Bifidobacterium longum CCFM1509 with autophagy promoting and colon aging resisting functions and application of bifidobacterium longum CCFM1509

Abstract

The invention discloses bifidobacterium longum CCFM1509 with autophagy promoting and colon aging resisting functions and application of the bifidobacterium longum CCFM1509, and belongs to the field of microorganisms. The bifidobacterium longum CCFM1509 provided by the invention can relieve colon tissue pathological damage caused by senescence, reduce the level of proinflammatory cytokines in colon tissues, reduce the oxidative stress level in the colon tissues, reduce the level of senescence-related senescence markers in the colon tissues and adjust the autophagy level in the colon tissues. Therefore, the bifidobacterium longum CCFM1509 disclosed by the invention can be widely applied to the field of medicines and is used for preparing pharmaceutical preparations for preventing or treating colon aging and related diseases; meanwhile, the bacillus subtilis can also be used for developing various functional fermented foods, health foods and formula foods for special medical purposes, provides a new microbial solution for promoting intestinal health and delaying colon senescence, and has important development value and wide market prospects.

Description

Technical Field

[0001] This invention relates to a strain of Bifidobacterium longum CCFM1509 with autophagy-promoting and anti-colonic aging functions and its applications, 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 challenge in the field of public health. The colon, as a vital digestive and immune organ, undergoes significant changes with age. For example, intestinal barrier function declines, oxidative stress increases, and levels of pro-inflammatory cytokines rise, leading to increased ROS production. This vicious cycle results in gut microbiota dysbiosis and increased intestinal permeability. Conversely, disruption of the intestinal barrier promotes endotoxin translocation, and endotoxins are closely related to the initiation of low-grade inflammation. Its aging process is closely associated with gut microbiota imbalance, decreased intestinal barrier function, and chronic inflammatory responses. Therefore, finding safe and effective intervention strategies to actively regulate the colonic health of the elderly is of great significance.

[0003] A core mechanism of colonic aging is known to be closely related to the dysfunction of autophagy. Autophagy, an evolutionarily conserved intracellular degradation pathway, is responsible for clearing damaged organelles, misfolded proteins, and exogenous pathogens, playing a crucial role in maintaining cellular homeostasis. With age, autophagy activity generally declines, leading to the accumulation of abnormal proteins and damaged organelles. This, in turn, induces oxidative stress, mitochondrial dysfunction, and persistent inflammatory responses, ultimately driving cellular aging and tissue degeneration. Therefore, targeted regulation of the autophagy pathway has become an important research direction for delaying colonic aging.

[0004] Currently, various intervention methods have been used to combat aging and related diseases, including pharmacological methods (such as autophagy inducers like rapamycin), daily behavioral regulation, and the application of probiotics and prebiotics. Among them, probiotics have become an important direction in colonic anti-aging research due to their good safety record, diverse health benefits, and colonization ability in the colon. Existing research has confirmed that certain probiotics can indirectly produce anti-aging effects through multiple pathways such as regulating the balance of gut microbiota, strengthening the intestinal barrier, and inhibiting inflammatory responses. For example, Bifidobacterium has received attention from the academic community due to its functions in immune regulation and anti-inflammation (Gut Microbes, 2023, 15(2)). Chinese patent CN120290356A points out that Bifidobacterium bifidum can effectively reduce cognitive deficits in aging mice, improve the antioxidant capacity of the brain and liver, and reduce colonic inflammation; another study reported that Bifidobacterium longum can promote bone healing in aging mice with fractures, and this effect is related to its role in maintaining the integrity of the intestinal barrier, inhibiting systemic inflammation, and regulating intestinal homeostasis (Aging Cell, 2023, 22: e13786). Nevertheless, current research on probiotic anti-aging primarily focuses on overall phenotypic improvement or metabolic regulation. However, there is a lack of strains and intervention programs specifically targeting the local aging process in the colon, particularly those based on autophagy gene regulation mechanisms. Evidence of strains directly acting on core aging pathways is also lacking. Therefore, this patent aims to fill this technological gap by providing a novel *Bifidobacterium longum* strain capable of activating autophagy genes, enhancing intestinal barrier function, and inhibiting colonic inflammation, as well as its application in anti-colon aging products. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a strain of Bifidobacterium longum CCFM1509 with autophagy-promoting and anti-colonic aging functions and its applications.

[0006] This invention provides a strain of Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1509 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66745.

[0007] In one embodiment of the present invention, the Bifidobacterium longum CCFM1509 was obtained from the feces of a 63-year-old woman in Nantong City, Jiangsu Province. The strain was sequenced and the sequenced sequence was compared with the nucleic acid sequence in NCBI Standard Nucleotide BLAST. The results showed that the nucleic acid sequence similarity with the genus Bifidobacterium longum was 100%. The results showed that the strain was Bifidobacterium longum and it was named Bifidobacterium longum CCFM1509.

[0008] In one embodiment of the present invention, the Bifidobacterium longum CCFM1509 has the following biological characteristics: (1) Bacterial characteristics: Gram-positive, does not form spores, and is a non-motile bacterium.

[0009] (2) Colony characteristics: They are round, raised, smooth, with neat edges, and are milky white or opaque white.

[0010] (3) Growth characteristics: Under constant temperature of 37°C in MRS medium, it reaches the logarithmic phase in about 18 hours.

[0011] The present invention also provides a microbial preparation containing the aforementioned Bifidobacterium longum CCFM1509.

[0012] In one embodiment of the present invention, the microbial preparation is a liquid preparation, a powder preparation, or a granular preparation.

[0013] In one embodiment of the present invention, the microbial preparation is lyophilized Bifidobacterium longum CCFM1509 powder.

[0014] In one embodiment of the present invention, the number of *Bifidobacterium longum* CCFM1509 cells is not less than 1 × 10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0015] The present invention provides a product containing the above-mentioned Bifidobacterium longum CCFM1509 or containing the above-mentioned microbial preparation.

[0016] In one embodiment of the present invention, the number of Bifidobacterium longum CCFM1509 cells in the above-mentioned product is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

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

[0018] In one embodiment of the present invention, the food is a fermented food, which is produced by fermentation using Bifidobacterium longum CCFM1509 and then pasteurized. The pasteurized food includes solid food, liquid food, and semi-solid food.

[0019] In one embodiment of the present invention, the food includes dairy products, soy products, or fruit and vegetable products, etc.

[0020] In one embodiment of the present invention, 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.

[0021] The present invention also provides the application of the aforementioned Bifidobacterium longum CCFM1509 in the preparation of a drug for anti-colonic aging.

[0022] In one embodiment of the present invention, the viable count of Bifidobacterium longum CCFM1509 in the functional product is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0023] In one embodiment of the present invention, the improvement of colonic aging symptoms includes, but is not limited to, the following aspects: (a) Improves pathological damage to colon tissue; (b) Reduce the level of pro-inflammatory cytokines in colon tissue; (c) Reduce oxidative stress levels in colonic tissue; (d) Reduces the activity of aging-related β-galactosidase in colon tissue; (e) Reduce the transcriptional levels of aging markers in colon tissue; (f) Adjust the level of autophagy in colon tissue.

[0024] The present invention also provides the use of the Bifidobacterium longum CCFM1509 or the microbial preparation in the preparation of anti-aging health products.

[0025] The present invention has the following beneficial effects: (1) The Bifidobacterium longum CCFM1509 of the present invention has good activity, which can alleviate the pathological damage of colon tissue caused by aging, reduce the level of pro-inflammatory cytokines in colon tissue, reduce the level of oxidative stress in colon tissue, reduce the activity of aging-related β-galactosidase in colon tissue, reduce the transcription level of aging markers in colon tissue, and adjust the level of autophagy in colon tissue.

[0026] (2) This invention can be regarded as an anti-colonic aging drug, and can also be applied to pharmaceuticals or some fermented foods and functional foods to exert its effect, and has great application prospects.

[0027] Preservation of biological materials A strain of Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1509, its taxonomic name is: Bifidobacterium longum subsp. longum It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66745. Attached Figure Description

[0028] Figure 1 The effect of Bifidobacterium longum on the level of FITC-glucan in mouse serum; Figure 2 The effect of Bifidobacterium longum on endotoxin levels in mouse serum; Figure 3 The effect of Bifidobacterium longum on colon length in mice; Figure 4 The effect of Bifidobacterium longum on H&E staining of mouse colon tissue; Figure 5 The effect of Bifidobacterium longum on AB-PAS staining of mouse colon tissue; Figure 6 The effect of Bifidobacterium longum on the content of pro-inflammatory cytokines in mouse colon tissue; Figure 7 The effect of Bifidobacterium longum on oxidative stress levels in mouse colon tissue; Figure 8 The effect of Bifidobacterium longum on the activity of β-galactosidase in mouse colon tissue; Figure 9 The effect of Bifidobacterium longum on Tunel staining of mouse colon tissue; Figure 10 The effect of Bifidobacterium longum on the transcriptional levels of p21 and p53, markers of colonic aging in mice; Figure 11 Transmission electron micrographs of colon tissue from different groups of mice; Figure 12 The effect of Bifidobacterium longum on the transcriptional levels of autophagy-related proteins Atg3 and Atg5 in mouse colon tissue; Figure 13 The effect of Bifidobacterium longum on the transcriptional levels of TFEB and HKDC1 in mouse colon tissue; Detailed Implementation To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0029] The culture medium preparations involved in the following examples are as follows: MRS fermentation medium ( / L): peptone 10 g, yeast extract 5 g, beef extract 10 g, glucose 20 g, sodium acetate 2 g, diammonium hydrogen citrate 2 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate heptahydrate 0.1 g, manganese sulfate monohydrate 0.05 g, Tween 80 1 mL, L-cysteine ​​hydrochloride 1 g, pH 6.5~6.8.

[0030] MRS solid medium ( / L): 15 g of agar powder was added to the MRS fermentation medium.

[0031] The strains and animals involved in the following examples are as follows: Bifidobacterium longum ( Bifidobacterium longum subsp. longum CCFM1509, its taxonomic name is: Bifidobacterium longum subsp. longum It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66745.

[0032] Male BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0033] The bacterial culture preparation methods involved in the following examples are as follows: Bifidobacterium longum CCFM1509 was streaked onto MRS solid medium and cultured in an anaerobic incubator at 37°C for 48 h. Single colonies were picked and cultured in 5 mL of MRS liquid medium at 37°C for 24 h. After at least three subcultures, activated bacterial culture was obtained. The activated bacterial culture was inoculated into MRS liquid medium at a 2% (v / v) fraction and cultured in an anaerobic incubator at 37°C for 24 h to obtain fermentation broth. After centrifugation, bacterial sludge was collected. The obtained wet bacterial sludge was washed three times with PBS buffer (pH 7.4) and resuspended in sterile physiological saline.

[0034] Example 1: Screening, identification and preservation of Bifidobacterium longum CCFM1509 A certain amount of fecal samples from healthy human subjects in Nantong City, Jiangsu Province, were serially diluted with PBS and spread onto MRS solid medium. The samples were then incubated in an anaerobic incubator at 37℃ for 48 h. Purified single colonies were picked and inoculated into MRS liquid medium, and incubated in an anaerobic incubator at 37℃ for 24 h to obtain strain CCFM1509. 1 mL of the bacterial suspension was centrifuged at 6000 r / min for 3 min, the supernatant was discarded, and the sample was washed three times with 1 mL of sterile water. The suspension was then resuspended in 1 mL of sterile water for 16S rDNA amplification. The obtained PCR product was sequenced (by Shanghai Sangon Biotech Co., Ltd.). The sequencing results were searched and compared using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and identified as *Bifidobacterium longum* (…). Bifidobacterium longum subsp. longum ).

[0035] For correctly identified bacterial strains, transfer 1 mL of bacterial solution to a 2 mL bacterial culture tube, centrifuge at 8000 r / min for 3 min, discard the supernatant, add 1 mL of 30% (v / v) sterile glycerol, vortex until homogeneous, and store at -80℃.

[0036] Example 2: Effects of Bifidobacterium longum CCFM1509 on the histopathological characteristics of colon tissue in aging mice Twenty-four healthy male BALB / c mice aged 8 weeks were randomly divided into four groups of six each: model group, blank group, Bifidobacterium longum CCFM1509 intervention group, and Bifidobacterium longum FBJ19M2 (self-screened strain) intervention group. The animal model was constructed using D-galactose. The experimental protocol and treatment methods for each group are shown in Table 1.

[0037] Table 1 Grouping of experimental animals

[0038] After the experiment ended on day 56, mice were fasted for 4 hours and then administered FITC-glucan (0.4 mg / g BW) by gavage. After 1.5 hours, 100 μL of blood was collected from the tail vein of the mice. The serum sample was collected after centrifugation. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm to obtain the FITC-glucan level in the serum. Results are shown below. Figure 1 In the control group, the serum FITC-glucan level of mice was 1.82 μg / mL, while the model group showed a significant increase in FITC-glucan level (> 6.00 μg / mL). Intervention with Bifidobacterium longum CCFM1509 and FBJ19M2 could reduce FITC-glucan level, and CCFM1509 could reduce it to 3.78 μg / mL, proving that CCFM1509 can alleviate the increase in colonic permeability.

[0039] After the experiment, mouse serum was collected, and the level of endotoxin in the mouse serum was determined using a kit (catalog number C0276S, purchased from Shanghai Beyotime Biotechnology Co., Ltd.). Results are shown below. Figure 2 The endotoxin level in the serum of mice in the blank control group was 0.79 EU / mL, while the endotoxin level in the model group was significantly increased (> 1.20 EU / mL). After intervention with FBJ19M2, the endotoxin level in the serum of mice was not significantly different from that in the model group. However, after intervention with Bifidobacterium longum CCFM1509, the endotoxin level was significantly reduced to 0.91 μg / mL, which further proves that CCFM1509 can alleviate the increase in colonic permeability.

[0040] After the mice were sacrificed, the entire colon was harvested, and its length was measured. Figure 3It can be seen that after modeling, the colon length of the model group was shortened to 8.55 cm, which was significantly lower than that of the blank group. The colon length of the FBJ19M2 group (9.25 cm) was slightly higher than that of the model group, but not significantly. CCFM1509 intervention significantly increased the colon length to 10.65 cm, indicating that CCFM1509 can effectively alleviate the phenomenon of colon shortening in aging mice.

[0041] After mice were sacrificed, 1 cm of distal colon tissue was collected, fixed, dehydrated, embedded, and stained with H&E. The H&E staining of colon tissue from different groups of mice was observed. Figure 4 It can be seen that the colon tissue of the model group mice showed a large number of inflammatory cell infiltrations, crypt disappearances and tissue edema; the intervention of CCFM1509 significantly improved the damage to the colon tissue. Although there were still a small number of inflammatory cell infiltrations, the overall tissue morphology was similar to that of the blank group colon, indicating that CCFM1509 can significantly improve the colonic mucosal damage in mice and the effect is better than that of FBJ19M2.

[0042] After sacrifice, 1 cm of distal colon tissue was taken for fixation, dewaxing, embedding, sectioning, AB-PAS staining (Alcian blue, GDS, Chevron staining), and mounting. The staining was then observed to determine the AB-PAS staining of colon tissue from different groups of mice. Figure 5 It can be seen that the number of goblet cells in the model group mice was significantly reduced and their distribution was uneven. FBJ19M2 intervention failed to improve this pathological phenomenon, while CCFM1509 intervention significantly increased the number of goblet cells in the colon tissue.

[0043] The above results indicate that the Bifidobacterium longum CCFM1509 of the present invention has a good alleviating effect on the histopathological symptoms of the colon in D-galactose-induced aging mice.

[0044] Example 3: Effects of Bifidobacterium longum CCFM1509 on pro-inflammatory cytokines in colon tissue of aging mice The modeling method was the same as in Example 2. After the mice were sacrificed, colon tissue was taken and PBS was added at a ratio of 1:9. The colon tissue was homogenized using a high-throughput homogenizer, and then centrifuged at 8000 g and 4℃ for 15 min. The supernatant was collected to obtain colon tissue supernatant. The levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 were measured using an ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.). The concentration of total protein in the colon tissue supernatant was determined using a BCA kit.

[0045] Depend on Figure 6It was found that the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 in the colon of mice in the model group were significantly increased. Bifidobacterium longum FBJ19M2 and CCFM1509 reduced IL-1β from 5.74 pg / mg in the model group to 4.83 pg / mg and 4.48 pg / mg, respectively; reduced IL-6 from 3.28 pg / mg in the model group to 2.77 pg / mg and 2.17 pg / mg, respectively; reduced IL-8 from 6.44 pg / mg in the model group to 5.16 pg / mg and 5.03 pg / mg, respectively; and reduced TNF-α from 38.28 pg / mg in the model group to 29.18 pg / mg and 27.32 pg / mg, respectively. Except for FBJ19M2, which significantly reduced the levels of these pro-inflammatory cytokines for IL-1β, the other groups showed the best effect, with the most significant reduction in inflammatory cytokine levels.

[0046] Example 4: Effects of Bifidobacterium longum CCFM1509 on oxidative stress levels in colonic tissue of aging mice The modeling method was the same as in Example 2. After the mice were sacrificed, colon tissue was taken and PBS was added at a ratio of 1:9. The colon tissue was homogenized using a high-throughput homogenizer to obtain a homogenate, and then centrifuged at 8000 g and 4℃ for 15 min. The supernatant was collected to obtain colon tissue supernatant. The levels of GSH, MDA, SOD, and CAT in the colon tissue supernatant were determined using a kit (Nanjing Jiancheng); the concentration of total protein in the colon tissue supernatant was determined using a BCA kit.

[0047] Experimental results are as follows Figure 7As shown, the MDA content (5.85 nmol / mg) in the colon of the model group mice was significantly increased compared with that in the control group (2.17 nmol / mg), the GSH (11.60 μmol / mg) was significantly decreased compared with that in the control group (20.82 μmol / mg), the SOD enzyme activity (31.33 U / mg) was significantly decreased compared with that in the control group (41.46 U / mg), and the CAT enzyme activity (57.68 U / g) was significantly decreased compared with that in the control group (97.36 U / g). This indicates that D-galactose aging modeling induced an increase in the level of oxidative stress in the colon of mice. In the intervention groups of *Bifidobacterium longum* FBJ19M2 and CCFM1509, the colonic MDA content (2.78 nmol / mg and 3.20 nmol / mg) was significantly decreased, while the GSH content (26.39 μmol / mg and 19.50 μmol / mg) was increased, SOD activity (34.71 U / mg and 38.28 U / mg) was increased, and CAT activity (92.36 U / mg and 86.94 U / mg) was significantly increased. The intervention with *Bifidobacterium longum* CCFM1509 showed the best effect. This indicates that CCFM1509 can significantly reduce oxidative stress levels in the mouse colon, exhibiting good antioxidant activity, and its effect is superior to that of *Bifidobacterium longum* FBJ19M2.

[0048] Example 5: Effects of Bifidobacterium longum CCFM1509 on aging-related β-galactosidase activity in colon tissue of aging mice The modeling method was the same as in Example 2. After the mice were sacrificed, colon tissue was taken and PBS was added at a ratio of 1:9. The colon tissue was homogenized using a high-throughput homogenizer to obtain a homogenate, which was then centrifuged at 8000 g and 4℃ for 15 min. The supernatant was collected to obtain the colon tissue supernatant. The β-galactosidase activity in the colon tissue supernatant was determined using a β-galactosidase activity kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.); the concentration of total protein in the colon tissue supernatant was determined using a BCA kit.

[0049] Experimental results are as follows Figure 8 As shown, the β-galactosidase activity in the colonic tissue of the model group mice (538.1 U / mg protein) was significantly higher than that in the control group (358.7 U / mg protein). Intervention with *Bifidobacterium longum* FBJ19M2 reduced β-galactosidase activity in the colonic tissue of aging mice (500.5 U / mg protein), but there was no significant difference compared to the model group. However, gavage administration of *Bifidobacterium longum* CCFM1509 significantly reduced β-galactosidase activity (398.2 U / mg protein). This indicates that CCFM1509 intervention can effectively reduce age-related β-galactosidase activity in colonic tissue, exerting an anti-colonial aging effect.

[0050] Example 6: Effects of Bifidobacterium longum CCFM1509 on apoptosis in colon tissue of aging mice The modeling method was the same as in Example 2. After the mice were sacrificed, 1 cm of distal colon tissue was taken for dewaxing and hydration, proteinase K repair, membrane permeation, incubation with Tunel reaction solution, nuclear staining with DAPI, and mounting with antifluorescein quencher. Tunel fluorescence of colon tissue from different groups of mice was observed.

[0051] Experimental results are as follows Figure 9 As shown, the aging model constructed using D-galactose revealed a significant increase in the number of apoptotic cells in the colon tissue of the model group compared to the control group, confirming the effectiveness of the model construction. The results further indicate that intervention with *Bifidobacterium longum* FBJ19M2 did not exhibit a significant positive regulation of colon cell apoptosis. Conversely, intervention with *Bifidobacterium longum* CCFM1509 significantly reduced the number of apoptotic cells, suggesting that this strain may exert its anti-aging effect by inhibiting aging-related apoptosis pathways.

[0052] Example 7: Effects of Bifidobacterium longum CCFM1509 on the transcriptional levels of p21 and p53, aging markers, in colonic tissue of aged mice. The modeling method was the same as in Example 2. After the mice were sacrificed, colon tissue was taken, and the transcriptional levels of aging markers p21 and p53 in the colon tissue were measured.

[0053] Total RNA was extracted from mouse colon tissue using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were calculated, and 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit. The reaction system was prepared using the ChamQ Universal SYBR qPCR MasterMix. β-actin was used as an internal control gene, based on 2... -△△Ct The expression level of the target gene was calculated using the method described in Table 2.

[0054] Table 2 Mouse primer sequences

[0055] p21 and p53 are classic biomarkers of aging. According to... Figure 10The results show that, using the p21 and p53 mRNA expression levels in the control group as a baseline (set as 1), the relative expression levels of these two markers in the model group were upregulated to 2.18 and 2.55, respectively, indicating significant aging-related upregulation. Under the treatment with *Bifidobacterium longum* FBJ19M2, the relative expression levels of p21 and p53 mRNA were 1.75 and 2.38, respectively, with no statistically significant difference compared to the model group. However, after intervention with *Bifidobacterium longum* CCFM1509, these indicators decreased to 0.94 and 1.82, respectively, showing significant differences. Therefore, CCFM1509 is significantly more effective than strain FBJ19M2 in reducing the transcriptional levels of the aging-related markers p21 and p53 in colonic tissue.

[0056] Example 8: Effects of Bifidobacterium longum CCFM1509 on autophagy levels in colonic tissue of aging mice The modeling method was the same as in Example 2. After the mice were sacrificed, colon tissue was collected, and autophagosomes were observed using a transmission electron microscope. Based on... Figure 11 The results show that, compared with the blank control group, the number of autophagosomes in the colonic tissue of the model group mice was significantly reduced. After intervention with *Bifidobacterium longum* FBJ19M2, the number of autophagosomes in the colon of aging mice did not recover significantly, indicating that this strain has limited regulatory effect on autophagy levels. In contrast, treatment with CCFM1509 significantly increased the number of autophagosomes in the colonic tissue, and effectively enhanced autophagy activity, indicating that this strain has a role in improving autophagy function under aging conditions.

[0057] The modeling method was the same as in Example 2. After sacrifice, mouse colon tissue was collected, and the transcriptional levels of autophagy-related proteins Atg3 and Atg5 in the colon tissue were measured. Total RNA was extracted from mouse colon tissue using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were calculated, and 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit. The reaction mixture was prepared using ChamQ UniversalSYBR qPCR Master Mix for real-time PCR. β-actin was used as an internal control gene, based on 2... -△△Ct The expression level of the target gene was calculated using the method described in Table 3.

[0058] Table 3 Mouse primer sequences

[0059] Atg3 and Atg5 are classic autophagy-related genes that play a crucial role in autophagy formation. According to Figure 12The results show that, using the Atg3 and Atg5 mRNA expression levels in the control group as a baseline (set as 1), the relative expression levels of these two genes in the model group decreased to 0.63 and 0.56, respectively, indicating a significant inhibition of the autophagy pathway. Under the treatment with *Bifidobacterium longum* FBJ19M2, the relative expression levels of Atg3 and Atg5 mRNA increased to 0.76 and 0.79, respectively, but there was no significant change compared to the model group. However, after intervention with *Bifidobacterium longum* CCFM1509, these two indicators increased to 1.22 and 1.30, respectively, significantly higher than the levels in the model group. Therefore, CCFM1509 can effectively restore the abnormal expression of autophagy-related genes caused by aging, and its regulatory effect is significantly better than that of the FBJ19M2 strain.

[0060] Example 9: Effects of Bifidobacterium longum CCFM1509 on the transcriptional levels of TFEB and HKDC1 in colon tissue of aging mice The modeling method was the same as in Example 2. After sacrifice, mouse colon tissue was collected, and the transcriptional levels of TFEB and HKDC1 in the colon tissue were measured. Total RNA was extracted from mouse colon tissue using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were calculated, and 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit. The reaction mixture was prepared using ChamQ Universal SYBR qPCRMaster Mix for quantitative real-time PCR. β-actin was used as an internal control gene, based on 2... -△△Ct The expression level of the target gene was calculated using the method described in Table 4.

[0061] Table 4 Mouse primer sequences

[0062] The successful completion of autophagy depends on the degradation process involving lysosomes. As the final execution unit of the autophagy pathway, lysosomes are responsible for the final breakdown of autophagic substrates. During the initiation phase of autophagy, enhancing lysosomal biosynthesis is indispensable to ensure the smooth fusion of autophagosomes and lysosomes and to meet the needs of subsequent degradation. This biological process is mainly regulated by the transcription factor TFEB, and its downstream target gene HKDC1 plays an important role in maintaining the homeostasis of the mitochondrial-lysosomal system, thereby effectively delaying the cellular senescence process.

[0063] according to Figure 13The experimental results, using the expression levels of TFEB and HKDC1 mRNA in the blank control group as a reference (normalized to 1), showed that the expression levels of these two key genes in the model group were significantly inhibited, decreasing to approximately 0.76 and 0.36, respectively. Treatment with *Bifidobacterium longum* FBJ19M2 did not significantly improve the transcriptional levels of TFEB and HKDC1; however, intervention with *Bifidobacterium longum* CCFM1509 increased these two indicators to 1.51 and 1.22, respectively. These results indicate that, compared to FBJ19M2, CCFM1509 can effectively promote the transcriptional activity of the TFEB-HKDC1 signaling pathway in colonic tissue, enhancing autophagy function by activating this autophagy regulatory axis, thus exhibiting a significant protective effect against colonic tissue aging.

[0064] 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 CCFM1509 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66745.

2. A microbial preparation containing the Bifidobacterium longum CCFM1509 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 CCFM1509 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 is a liquid preparation, a powder preparation, or a granular preparation.

5. A product containing the Bifidobacterium longum CCFM1509 of claim 1 or any of the microbial preparations of claims 2 to 4.

6. The product as described in claim 5, characterized in that, The products include food, medicine, or health products.

7. The product as described in claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients; the excipients include diluents, adjuvants and / or carriers.

8. The use of the Bifidobacterium longum CCFM1509 of claim 1 or any of the microbial preparations of claims 2 to 4 in the preparation of a medicament for anti-colonic aging.

9. The application as described in claim 8, characterized in that, The drug has at least one of the following functions: (a) Improves pathological damage to colon tissue; (b) Reduce the level of pro-inflammatory cytokines in colon tissue; (c) Reduce oxidative stress levels in colonic tissue; (d) Reduces the activity of aging-related β-galactosidase in colon tissue; (e) Reduce the transcriptional levels of aging markers in colon tissue; (f) Adjust the level of autophagy in colon tissue.

10. The use of the Bifidobacterium longum CCFM1509 of claim 1 or the microbial preparation of any one of claims 2 to 4 in the preparation of anti-aging health products.

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