mice to 3.97 μg / mL and 0.94 EU / mL, respectively.

[0041] Example 4: Effect of Bifidobacterium longum CCFM1508 on colon length in D-galactose-induced aging mice The modeling method was the same as in Example 3. After the experiment, the mice were euthanized, and the entire colon, from the end of the cecum to the anus, was harvested to measure the colon length.

[0042] like Figure 3 As shown, the colon length in the model group was significantly shorter than that in the control group, and this phenomenon was significantly improved after intervention with Bifidobacterium longum CCFM1508. Therefore, CCFM1508 can effectively alleviate age-related changes in colon length.

[0043] Example 5: Effect of Bifidobacterium longum CCFM1508 on β-galactosidase activity in colon tissue of D-galactose-induced aging mice The modeling method was the same as in Example 3. After the experiment, the mice were euthanized, and their colonic tissue was weighed. PBS was added at a ratio of 1:9, and the colonic tissue was homogenized using a high-throughput tissue homogenizer. The homogenate was then centrifuged at 8000 g and 4℃ for 15 min, and the supernatant was collected. The β-galactosidase activity in the colonic tissue supernatant was determined using a kit (catalog number BC2580, purchased from Beijing Solarbio Science & Technology Co., Ltd.); the protein concentration in the supernatant was determined using a BCA protein concentration assay kit.

[0044] Age-related β-galactosidase is one of the most widely used biological markers of aging. For example... Figure 4 As shown, after D-galactose modeling, the activity of β-galactosidase in mouse colon tissue was significantly increased (538.10 U / mg protein). p The value <0.05 indicates the success of the D-galactose aging model. The figure shows that *Bifidobacterium longum* FJSWXJ14M1 can reduce β-galactosidase activity in mouse colon tissue, but the difference is not significant. However, after intervention with *Bifidobacterium longum* CCFM1508, β-galactosidase activity was significantly reduced to 391.80 U / mg protein, comparable to the control group (358.72 U / mg protein), indicating that CCFM1508 intervention can improve the increase in β-galactosidase activity caused by aging.

[0045] Example 6: Effect of Bifidobacterium longum CCFM1508 on apoptosis in D-galactose-induced aging mouse colon tissue The modeling method was the same as in Example 3. After the experiment, the mice were sacrificed, and the colon tissue was collected. Paraffin sections of the mouse colon were prepared, and the sections were dewaxed and hydrated, repaired with proteinase K, permeabilized, incubated with Tunel reaction solution, stained with DAPI, mounted with antifluorescein quencher, and examined under a microscope to observe the Tunel fluorescence of the colon of different groups of mice.

[0046] In late-stage apoptotic cells, double-strand or single-strand breaks in chromosomal DNA create sticky 3'-OH ends. Catalyzed by deoxyribonucleotide terminal transferase, dUTPs carrying luciferin molecules are labeled to the 3'-ends of the DNA. This labeling, observed and imaged using a fluorescence microscope, allows for the detection of apoptotic cells. Figure 5 As shown, the number of apoptotic cells increased after D-galactose modeling compared to the control group, demonstrating the success of the D-galactose aging model. The figure also shows that *Bifidobacterium longum* FJSWXJ14M1 did not have a positive regulatory effect on apoptosis in colon tissue. However, intervention with *Bifidobacterium longum* CCFM1508 significantly reduced the number of apoptotic cells, indicating that CCFM1508 intervention can improve aging-induced apoptosis.

[0047] Example 7: Effects of Bifidobacterium longum CCFM1508 on pro-inflammatory cytokines in D-galactose-induced aging mouse colon tissue The modeling method was the same as in Example 3. After the experiment, the mice were euthanized, and their colon tissue was weighed. PBS was added at a ratio of 1:9, and the colon tissue was homogenized using a high-throughput tissue homogenizer. The homogenate was then centrifuged at 8000 g and 4℃ for 15 min, and the supernatant was collected. The levels of pro-inflammatory cytokines TNF-α (catalog number YJ002095, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.), IL-1β (catalog number YJ301814, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.), IL-6 (catalog number YJ098430, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.), and IL-8 (catalog number YJ058632, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.) in the colon tissue supernatant were measured using a kit. The protein concentration in the supernatant was measured using a BCA protein concentration assay kit.

[0048] Depend on Figure 6 The results showed that D-galactose modeling significantly increased the levels of pro-inflammatory cytokines TNF-α (38.28 pg / mg protein), IL-1β (5.74 pg / mg protein), IL-6 (3.28 pg / mg protein), and IL-8 (6.44 pg / mg protein) in colonic tissue. In contrast, the levels of pro-inflammatory cytokines TNF-α in the colonic tissue of mice in the Bifidobacterium longum FJSWXJ14M1 and CCFM1508 intervention groups decreased to 28.40 pg / mg protein and 24.45 pg / mg protein, respectively; the levels of pro-inflammatory cytokines IL-1β decreased to 4.90 pg / mg protein and 4.08 pg / mg protein, respectively; the levels of pro-inflammatory cytokines IL-6 decreased to 2.96 pg / mg protein and 2.55 pg / mg protein, respectively; and the levels of pro-inflammatory cytokines IL-8 decreased to 5.44 pg / mg protein and 5.24 pg / mg protein, respectively. Bifidobacterium longum FJSWXJ14M1 did not significantly alter the levels of pro-inflammatory cytokines in the mouse colon, while CCFM1508 significantly reduced the levels of all four pro-inflammatory cytokines. p <0.05).

[0049] Example 8: Effects of Bifidobacterium longum CCFM1508 on D-galactose-induced oxidative stress in colonic tissue of aging mice The modeling method was the same as in Example 3. After the experiment, the mice were euthanized, and their colon tissue was weighed. PBS was added at a ratio of 1:9, and the colon tissue was homogenized using a high-throughput tissue homogenizer. The homogenate was then centrifuged at 8000 g and 4℃ for 15 min, and the supernatant was collected. The levels of GSH (catalog number A006-2-1, purchased from Nanjing Jiancheng), MDA (catalog number A003-1-2, purchased from Nanjing Jiancheng), SOD (catalog number A001-3-2, purchased from Nanjing Jiancheng), and CAT (catalog number A007-2-1, purchased from Nanjing Jiancheng) in the colon tissue supernatant were determined using a kit; the protein concentration in the supernatant was determined using a BCA protein concentration assay kit.

[0050] GSH is one of the most important antioxidants in cells. When tissues face oxidative stress, GSH levels decrease to neutralize excess reactive oxygen species, thereby protecting cells from oxidative damage. Therefore, a decrease in GSH levels is often a marker of increased oxidative damage. Figure 7 The results showed that the GSH content in the model group was significantly reduced in the colon tissue. p The concentration of GSH in the *Bifidobacterium longum* FJSWXJ14M1 and CCFM1508 intervention groups was <0.05%, indicating the success of the D-galactose aging model. The GSH content in these groups increased by 96.72% and 45.95% respectively compared to the model group. The MDA content in colonic tissue can indirectly reflect the degree of oxidative damage in colonic tissue. Figure 7 The results showed that the MDA content in the colon tissue of the model group was significantly increased. p <0.05), while the GSH content in the Bifidobacterium longum FJSWXJ14M1 and CCFM1508 intervention groups decreased by 26.84% and 35.74% respectively compared with the model group, thus mitigating the degree of oxidative stress damage to cells and delaying aging. In addition, the activity levels of SOD and CAT were significantly reduced in the model group ( p The levels of SOD and CAT in the CCFM1508 intervention group were <0.05, showing no significant difference compared to FJSWXJ14M1. However, the activities of SOD and CAT in the CCFM1508 intervention group reached 36.94 U / mg protein and 93.00 U / g protein, respectively. This demonstrates that CCFM1508 in this invention can reduce oxidative damage in colonic tissue and improve the tissue's antioxidant capacity.

[0051] Example 9: Effects of Bifidobacterium longum CCFM1508 on colonic histopathology in D-galactose-induced aging mice The modeling method was the same as in Example 3. After the experiment, the mice were sacrificed, and 1 cm of distal colon tissue was taken and fixed in 4% paraformaldehyde solution for 48 h. After dehydration, embedding, sectioning, and H&E staining, the pathological condition of the colon tissue of mice in different groups was observed.

[0052] Depend on Figure 8 It can be seen that the colon tissue of the model group mice showed obvious damage, including severe inflammatory cell infiltration, destruction of crypt and gland structures, etc. The damage to the colon tissue of the FJSWXJ14M1 intervention group was milder than that of the model group, but there was still a lot of inflammatory cell infiltration. The colon tissue structure of the CCFM1508 intervention group was similar to that of the blank group, with relatively intact glands and crypts and only a small amount of inflammatory cell infiltration.

[0053] Example 10: Effects of Bifidobacterium longum CCFM1508 on goblet cells in colon tissue of D-galactose-induced aging mice The modeling method was the same as in Example 3. After the experiment, the mice were euthanized, and 1 cm of distal colon tissue was taken. After fixation, dewaxing, embedding, sectioning, Alcian blue staining, GDS staining, Chevron staining, dehydration and mounting, the goblet cell status of the colon tissue of mice in different groups was observed.

[0054] Depend on Figure 9 It can be seen that the colonic crypt morphology of mice in the control group was normal, with goblet cells evenly distributed and abundant. However, in the model group, this normal structure was disrupted, manifested as a decrease in the number of goblet cells and accompanying mucin depletion. CCFM1508 intervention significantly improved this pathological change and effectively increased the number of colonic goblet cells, while the FJSWXJ14M1 intervention group showed no significant improvement. In conclusion, CCFM1508 has a clear effect on alleviating age-induced goblet cell loss in mice.

[0055] Example 11: Effects of Bifidobacterium longum CCFM1508 on the transcriptional levels of p21 and p53, aging markers, in the colon tissue of D-galactose-induced aging mice. The modeling method was the same as in Example 3. After the experiment, the mice were sacrificed, and their colon tissue was collected to determine the transcriptional levels of aging markers p21 and p53 in the colon tissue.

[0056] The assay method is as follows: Primer sequences for p21, p53, and β-actin were synthesized, and primer information is shown in Table 2. Total RNA was extracted from mouse colon tissue using the Trizol method. The concentration of extracted RNA and OD were determined using a micro-spectrophotometer. 260 / OD 280The quality was acceptable between 1.9 and 2.0. Using the extracted, high-quality total RNA as a template, cDNA was synthesized according to the reverse transcription kit instructions. The cDNA obtained from reverse transcription was then subjected to qRT PCR. The PCR system consisted of: 5 μL LSYBR Green Supermix, 3 μL deionized water, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), and 1 μL cDNA template (100 ng / μL). The qRT PCR program was set as follows: 94℃, 2 min, (94℃, 30 s; 61℃, 30 s; 72℃, 20 s) for 39 cycles; β-actin was used as an internal control gene, and 2... -△△Ct The method was used to perform relative gene expression analysis.

[0057] Table 2 Mouse primer sequences

[0058] p21 and p53 are core regulators in the aging process. They upregulate their transcription or activity in response to various stress signals (especially DNA damage), a key characteristic of aging. Figure 10 The results showed that, with the expression levels of p21 and p53 mRNA in the control group as 1, the expression levels in the model group increased by 2.18-fold and 2.55-fold, respectively, significantly higher than the control group. After intervention with *Bifidobacterium longum* FJSWXJ14M1, the expression levels of p21 and p53 mRNA did not change significantly compared to the model group. After intervention with *Bifidobacterium longum* CCFM1508, the expression levels of p21 and p53 mRNA were 1.23 and 1.53, respectively. Therefore, compared with *Bifidobacterium longum* FJSWXJ14M1, CCFM1508 significantly reduced the transcriptional levels of the aging markers p21 and p53 in colonic tissue.

[0059] Example 12: Effects of Bifidobacterium longum CCFM1508 on the transcriptional level of tight junction protein in the colon tissue of D-galactose-induced aging mice The modeling method was the same as in Example 3. After the experiment, the mice were sacrificed, and their colon tissue was collected to determine the transcriptional levels of the tight junction proteins Claudin-1 and ZO-1 in the colon tissue.

[0060] The determination method is the same as in Example 11, and the primer information is shown in Table 3.

[0061] Table 3 Mouse primer sequences

[0062] Transcriptional levels of tight junction proteins Claudin-1 and ZO-1. Figure 11The results showed that, with the expression levels of Claudin-1 and ZO-1 in the control group as 1, the expression levels in the model group decreased by 52.50% and 50.72%, respectively, significantly lower than the control group. After intervention with *Bifidobacterium longum* FJSWXJ14M1, the expression levels of Claudin-1 and ZO-1 mRNA did not change significantly compared to the model group. After intervention with *Bifidobacterium longum* CCFM1508, the expression levels of Claudin-1 and ZO-1 mRNA were 1.00 and 0.64, respectively. Therefore, compared with *Bifidobacterium longum* FJSWXJ14M1, CCFM1508 significantly increased the transcriptional levels of the tight junction proteins Claudin-1 and ZO-1 in colonic tissue, improving the colonic mechanical barrier in aging mice.

[0063] Example 13: Effects of Bifidobacterium longum CCFM1508 on D-galactose-induced autophagy in the colon of aging mice The modeling method was the same as in Example 3. After the experiment, the mice were euthanized, and their colon tissue was collected for observation of autophagosomes using a transmission electron microscope.

[0064] The method for determining the transcriptional levels of autophagy-related proteins Atg3 and Atg5 is the same as in Example 11, and the primer information is shown in Table 4.

[0065] Table 4 Mouse primer sequences

[0066] from Figure 12 It can be seen that the number of autophagosomes in the colon of the model group mice was significantly less than that in the control group. Intervention with *Bifidobacterium longum* FJSWXJ14M1 had no effect on improving the autophagy level in the colon of aging mice, while CCFM1508 significantly increased the number of autophagosomes in the colonic tissue and improved the autophagy level in the colonic tissue. Furthermore, from... Figure 13 It can be seen that after CCFM1508 intervention, the expression levels of autophagy-related proteins Atg3 and Atg5 mRNA returned to normal levels, proving that CCFM1508 intervention can significantly adjust the autophagy level in the colon of aging mice, thereby exerting an anti-colon aging effect.

[0067] Example 14: Effects of Bifidobacterium longum CCFM1508 on TFEB and HKDC1 transcription levels in colon tissue of D-galactose-induced aging mice The modeling method was the same as in Example 3. After the experiment, the mice were sacrificed, and their colon tissue was collected to determine the transcriptional levels of tight junction proteins TFEB and HKDC1 in the colon tissue.

[0068] The determination method is the same as in Example 11, and the primer information is shown in Table 5.

[0069] Table 5 Mouse primer sequences

[0070] Autophagic degradation depends on lysosomes, which are the terminal components of autophagy and contain more than 50 acidic hydrolases. Increased lysosomal biogenesis is necessary during autophagy induction to meet the requirements for fusion with autophagosomes and subsequent autophagic degradation. Transcriptional regulation of lysosomal biogenesis genes is mediated by TFEB, and HKDC1 is a target of TFEB, which can maintain mitochondrial and lysosomal homeostasis and prevent cellular senescence. Figure 14 As can be seen, with the expression levels of TFEB and HKDC1 mRNA in the control group mice as 1, the expression levels in the model group decreased by 24.19% and 65.29%, respectively, which were significantly lower than those in the control group. After intervention with *Bifidobacterium longum* FJSWXJ14M1, the expression levels of TFEB and HKDC1 mRNA did not change significantly compared to the model group. After intervention with *Bifidobacterium longum* CCFM1508, the expression levels of TFEB and HKDC1 mRNA were 1.00 and 0.91, respectively. Therefore, compared with *Bifidobacterium longum* FJSWXJ14M1, CCFM1508 can significantly increase the transcriptional levels of TFEB and HKDC1 in colonic tissue, activate the TFEB-HKDC1 autophagy pathway, and thus maintain the autophagy level in colonic tissue, exerting an anti-colonial aging effect.

[0071] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1508 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66744.

2. A microbial preparation containing the Bifidobacterium longum CCFM1508 as described in claim 1.

3. The microbial preparation as described in claim 2, characterized in that, In the microbial preparation, the number of Bifidobacterium longum CCFM1508 cells is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

4. The microbial preparation as described in claim 3, characterized in that, The microbial preparation also contains a protectant; the protectant includes one or more of skim milk, glycerin, maltodextrin, trehalose, and monosodium glutamate (MSG).

5. The microbial preparation as described in claim 4, characterized in that, The microbial preparation is a liquid preparation, a powder preparation, or a granular preparation.

6. A product containing the Bifidobacterium longum CCFM1508 of claim 1 or any of the microbial preparations of claims 2 to 5.

7. The product as described in claim 6, characterized in that, The products include food, medicine, or health products.

8. The product as described in claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients; the excipients include diluents, adjuvants and / or carriers.

9. The use of the Bifidobacterium longum CCFM1508 of claim 1 or the microbial preparation of any one of claims 2 to 5 in the preparation of a medicament for anti-colonic aging.

10. The use of the Bifidobacterium longum CCFM1508 of claim 1 or the microbial preparation of any one of claims 2 to 5 in the preparation of anti-aging health products.

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