Probiotic composition with anti-aging and life-prolonging functions and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a probiotic composition capable of resisting aging and prolonging the service life and application thereof.The composition comprises Bifidobacterium longum subsp. Longum FMBL B250365 MQ, Bifidobacterium adolescentis FMBL B250316 MQ and Lacticaseibacillus rhamnosus FMBL L25209 MQ, the composition is preserved in the China Center for Type Culture Collection (CCTCC) on September 18, 2025, the preservation numbers are CCTCC M 20252060, CCTCC M 20252061 and CCTCC M 20252059, the preservation number is CCTCC M 20252060, the preservation number is CCTCC M 20252061, and the preservation number is CCTCC M 20252059. The life of the nematodes can be remarkably prolonged, the senescence-related physiological indexes can be improved, the exercise ability can be enhanced, the resistance to external oxidative stress can be improved, and the nematodes can be used for developing health-care products, functional foods or medicines with the senescence-delaying and anti-oxidation functions and have wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a probiotic composition with anti-aging and life-extending effects and its applications. Background Technology

[0002] Aging is a complex physiological process in which bodily functions gradually decline with age. As a key risk factor, it is closely related to the development of various chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and metabolic syndrome. Among the many theories explaining the mechanisms of aging, the oxidative stress theory has been widely accepted. This theory states that when the body's production of reactive oxygen species (ROS) exceeds its scavenging capacity due to internal and external factors, an oxidative stress state is triggered, leading to irreversible oxidative damage to key biomolecules such as proteins, lipids, and DNA. This cumulative damage is the core link driving cellular senescence, tissue functional decline, and even the overall aging process of the body. Therefore, enhancing the body's antioxidant defense capabilities and mitigating oxidative stress damage through effective strategies has become one of the most promising intervention directions in the field of anti-aging research.

[0003] With the deepening research in microbiome science, the use of probiotics to regulate the body's oxidative stress levels shows promising application prospects. Current technologies indicate that specific probiotics, especially Lactobacillus (Lactobacillus spp.), can effectively regulate oxidative stress levels. Lactobacillus ) and Bifidobacterium spp. Bifidobacterium Some strains of *Bifidobacterium longum* exhibit antioxidant activity in in vitro models by directly scavenging DPPH and hydroxyl radicals. Further in vivo studies suggest that these strains may help alleviate cellular oxidative damage and regulate gut microbiota dysbiosis associated with aging. For example, patent CN118146998B discloses a strain of *Bifidobacterium longum* subsp. KS1, which claims to have anti-inflammatory, gut microbiota-improving, and antioxidant functions; another patent CN117286045B discloses *Bifidobacterium longum* subsp. KS2, reporting its potential activity in promoting protein digestion and anti-inflammation. These findings provide preliminary theoretical and practical evidence for using probiotics to intervene in age-related oxidative stress.

[0004] While existing technologies demonstrate the antioxidant potential of probiotics, their application in systemic anti-aging remains significantly limited. This limitation stems primarily from the following: current technologies largely focus on preliminary evaluations of one or a few local physiological functions of the strains (such as anti-inflammation and gut regulation), without addressing or rigorously validating their direct effects on extending healthy lifespan or maximum lifespan through biological models. This makes it difficult to accurately assess and confirm the actual anti-aging value of the strains. Different probiotic strains exhibit varying and unstable antioxidant activity, gastrointestinal tolerance, and colonization and efficacy in complex in vivo environments due to significant differences in origin, genetic background, and living environment. More critically, existing research often focuses on single strains, resulting in relatively limited mechanisms of action and targets. They typically function only in single areas such as free radical scavenging, failing to achieve synergistic intervention in multiple key pathophysiological processes, such as enhancing the activity of endogenous antioxidant enzymes (e.g., SOD, GSH-Px), repairing existing oxidative damage, and regulating age-related immune imbalances. Aging is a systemic physiological degeneration process driven by multiple factors and pathways. Existing single-strain application strategies, due to the limitations of their targets and mechanisms, cannot achieve efficient and synergistic regulation of the complete chain of anti-oxidative stress and aging, namely "scavenging ROS—activating the endogenous antioxidant system—repairing macromolecular damage—regulating immune homeostasis," and therefore the intervention effect is limited and unstable.

[0005] To address the aforementioned technical problems, developing a probiotic composition that can effectively compensate for these deficiencies, has been scientifically verified to have a clear lifespan-extending effect, and can exert a systemic anti-aging effect through the synergistic action of multiple strains in scavenging free radicals, enhancing endogenous antioxidant defense, repairing damage, and regulating immunity has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] The primary objective of this invention is to provide a probiotic composition with anti-aging and life-extending effects, wherein the probiotic composition comprises *Bifidobacterium longum* (… Bifidobacterium longum subsp. longum FMBLB250365 MQ, Bifidobacterium adolescentis ( Bifidobacterium adolescentis FMBL B250316 MQ and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The following samples were deposited on September 18, 2025, at the China Center for Type Culture Collection (CCTCC) with accession numbers CCTCC M 20252060, CCTCC M20252061, and CCTCC M 20252059, respectively.

[0007] A second objective of the present invention is to provide a probiotic agent containing the aforementioned probiotic composition.

[0008] A third objective of this invention is to provide the application of the probiotic composition or the bacterial agent in the preparation of anti-aging products.

[0009] A fourth objective of this invention is to provide the application of the probiotic composition or the probiotic agent in the preparation of products that extend lifespan.

[0010] The fifth objective of this invention is to provide the application of the probiotic composition or the bacterial agent described herein in the preparation of products that promote growth and development.

[0011] The sixth objective of this invention is to provide the application of the probiotic composition or the bacterial agent in the preparation of stress-resistant products, wherein the stress resistance refers to heat resistance or oxidation resistance.

[0012] The seventh objective of this invention is to provide the application of the probiotic composition or the bacterial agent in the preparation of antioxidant products.

[0013] The eighth objective of this invention is to provide an anti-aging drug, comprising an anti-aging active ingredient, wherein the anti-aging active ingredient comprises the probiotic composition or the bacterial agent.

[0014] The ninth objective of this invention is to provide a product for extending lifespan, comprising a lifespan-extending active ingredient, wherein the lifespan-extending active ingredient includes the probiotic composition or the bacterial agent.

[0015] The tenth objective of this invention is to provide an antioxidant product, comprising the probiotic composition or the bacterial agent described above.

[0016] Beneficial effects of the present invention: The present invention provides a probiotic composition with anti-aging and life-extending effects, wherein the probiotic composition comprises *Bifidobacterium longum* (… Bifidobacterium longum subsp. longum FMBL B250365 MQ, Bifidobacterium adolescentis ( Bifidobacterium adolescentis FMBL B250316 MQ and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosusThe *Bifidobacterium longum* FMBL L25209 MQ, *Bifidobacterium adolescentis* FMBL B250365 MQ, and *Lactobacillus rhamnosus* FMBL L25209 MQ were deposited at the China Center for Type Culture Collection (CCTCC) on September 18, 2025, with accession numbers CCTCC M 20252060, CCTCC M 20252061, and CCTCC M 20252059, respectively. Evaluation using a *C. elegans* model revealed that this composition significantly extended the average lifespan of nematodes by 37.29%; it also effectively improved their physiological indicators, including significantly increased body length, improved body swaying frequency and motor ability, and enhanced tolerance to heat and oxidative stress. In terms of biochemical indicators, this composition reduced the fluorescence intensity of lipofuscin in the nematode intestine by 24.25%, decreased the level of reactive oxygen species (ROS) by 18.60%, and increased the mitochondrial membrane potential by 0.7 times. Furthermore, it significantly reduced malondialdehyde (MDA) content by 38.59% (p<0.0001) and increased the activities of superoxide dismutase (SOD) and catalase (CAT) to 2.71 times and 28.83% of the control group, respectively (p<0.0001). In conclusion, this probiotic composition possesses anti-aging and antioxidant functions and can be used as a high-quality probiotic resource for the development of health products, functional foods, or pharmaceuticals, showing broad application prospects. Attached Figure Description

[0017] Figure 1 A diagram showing that a probiotic combination prolongs the lifespan of *C. elegans*. Figure 2 Effect of probiotic composition on body length of Caenorhabditis elegans; Figure 3 Effect of probiotic composition on the rate of body undulation in Caenorhabditis elegans; Figure 4 Effects of probiotic compositions on the motility of Caenorhabditis elegans; Figure 5 The effect of probiotic compositions on the heat tolerance of *C. elegans*; Figure 6 The effect of probiotic compositions on the antioxidant capacity of *C. elegans*; Figure 7 Effect of probiotic composition on lipofuscin content in Caenorhabditis elegans; Figure 8 Effect of probiotic composition on ROS content in Caenorhabditis elegans; Figure 9 Effects of probiotic compositions on the mitochondrial potential membrane of Caenorhabditis elegans; Figure 10Effects of probiotic composition on MDA, GSH content and SOD activity of Caenorhabditis elegans. Detailed Implementation

[0018] The following embodiments are provided to facilitate a better understanding of the present invention, but are not limited to it. Unless otherwise specified, the experimental methods in the following embodiments are conventional laboratory methods. Unless otherwise specified, the experimental materials used in the following embodiments are conventional biochemical reagents that can be purchased commercially. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0019] The culture medium formulations used in the following examples are as follows: NGM (Nematode growth medium) solid medium: 3.03 g NaCl, 2.30 g K2HPO4, 17.02 g KH2PO4, 2.71 g peptone, 1 L deionized water. Mix well, then add 17.14 g agar. Autoclave at 121℃ for 20 min. When pouring plates, add 1 mL each of 1 M CaCl2 solution, 1 M MgSO4 solution, and 5 mg / mL cholesterol solution. Mix thoroughly after each addition to prevent precipitation. LB (Luria-Bertani) solid medium: 10 g NaCl, 10 g peptone, 5 g yeast extract, 1 L deionized water, shake to dissolve, add 17 g agar, autoclave at 121℃ for 20 min and set aside for use.

[0020] LB (Luria-Bertani) liquid medium: 10 g NaCl, 10 g peptone, 5 g yeast extract, 1 L deionized water, shake to dissolve, autoclave at 121℃ for 20 min and set aside for use.

[0021] The solution formulations used in the following examples are as follows: 1M CaCl2 solution: Dissolve 111 g CaCl2 in 1 L of deionized water, autoclave at 121℃ for 20 min and set aside for use.

[0022] 1M MgSO4 solution: Dissolve 120 g MgSO4 in 1 L of deionized water, autoclave at 121℃ for 20 min and set aside for use.

[0023] 5 mg / mL cholesterol solution: Dissolve 5 g cholesterol in 1 L of anhydrous ethanol by sonication. After complete dissolution, aliquot into centrifuge tubes and store at 4°C for later use.

[0024] 1M NaOH solution: Dissolve 40 g NaOH in 1 L of deionized water and let stand at room temperature until ready to use.

[0025] 30% glycerol: Mix 300 mL glycerol and 700 mL deionized water, autoclave at 121℃ for 20 min and set aside for use.

[0026] 40% glycerol: Mix 400 mL glycerol and 700 mL deionized water, autoclave at 121℃ for 20 min and set aside for use.

[0027] M9 buffer solution: Dissolve 4.97 g NaCl, 20.19 g Na2HPO4·12 H2O, 2.99 g KH2PO4, 0.25 g MgSO4·7 H2O in 1 L of deionized water, autoclave at 121℃ for 20 min.

[0028] S base: Dissolve 5.85 g NaCl, 1.00 g K2HPO4, 6.00 g KH2PO4 in 1 L of deionized water, autoclave at 121℃ for 20 min.

[0029] Nematode lysis buffer: 5 mL 1 M NaOH solution and 4.68 mL M 9 buffer + 320 μL NaClO, mix by pipetting. Mix 1:1 before use.

[0030] Unless otherwise specified, the reagents and consumables used in the following examples can be purchased from the market.

[0031] Unless otherwise specified, the methods used in the following embodiments are conventional methods and can be obtained by referring to the corresponding literature.

[0032] In the following examples, the probiotic composition consisting of *Bifidobacterium longum* subsp. *longum* FMBL B250365 MQ, *Bifidobacterium adolescentis* FMBLB250316 MQ, and *Lactobacillus rhamnosus* FMBL L25209 MQ is abbreviated as "probiotic composition"; *Bifidobacterium longum* subsp. *longum* FMBL B250365 MQ is abbreviated as BL; *Bifidobacterium adolescentis* FMBL B250316 MQ is abbreviated as BA; *Lactobacillus rhamnosus* FMBL L25209 MQ is abbreviated as LR; *Lactobacillus rhamnosus* ( Lacticaseibacillus rhamnosus )GG was purchased from the China Industrial Microbial Culture Collection Center (No.: CICC 6141), abbreviated as LGG.

[0033] Example 1: Preparation of a probiotic composition and its effect on the lifespan of *C. elegans* 1. Cultivation and preparation of test strains Bifidobacterium longum subsp. MQ, frozen at -80℃, was used to store the following samples: Bifidobacterium longum subsp. longum Bifidobacterium adolescentis FMBL B250316 MQ ( Bifidobacterium adolescentis Lactobacillus rhamnosus FMBL L25209 MQ Lacticaseibacillus rhamnosus ) and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus After thawing, the GG strains were inoculated into MRS liquid medium and cultured anaerobically at 37°C (48 h for Bifidobacterium and 24 h for Lactobacillus). Subsequently, they were streaked onto MRS agar plates and cultured anaerobically at 37°C. Single colonies were then transferred to MRS liquid medium and cultured anaerobically at 37°C until the logarithmic growth phase. The cells were collected by centrifugation at 6000 rpm for 4 min, washed three times with sterile M9 buffer, and finally adjusted to an OD600 of 0.8 (approximately 1 × 10⁻⁶). 9 (CFU / mL), as a bacterial supply.

[0034] 2. Culture of Caenorhabditis elegans (1) The recovery of Caenorhabditis elegans Remove the frozen nematodes from -80°C and place them in a 37°C water bath for 1-2 minutes until completely thawed, avoiding damage from ice crystals. Transfer the thawed nematode suspension to OP50 (450 μL, OD50) prepared 24 hours in advance. 600 Inverted culture was carried out on NGM plates with a concentration of 0.8 g / cm³ on a 20°C, light-proof incubator.

[0035] (2) Transmission of Caenorhabditis elegans Inside the clean bench, a piece of NGM plate containing nematodes, about one square centimeter in size, was cut off with a cauterized scalpel. The plate was then inverted onto a new NGM plate coated with OP50 and placed in a 20°C incubator for culture.

[0036] (3) Cryopreservation of Caenorhabditis elegans The nematodes in the larval stage were starved, then washed off the culture medium with M9 buffer, centrifuged three times with a hand centrifuge and the supernatant was discarded. The collected nematodes were mixed with sterilized S basal solution and aliquoted into cryovials. An equal volume of 30% glycerol was added to each cryovial, the strain time was marked, and the cryovials were stored at -80℃.

[0037] (4) Synchronization of Caenorhabditis elegans To ensure that the nematodes were at the same growth stage as possible in the experiment, nematodes in the oviposition stage were rinsed off the culture dish with M9 buffer and collected into 15 mL centrifuge tubes. The tubes were allowed to stand to precipitate the nematodes, and the supernatant was discarded. 6 mL of nematode synchronization lysis buffer was added, and the tubes were vortexed for 6-8 min until the nematodes broke apart and released eggs. The lysate was quickly aliquoted into 1.5 mL centrifuge tubes, centrifuged at 5000 rpm at room temperature for 1 min, and the supernatant was discarded. M9 buffer was added, and the tubes were gently vortexed and centrifuged at 5000 rpm at room temperature for 2 min. The supernatant was carefully aspirated, and M9 buffer was added again, and the tubes were gently vortexed and centrifuged at 5000 rpm at room temperature for 3 min. The supernatant was aspirated, and a small amount of precipitate and liquid were retained and combined into one centrifuge tube for subculturing or experimentation.

[0038] 3. Effects of strains on the lifespan of *C. elegans* Caenorhabditis elegans synchronized to stage L4 was randomly divided into three replicates per group. Approximately 35 nematodes per plate were cultured on NGM plates containing 0.1 mg / mL 5-fluorodeoxyuridine (20℃). Nematode survival was assessed and recorded every 24 h, and NGM plates were replaced after 48 h. Mortality was defined as cessation of the pharyngeal pump and lack of response to mechanical stimulation, excluding individuals that died from desiccation, accidental death, loss, or produced offspring. Temperature (20±0.5℃) and bacterial concentration (OD) were controlled throughout the experiment. 600 =0.5±0.05), to ensure data reliability.

[0039] 4. Results Feeding single group of Caenorhabditis elegans ( Figure 1 A) Two-strain complex group ( Figure 1 B) and probiotic composition group ( Figure 1 The result of C) is as follows Figure 1 As shown in Table 1, compared with the blank control group fed with *Escherichia coli* OP50, the positive control group fed with *Lactobacillus rhamnosus* GG, and other single or combined bacterial experimental groups, the probiotic composition treatment group of the present invention showed the best performance in both the average lifespan and maximum lifespan of nematodes. The specific lifespan statistics in Table 1 further confirm the anti-aging potential of this composition. Compared with the OP50 control group, the average lifespan of *C. elegans* fed with the probiotic composition of the present invention was significantly extended by 37.29%. Furthermore, the longest lifespan of the nematodes in this treatment group reached 28 days, fully demonstrating the outstanding advantages of the composition of the present invention in promoting healthy aging and extending lifespan. The probiotic composition provided by the present invention exhibits significantly better efficacy than single strains and combinations of other strains in extending the lifespan of nematodes.

[0040] Table 1. Results of probiotic composition on the lifespan of Caenorhabditis elegans.

[0041] Note: Different letters indicate significant differences between groups.

[0042] Example 2: Effects of probiotic composition on physiological indicators and muscle function decline in *C. elegans* 1. Size of nematodes Twenty L4 nematodes were randomly selected and cultured on plates from different treatment groups. Their body size was measured on days 5 and 10 after bacterial feeding. The nematodes were fixed on 2% agarose gel pads, then anesthetized with 25 mM levamisole and straightened. The nematodes were photographed using a bright-field fluorescence microscope and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The body length of the nematodes was measured and used as an indicator of their body size.

[0043] 2. The speed of the nematode's body swaying Twenty 6-14 nematodes were randomly selected and their oscillation rate was measured using an SMZ-168 stereomicroscope (Motic). The test nematodes were placed in M9 buffer and allowed to swim freely for 30 seconds to acclimatize before the test began. One complete oscillation of the nematode's head or tail was recorded as one oscillation, and the time was recorded for 1 minute.

[0044] 3. Nematode locomotion ability Ninety nematodes of 8–16 days were randomly selected and their motility was measured using an SMZ-168 stereomicroscope (Motic). Based on their motility, they were divided into four categories: Category A – spontaneous and / or regular sinusoidal movements; Category B – irregular and / or uncoordinated movements; Category C – only the head and / or tail moved when gently touched with a platinum wire picker; and Category D – nematodes that died.

[0045] 4. Results like Figure 2 It was found that at 5 days, there was no significant difference in body length between the OP50 group and the probiotic composition group; however, at 10 days, the body length of the probiotic composition group was significantly higher than that of the OP50 group (p<0.01). This indicates that over time, the probiotic composition demonstrates an advantage in promoting nematode body length growth and is more beneficial to supporting the growth and development of nematodes compared to OP50.

[0046] like Figure 3As shown, during the 12-day intervention period, compared with the OP50 control group, the probiotic composition significantly increased the body movement frequency of *C. elegans* after intervention. The probiotic composition showed a better promoting effect on day 8 of the initial intervention, with a higher movement frequency than the control group; as the intervention time increased, the improvement was more significant on day 12, with a movement frequency significantly better than the OP50 group (p<0.05). The results indicate that the probiotic composition has a sustained and enhanced promoting effect on maintaining the body's motor capacity, suggesting its potential application value in improving physical performance.

[0047] Depend on Figure 4 It is evident that the proportions of nematode motility classifications differed significantly at different time points (4, 8, 12, and 16 days). At 4 days, almost all nematodes treated with the OP50 group and the probiotic composition group belonged to category A. As time progressed, the proportion of category A gradually decreased, while the proportions of categories B, C, and D gradually increased. At each time point, the proportion of category A in the probiotic composition group was higher than that in the OP50 group, while the proportion of category D was lower. Particularly at 16 days, the probiotic composition group maintained a high proportion of category A and a significantly lower proportion of category D. This indicates that the probiotic composition can maintain the motility of nematodes for a longer period, delaying the onset of age-related motility decline such as irregular and uncoordinated movement and death, thus exhibiting a certain anti-aging effect and positively contributing to the maintenance of nematode physiological function.

[0048] Example 3: Effect of probiotic composition on the stress resistance of Caenorhabditis elegans 1. Analysis of thermal stress capacity Nematodes synchronized to the L4 stage were cultured in groups of three plates. Each plate contained 30 nematodes. After 5 days of culture, the nematodes were transferred to a constant temperature of 37℃ for 2 hours, and then placed in a constant temperature of 20℃ for 12 hours to recover. After the recovery period, the survival of the nematodes was observed.

[0049] 2. Analysis of oxidative stress capacity The nematodes were transferred to NGM medium containing 1 mL of 0.2% H2O2. The survival status of each group of nematodes was observed and recorded every 30 min until all nematodes died. The average lifespan of each group was calculated and survival curves were plotted.

[0050] 3. Results like Figure 5 As shown, after heat stress, the heat-resistant survival rate of *Caenorhabditis elegans* OP50 group was 58.33%; the heat-resistant survival rate of nematodes in the probiotic composition group was 74.09%, significantly higher than that of the OP50 group (p<0.01), indicating that the probiotic composition can effectively enhance the nematode's ability to cope with heat stress; Figure 6 The results showed that the antioxidant survival rates of both the OP50 group and the probiotic composition group decreased over time. However, at the same time point, the antioxidant survival rate of the probiotic composition group was consistently higher than that of the OP50 group, and the rate of decline was slower. In summary, the probiotic composition not only enhances the heat stress resistance of nematodes but also prolongs their antioxidant survival time, demonstrating a positive effect on improving the stress resistance and maintaining physiological functions of nematodes.

[0051] Example 4: Effect of probiotic composition on lipofuscin accumulation in the intestine of *C. elegans* Several 10-15 antennae were randomly selected from different treatment groups to determine their lipofuscin accumulation levels. The test nematodes were washed three times with M9 buffer, then fixed with 25 mM levamisole onto levamisole-anesthetized nematodes, and transferred to 2% agarose gel slides. Images were then captured and recorded under an upright fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ software. The lipofuscin accumulation levels of 10-15 antennae were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0052] like Figure 7 As shown, to systematically evaluate the effect of the probiotic composition of the present invention on delaying cellular senescence, this study quantitatively analyzed its effect on the accumulation of lipofuscin, a senescence marker, in *C. elegans* using fluorescence microscopy. During the normal aging process, due to decreased autophagy and accumulated oxidative damage, lipofuscin (a typical senescence-related fluorescent pigment) in nematodes exhibits a continuous deposition characteristic with increasing age, and its accumulation level is significantly positively correlated with the aging progress. The results indicate that the probiotic composition of the present invention can effectively delay the age-related pigment deposition process and significantly regulate the accumulation of senescence markers, demonstrating good application potential for the composition in delaying functional decline and promoting healthy aging. Experimental results showed that, compared with the control group fed *Escherichia coli* OP50, the experimental group of nematodes fed the probiotic composition of the present invention exhibited a significant anti-aging phenotype. On day 3 of intervention, quantitative fluorescence intensity analysis revealed that the fluorescence intensity of lipofuscin in the intestines of the probiotic group was significantly reduced by 24.25% compared with the OP50 control group (p<0.05), indicating that the composition can effectively inhibit senescence-related pigment deposition. The above results indicate that the probiotic composition provided by the present invention can effectively delay the accumulation of aging-related biomarkers by regulating cellular metabolic processes, which provides application value for revealing its anti-aging mechanism and developing related anti-aging products.

[0053] Example 5: Effect of probiotic composition on reactive oxygen species (ROS) levels in *C. elegans* L4 stage nematodes were transferred to NGM medium in different treatment groups and cultured until day 5. The ROS accumulation level in the nematodes was determined using the DCFH-DA fluorescent dye method. Forty nematodes from each group were picked and placed in EP tubes containing M9 buffer. After centrifugation and washing, the supernatant was removed, and then 1 mL of 10 μmol / L DCFH-DA solution was added. The tubes were stored at room temperature in the dark for 2 hours. After 2 hours, the tubes were washed five times with M9 buffer, and the nematode suspension was dropped onto a glass slide and observed and photographed under an upright fluorescence microscope. The relative fluorescence intensity was analyzed using ImageJ software.

[0054] like Figure 8 As shown, to verify the effect of the probiotic composition of this invention in regulating oxidative stress, this study detected the level of ROS accumulation in *C. elegans* during aging using a specific fluorescent probe for reactive oxygen species (ROS). The results showed that during natural aging, the level of ROS in the nematodes increased significantly with age, reflecting the gradual aggravation of oxidative damage with aging. Quantitative analysis showed that, compared with the OP50 control group, the experimental group fed with the probiotic composition of this invention exhibited a significant antioxidant effect. On day 3 of intervention, the average fluorescence intensity of ROS in the experimental group was reduced by 18.60% compared with the control group (p<0.05), indicating an enhanced antioxidant protective effect. In other words, the probiotic composition of this invention can effectively inhibit the accumulation of reactive oxygen species during aging and significantly reduce age-related oxidative damage, demonstrating its good potential for application in the preparation of functional foods or microecological preparations for alleviating oxidative stress and delaying age-related metabolic decline.

[0055] Example 6: Effect of probiotic composition on mitochondrial membrane potential of Caenorhabditis elegans L4 stage nematodes were transferred to NGM medium in different treatment groups and cultured until day 3. Nematodes were randomly selected and tested using the JC-1 mitochondrial membrane potential assay kit. Specifically, M9-washed nematodes were placed in 500 μL of freshly prepared JC-1 staining working solution in a 24-well plate and incubated at 20°C in the dark for 2 h. The nematodes were washed three times with M9 buffer to remove stain from the surface. *C. elegans* were observed under a fluorescence microscope using red and green fluorescence, and photographs were taken. The intensity of red and green fluorescence in *C. elegans* was measured using ImageJ, and the ratio was calculated. In damaged or apoptotic cells, the red-green fluorescence ratio decreased.

[0056] like Figure 7As shown, to investigate the regulatory effect of the probiotic composition of this invention on mitochondrial function, this study systematically evaluated the dynamic changes in mitochondrial membrane potential (ΔΨm) during the aging process of *C. elegans* using the JC-1 fluorescent probe method. Mitochondrial membrane potential is a key indicator for evaluating mitochondrial functional integrity and cellular energy metabolism status. The experimental results showed that during the natural aging process, the mitochondrial membrane potential of OP50 control group nematodes showed a significant decreasing trend with increasing age, indicating that their mitochondrial function gradually deteriorates with aging. In contrast, feeding with the probiotic composition of this invention maintained a significantly higher mitochondrial membrane potential level throughout the observation period (p<0.05). Quantitative fluorescence analysis showed that the fluorescence intensity of mitochondrial membrane potential in the somatic cells of nematodes in the compound bacterial group was approximately 0.7 times higher than that in the control group. The probiotic composition of this invention can significantly maintain mitochondrial membrane potential stability and effectively delay age-related mitochondrial functional decline, demonstrating its good potential for application in the preparation of functional foods, special diets, or microecological preparations for improving energy metabolism and delaying age-related functional decline.

[0057] Example 7: Effects of probiotic composition on MDA, GSH content and SOD activity of Caenorhabditis elegans L4 stage nematodes were transferred to NGM medium in different treatment groups and cultured until day 3. Approximately 1500 nematodes were taken from each group, washed with M9 buffer, and collected into EP tubes. The tubes were then ground into a paste using a tissue homogenizer at low temperature until homogenized. After centrifugation, the supernatant was collected, and the activities of superoxide dismutase (SOD), catalase (CAT), and MDA content were measured according to the kit instructions.

[0058] like Figure 10As shown, this study systematically evaluated the effects of a probiotic composition on oxidative stress-related indicators during the aging process of *C. elegans* using biochemical analysis methods. The results showed that, compared with the conventional feeding group (OP50 control group), the probiotic composition of this invention significantly improved the body's antioxidant defense system. The results showed that, during natural aging, compared with the OP50 control group, the probiotic composition intervention significantly reduced the content of malondialdehyde (MDA), a marker of oxidative damage, by 38.59% (p<0.0001), indicating that lipid peroxidation damage was effectively curbed. Simultaneously, it significantly increased superoxide dismutase (SOD) activity to 2.71 times that of the control group, significantly enhancing the ability to scavenge superoxide anion free radicals. Furthermore, it significantly increased catalase (CAT) activity by 28.83% (p<0.0001), strengthening the efficiency of hydrogen peroxide scavenging. The probiotic composition of this invention can synergistically upregulate the activities of superoxide dismutase and catalase while effectively reducing MDA content, comprehensively enhancing the body's antioxidant defense capabilities and significantly alleviating oxidative damage during aging. This demonstrates its promising potential for application in the preparation of functional foods or microecological preparations for regulating redox balance and delaying oxidative stress-related aging processes.

[0059] In summary, this invention provides a probiotic composition with anti-aging and life-extending effects, wherein the probiotic composition comprises *Bifidobacterium longum* (… Bifidobacterium longum subsp. longum FMBLB250365 MQ, Bifidobacterium adolescentis ( Bifidobacterium adolescentis FMBL B250316 MQ and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosusThe *Bifidobacterium longum* subspecies *FMBL*B250365 MQ, *Bifidobacterium adolescentis* FMBL B250316 MQ, and *Lactobacillus rhamnosus* FMBL L25209 MQ were deposited at the China Center for Type Culture Collection (CCTCC) on September 18, 2025, with accession numbers CCTCC M 20252060, CCTCC M20252061, and CCTCC M 20252059, respectively. Evaluation using a *Caenorhabditis elegans* model revealed that this composition significantly extended the average lifespan of nematodes by 37.29%; it also effectively improved their physiological indicators, including significantly increased body length, improved body swaying frequency and motor ability, and enhanced tolerance to heat and oxidative stress. In terms of biochemical indicators, this composition reduced the fluorescence intensity of lipofuscin in the nematode intestine by 24.25%, decreased the level of reactive oxygen species (ROS) by 18.60%, and increased the mitochondrial membrane potential by 0.7 times. Furthermore, it significantly reduced malondialdehyde (MDA) content by 38.59% (p<0.0001) and increased the activities of superoxide dismutase (SOD) and catalase (CAT) to 2.71 times and 28.83% of the control group, respectively (p<0.0001). This probiotic composition possesses anti-aging and antioxidant functions and can be used as a high-quality probiotic resource for the development of health products, functional foods, or pharmaceuticals, showing broad application prospects.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probiotic composition with anti-aging and life-extending effects, said probiotic composition comprising *Bifidobacterium longum* (… Bifidobacterium longum subsp. longum FMBL B250365 MQ, Bifidobacterium adolescentis ( Bifidobacterium adolescentis FMBL B250316 MQ and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus FMBL L25209 MQ, characterized in that, The aforementioned *Bifidobacterium longum* subspecies FMBL B250365 MQ, *Bifidobacterium adolescentis* FMBL B250316 MQ, and *Lactobacillus rhamnosus* FMBL L25209 MQ were deposited at the China Center for Type Culture Collection on September 18, 2025, with accession numbers CCTCC M 20252060, CCTCC M 20252061, and CCTCC M 20252059, respectively.

2. A microbial agent, characterized in that, The bacterial agent contains the probiotic composition of claim 1.

3. The use of the probiotic composition as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of anti-aging products.

4. The use of the probiotic composition as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of products that extend product lifespan.

5. The use of the probiotic composition of claim 1 or the bacterial agent of claim 2 in the preparation of products that promote growth and development.

6. The application of the probiotic composition of claim 1 or the bacterial agent of claim 2 in the preparation of stress-resistant products, wherein the stress resistance refers to heat resistance or oxidation resistance.

7. The use of the probiotic composition of claim 1 or the bacterial agent of claim 2 in the preparation of antioxidant products.

8. An anti-aging drug, characterized in that, It includes anti-aging active ingredients, which include the probiotic composition of claim 1 or the bacterial agent of claim 2.

9. A product for extending lifespan, characterized in that, It includes a life-extending active ingredient, which includes the probiotic composition of claim 1 or the bacterial agent of claim 2.

10. An antioxidant product, characterized in that, Includes the probiotic composition of claim 1 or the bacterial agent of claim 2.

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