Bifidobacterium bifidum capable of relieving aging and application of bifidobacterium bifidum

Abstract

The invention discloses a Bifidobacterium bifidum CCFM1424 capable of relieving senescence and an application of the Bifidobacterium bifidum CCFM1424, and belongs to the technical field of microorganisms. The bifidobacterium bifidum CCFM1424 disclosed by the invention can be used for effectively relieving cognitive defects of aged mice, improving the oxidation resistance (SOD, CAT, MDA and GSH-Px) of the brain and the liver, reducing the levels of proinflammatory factors TNF-alpha, IL-1beta and IL-6 in serum and relieving colitis. In addition, the intervention of the bifidobacterium bifidum CCFM1424 can maintain muscle health, and can improve the change of intestinal flora and intestinal metabolites related to aging. According to the bifidobacterium bifidum, the application range of the bifidobacterium bifidum as probiotics is expanded, and the bifidobacterium bifidum has a huge application prospect in the aspect of relieving senescence.

Description

Technical Field

[0001] The present invention relates to a Bifidobacterium bifidum strain capable of alleviating aging and its application, belonging to the field of microbial technology. Background Art

[0002] The global elderly population is showing a rapid growth trend, and population aging is often accompanied by the occurrence of chronic diseases such as sarcopenia, diabetes, cardiovascular diseases, and cognitive impairment, which seriously affect the quality of life of the elderly in their later years. Aging can lead to a decline in the immune system function of the elderly, usually manifested as an increase in the levels of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α), an increase in oxidative stress damage to the body, etc. Research shows that long-term exposure to inflammatory factors and persistent oxidative stress will exacerbate neurodegeneration, damage cognitive function, and trigger intestinal inflammation caused by intestinal cell aging. And the intestinal flora dysregulation and the destruction of intestinal barrier integrity caused by aging will in turn further exacerbate the systemic inflammation of the elderly. Therefore, it is of great significance to find safe and effective intervention strategies to actively regulate the health status of the elderly population and extend the healthy lifespan of the aging population.

[0003] So far, a wide range of intervention measures have been used to alleviate aging or aging-related diseases, including dietary regulation, calorie restriction, probiotic or prebiotic intervention, etc. Among them, probiotic intervention can not only shift the intestinal flora in a beneficial direction, but also delay aging by enhancing the body's antioxidant capacity, reducing inflammation, regulating the body's immunity, and improving cognitive ability. For example, research shows that Lactobacillus plantarum LLY-606 can effectively extend the lifespan of mice by reducing the levels of inflammatory factors in the body, increasing antioxidant capacity, alleviating intestinal barrier dysfunction, and improving cognitive impairment in mice (Journal of Agricultural and Food Chemistry, 2024, 72(8): 4049-4062); Bifidobacterium longum has been reported to accelerate the healing of aging mice with fractures, mainly attributable to the role of Bifidobacterium longum in maintaining the integrity of the intestinal barrier, inhibiting systemic inflammation, and maintaining intestinal homeostasis (Aging Cell, 2023, 22(4): e13786); it has also been reported that Bifidobacterium longum and Bifidobacterium animalis and their combinations can effectively alleviate D-galactose-induced age-related cognitive impairment by inhibiting neuroinflammation and oxidative stress (Journal of Functional Foods, 2020, 69: 103938).

[0004] Bifidobacterium is a common probiotic that has many functions, such as improving intestinal health, regulating immunity, promoting digestion and absorption, and lowering cholesterol. Among them, Bifidobacterium bifidum has attracted widespread attention due to its properties of regulating host immunity and anti-inflammatory (Microorganisms, 2019, 7(11): 544). In the prior art, patent CN118562643A discloses that Bifidobacterium bifidum can improve colon damage and colon inflammation in mice with ulcerative colitis caused by DSS; patent CN117821305A discloses that Bifidobacterium bifidum CCFM1359 can play a role in repairing the enteric nervous system by increasing the level of BDNF and its specific receptor TrkB in the mouse colon and increasing the number of enteric glial cells; another patent CN117835838A discloses that the combined use of Bifidobacterium bifidum BGN4 (KCCM12754P) and Bifidobacterium longum BORI (KCCM-10492) can effectively improve the neuroinflammatory response in the brain of Alzheimer's mice and improve the cognitive impairment of mice. However, so far, it is unknown whether Bifidobacterium bifidum can simultaneously alleviate cognitive impairment, colon inflammation, and muscle damage caused by aging, and its effect on improving aging-related intestinal flora and metabolism is also unclear. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Bifidobacterium bifidum that can alleviate aging and its application. Currently, it is unknown whether Bifidobacterium bifidum can simultaneously alleviate cognitive impairment, colon inflammation, and muscle damage caused by aging, and its effect on improving aging-related intestinal flora and metabolism is also unclear. It is necessary to develop a new probiotic for aging.

[0006] The first technical solution provided by the present invention is a strain of Bifidobacterium bifidum CCFM1424, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a preservation number of GDMCC No: 65151, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In some embodiments, the Bifidobacterium bifidum CCFM1424 is isolated from human feces. In some embodiments, the growth characteristics of the Bifidobacterium bifidum CCFM1424 are as follows: the strain is a facultative anaerobe, inoculated into a culture medium, and cultured in an anaerobic chamber at 37°C for at least 24 hours.

[0008] In some embodiments, the culture medium is MRS medium. The formulation of the MRS medium is as follows: peptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, anhydrous glucose 20 g / L, anhydrous sodium acetate 2 g / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.5 g / L, manganese sulfate monohydrate (MnSO4·H2O) 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.6 g / L, Tween 80 1 mL / L.

[0009] In some embodiments, the culture temperature is 37 °C. The culture time is at least 24 h.

[0010] In some embodiments, the colony characteristics of Bifidobacterium bifidum CCFM1424 are as follows: milky white, round and convex, with neat and smooth edges on the MRS solid medium.

[0011] The second technical solution provided by the present invention is a microbial preparation containing the Bifidobacterium bifidum CCFM1424 described in the first technical solution.

[0012] In some embodiments, in the microbial preparation, the concentration of Bifidobacterium bifidum CCFM1424 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0013] Preferably, in the microbial preparation, the concentration of Bifidobacterium bifidum CCFM1424 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0014] The third technical solution provided by the present invention is a product containing the Bifidobacterium bifidum CCFM1424 described in the first technical solution or the microbial preparation described in the second technical solution.

[0015] In some embodiments, in the product, the addition amount of Bifidobacterium bifidum CCFM1424 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0016] Preferably, in the product, the addition amount of Bifidobacterium bifidum CCFM1424 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0017] In some embodiments, the product comprises food or medicine.

[0018] In some embodiments, the food is a dairy product, a soy product or a fruit and vegetable product fermented with Bifidobacterium bifidum CCFM1424 described in the first technical solution or the microbial preparation described in the second technical solution.

[0019] In some embodiments, the food is a beverage or a snack containing Bifidobacterium bifidum CCFM1424 described in the first technical solution or the microbial preparation of the second technical solution.

[0020] The fourth technical solution provided by the present invention is the application of Bifidobacterium bifidum CCFM1424 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a product for alleviating aging.

[0021] In some embodiments, the alleviation of aging includes the effects of at least one of the following (a) to (k):

[0022] (a) Can significantly alleviate the decline in cognitive ability caused by aging;

[0023] (b) Improve the morphology of astrocytes in the brain tissue of aging individuals;

[0024] (c) Significantly alleviate oxidative stress in the brain and liver of elderly individuals, and the antioxidant indicators include at least one of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) or glutathione peroxidase (GSH-Px);

[0025] (d) Reduce the levels of pro-inflammatory factors in the serum of elderly individuals, and the pro-inflammatory factors include at least one of TNF-α, IL-1β or IL-6;

[0026] (e) Improve the muscle tissue morphology of aging individuals;

[0027] (f) Increase the grasping force of elderly individuals;

[0028] (g) Reduce the infiltration of inflammatory cells and the number of goblet cells in the colon tissue of elderly individuals;

[0029] (h) Reduce the levels of pro-inflammatory factors in the colon tissue of elderly individuals, and the pro-inflammatory factors include at least one of TNF-α, IL-1β or IL-6;

[0030] (i) Increase the levels of anti-inflammatory factors in the colon tissue of elderly individuals, and the anti-inflammatory factors include IL-10;

[0031] (j) Improve the intestinal microbiota disorder caused by aging;

[0032] (k) Improve intestinal metabolite disorders caused by aging.

[0033] In certain embodiments, in the product, the addition amount of Bifidobacterium bifidum CCFM1424 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0034] Preferably, in the product, the addition amount of Bifidobacterium bifidum CCFM1424 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0035] In certain embodiments, the product comprises food or medicine.

[0036] In certain embodiments, the medicine further contains a pharmaceutical carrier and / or pharmaceutical excipients.

[0037] In certain embodiments, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0038] In certain embodiments, the pharmaceutical excipients comprise excipients and / or additives.

[0039] In certain embodiments, the excipients comprise binders, fillers, disintegrants and / or lubricants.

[0040] In certain embodiments, the additives comprise solubilizers, cosolvents, latent solvents and / or preservatives.

[0041] In certain embodiments, the dosage form of the medicine is powder, granule, capsule, tablet, pill or oral liquid.

[0042] The fifth technical solution provided by the present invention is the application of Bifidobacterium bifidum CCFM1424 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a product for improving intestinal microbiota and intestinal metabolite disorders caused by aging.

[0043] In some embodiments, the improvement of gut microbiota disorders caused by aging refers to upregulating the relative abundances of beneficial bacteria such as Anaerotruncus, Prevotellaceae UCG-001, Eubacterium ruminantium group, and Dubosiella, and downregulating the relative abundances of opportunistic pathogenic bacteria such as Defluviitaleaceae UCG-011 and Enterococcus.

[0044] In some embodiments, the improvement of gut metabolite disorders caused by aging refers to upregulating the contents of spermidine and / or D-sphingosine and downregulating the content of adenine.

[0045] The technical effects of the present invention are as follows:

[0046] The present invention provides a Bifidobacterium bifidum CCFM1424 that can alleviate cognitive impairment, colon inflammation, and muscle damage caused by aging, and improve the changes / dysregulation of gut microbiota and gut metabolites caused by aging. After the Bifidobacterium bifidum CCFM1424 provided by the present invention acts on aging mice, it can significantly alleviate the cognitive impairment, colon inflammation, and muscle damage of aging mice, and improve the changes of gut microbiota and gut metabolites caused by aging. Specifically, compared with the model group:

[0047] (1) The cognitive function of aging mice is improved;

[0048] (2) The morphology of astrocytes in the brains of aging mice is improved;

[0049] (3) The SOD activity in the brains of aging mice increases from 160.82 ± 15.37 U / mg protein to 244.11 ± 51.78 U / mg protein;

[0050] (4) The CAT activity in the brains of aging mice increases from 12.42 ± 4.42 U / mg protein to 28.76 ± 3.04 U / mg protein;

[0051] (5) The GSH-Px activity in the brains of aging mice increases from 31.3 ± 1.05 U / mg protein to 43.15 ± 3.62 U / mg protein;

[0052] (6) The MDA concentration in the brains of aging mice decreases from 2.07 ± 0.08 nmol / mg protein to 1.15 ± 0.14 nmol / mg protein;

[0053] (7) The SOD activity in the liver tissue of senescent mice increased from 158.42 ± 4.81 U / mg protein to 223.02 ± 10.29 U / mg protein;

[0054] (8) The CAT activity in the liver tissue of senescent mice increased from 18.14 ± 1.37 U / mg protein to 27.67 ± 6.69 U / mg protein;

[0055] (9) The GSH-Px activity in the liver tissue of senescent mice increased from 232 ± 30.99 U / mg protein to 346.21 ± 23.79 U / mg protein;

[0056] (10) The MDA concentration in the liver tissue of senescent mice decreased from 4.14 ± 1.0 nmol / mg protein to 2.49 ± 0.2 nmol / mg protein;

[0057] (11) Improve the state of muscle tissue;

[0058] (12) Increase the grasping force of elderly individuals, from 103.03 ± 7.82 N to 131.03 ± 5.35 N;

[0059] (13) The concentration of inflammatory factor TNF-α in the serum of senescent mice decreased from 268.98 ± 10.77 ng / L to 211.41 ± 9.47 ng / L;

[0060] (14) The concentration of inflammatory factor IL-1β in the serum of senescent mice decreased from 56.4 ± 1.33 ng / L to 45.73 ± 1.27 ng / L;

[0061] (15) The concentration of inflammatory factor IL-6 in the serum of senescent mice decreased from 25.92 ± 1.12 pg / mL to 20.59 ± 1.85 pg / mL;

[0062] (16) The inflammatory infiltration in the colon tissue of senescent mice decreased;

[0063] (17) The number of goblet cells in the colon tissue of senescent mice increased;

[0064] (18) The concentration of inflammatory factor TNF-α in the colon tissue of senescent mice decreased from 23.68 ± 5.76 ng / mg protein to 12.83 ± 1.78 ng / mg protein;

[0065] (19) The concentration of inflammatory factor IL-1β in the colon tissue of senescent mice decreased from 6.11 ± 0.14 ng / mg protein to 3.84 ± 0.23 ng / mg protein;

[0066] (20) The concentration of inflammatory factor IL-6 in the colon tissue of senescent mice decreased from 3.42 ± 0.64 pg / mg protein to 2.09 ± 0.14 pg / mg protein;

[0067] (21) The concentration of anti-inflammatory factor IL-10 in the colon tissue of senescent mice increased from 18.68 ± 1.27 pg / mg protein to 39.78 ± 5.42 pg / mg protein;

[0068] (22) Regulate the intestinal microbiota disorder caused by aging, increase the relative abundance of beneficial bacteria including short-chain fatty acid-producing bacteria, and reduce the relative abundance of harmful bacteria, so that the intestinal microbiota returns to a healthy state.

[0069] (23) Improve and relieve the levels of intestinal metabolites in aging or aging-related diseases.

[0070] Therefore, Bifidobacterium bifidum CCFM1424 has great application prospects in the preparation of products for relieving aging.

[0071] Biological material preservation

[0072] Bifidobacterium bifidum CCFM1424, taxonomically named Bifidobacterium bifidum, was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 20, 2024, with the deposit number GDMCC No: 65151, and the deposit address is the 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. Description of the drawings

[0073] Figure 1 It is a flow chart of animal experiments.

[0074] Figure 2 It shows the performance of experimental mice in the open field behavior test of different groups: (A) Representative movement trajectories in the open field; (B) Time spent staying in the central area of the open field.

[0075] Figure 3 It shows the performance of experimental mice in the water maze behavior test of different groups: (A) Escape latency in the positioning navigation experiment of the water maze test for 4 consecutive days; (B) Heat map of representative swimming trajectories in the spatial exploration test on the 5th day; (C) Escape latency in the spatial exploration test on the 5th day; (D) Number of times crossing the platform in the spatial exploration test on the 5th day; (E) Total distance in the target quadrant in the spatial exploration test on the 5th day; (F) Cumulative duration in the target quadrant in the spatial exploration test on the 5th day.

[0076] Figure 4 It is the GFAP staining results of the brain tissue of experimental mice in different groups.

[0077] Figure 5 Antioxidant indexes in the brains of experimental mice in different groups: (A) SOD activity; (B) CAT activity; (C) GSH-Px activity; (D) MDA concentration.

[0078] Figure 6 Antioxidant indexes in the liver tissues of experimental mice in different groups: (A) SOD activity; (B) CAT activity; (C) GSH-Px activity; (D) MDA concentration.

[0079] Figure 7 Grip strength results of the forelimbs of experimental mice in different groups.

[0080] Figure 8 H&E staining results of muscle tissues of experimental mice in different groups.

[0081] Figure 9 Serum inflammatory factor levels of experimental mice in different groups: (A) TNF-α level; (B) IL-1β level; (C) IL-6 level.

[0082] Figure 10 H&E staining results of colon tissues of experimental mice in different groups.

[0083] Figure 11 AB-PAS staining results of colon tissues of experimental mice in different groups.

[0084] Figure 12 Count of goblet cells in the colon of experimental mice in different groups.

[0085] Figure 13 Levels of inflammatory and anti-inflammatory factors in the colon tissues of experimental mice in different groups; (A) TNF-α level; (B) IL-1β level; (C) IL-6 level; (D) IL-10 level.

[0086] Figure 14 Alpha diversity and beta diversity of fecal microbiota in mice of the blank group, model group, and CCFM1424 group.

[0087] Figure 15 Stacked plots of fecal microbiota at the phylum and genus levels in mice of the blank group, model group, and CCFM1424 group.

[0088] Figure 16 LDA scores for differential analysis of fecal microbiota between the blank group and the model group, and LDA scores for differential analysis of fecal microbiota between the CCFM1424 group and the model group.

[0089] Figure 17 sPLS-DA plots of fecal metabolites of experimental mice in different groups.

[0090] Figure 18 Volcano plot analysis of fecal metabolites in blank group and model group mice.

[0091] Figure 19 Volcano plot analysis of fecal metabolites in model group and CCFM1424 group mice. Specific implementation manners

[0092] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0093] The C57BL / 6J male mice involved in the following embodiments were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The ELISA kits for detecting TNF-α, IL-1β, IL-6, and IL-10 were purchased from Nanjing Senbeiga Biotechnology Co., Ltd. The kits for detecting SOD, CAT, GSH-Px, and MDA were purchased from Nanjing Jiancheng. The D-galactose involved in the following embodiments was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0094] The preparation method of the D-galactose solution involved in the following embodiments is as follows:

[0095] Each mouse was subcutaneously injected with 1000 mg / kg BW D-galactose solution, and the D-galactose solution was mixed with physiological saline.

[0096] The culture media involved in the following embodiments are as follows:

[0097] MRS liquid medium ( / L): peptone 10 g, beef extract 10 g, yeast powder 5 g, anhydrous glucose 20 g, anhydrous sodium acetate 2 g, magnesium sulfate (MgSO4·7H2O) 0.5 g, manganese sulfate (MnSO4·H2O) 0.25 g, diammonium citrate 2 g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.6 g, Tween 80 1 mL, pH value 7.2 - 7.4.

[0098] MRS solid medium formula ( / L): peptone 10 g, beef extract 10 g, yeast powder 5 g, anhydrous glucose 20 g, anhydrous sodium acetate 2 g, magnesium sulfate (MgSO4·7H2O) 0.5 g, manganese sulfate (MnSO4·H2O) 0.25 g, diammonium citrate 2 g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.6 g, Tween 80 1 mL, agar 15 g, pH value 7.2 - 7.4.

[0099] Example 1 Screening and identification of Bifidobacterium bifidum CCFM1424

[0100] (1) Screening of Bifidobacterium bifidum CCFM1424:

[0101] Using the feces of healthy human bodies from the Chaoyang area of Beijing as samples, a certain amount of the samples was serially diluted with 5 mL of PBS (added with 0.05% cysteine). Dilution solutions with concentrations of 10 -7 ~10 -9 were spread on MRS solid medium containing 1% nystatin and mupirocin, and cultured at 37 °C for 48 h under anaerobic conditions. The colony morphology was observed and recorded. Single colonies were picked and streaked on MRS solid medium for purification, and then cultured in an inverted position in a 37 °C anaerobic incubator for 48 h. Several single colonies were picked and inoculated into 5 mL of MRS liquid medium. After culturing in a 37 °C anaerobic incubator for 24 h, 0.6 mL of the bacterial solution was taken and added to a preservation tube together with 0.6 mL of 60% sterile glycerol for preservation. At the same time, a 1.5 mL aliquot of the bacterial cells was centrifuged, the supernatant was removed, and the bacterial pellet was washed with 1.5 mL of sterile water. After centrifugation under the same conditions to remove the supernatant, 1 mL of sterile water was added for resuspension to prepare a bacterial suspension template for strain identification.

[0102] (2) Strain species identification

[0103] The above bacterial suspension template was used as the PCR template. The 50 μL reaction system for bacterial 16S rDNA: 2.5 μL of each of the 27F forward primer and 1492R reverse primer, 25 μL of Taq enzyme, 10 μL of the bacterial suspension template, and 10 μL of sterile water. PCR reaction conditions: ① 94 °C, 5 min; ② 94 °C, 30 s; ③ 55 °C, 30 s; ④ 72 °C, 1 min; ⑤ Steps 2 - 4 were repeated 30 cycles; ⑥ 72 °C, 10 min; ⑦ 12 °C, 2 min. The PCR amplification product was sent to a professional sequencing company and sequenced using a next-generation sequencer. The obtained sequence results were compared with nucleic acid sequences using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the similarity of the nucleic acid sequence with Bifidobacterium bifidum was as high as 99%, and it was named Bifidobacterium bifidum CCFM1424. The strain was stored at -80 °C in a refrigerator for standby.

[0104] Example 2: Cultivation of Bifidobacterium bifidum CCFM1424

[0105] The specific steps are as follows:

[0106] First, Bifidobacterium bifidum CCFM1424 and Bifidobacterium longum subsp. longum FSHHK13M1 (disclosed in Human gut-derived B. longum subsp. longum strains protect against aging in a D-galactose-induced aging mouse model [J]. Microbiome, 9(1):180 [2025-02-17]. DOI: 10.1186 / s40168-021-01108-8.) were activated in the first generation. They were respectively inoculated into MRS liquid medium at an inoculation amount of 4% and cultured in an anaerobic chamber at 37°C for 24 h. Then, fresh bacterial liquid was aspirated again at an inoculation amount of 4% and transferred into fresh MRS liquid medium for the second-generation culture. They were cultured for 24 h under the same conditions, and the bacterial cells were centrifuged at 8000×g for 10 min. After washing the bacterial cells with 0.9% physiological saline, they were centrifuged again at 8000×g for 10 min. The bacterial cells were collected, resuspended with 30% glycerol solution, and stored in a -80°C refrigerator for later use.

[0107] Preparation of the bacterial suspension for gavage: When Bifidobacterium bifidum and Bifidobacterium longum subsp. longum are used for gavage of mice, they are taken out from the -80°C refrigerator and centrifuged at 8000×g at 4°C for 10 min. The supernatant is discarded, and they are resuspended with 0.9% sterile physiological saline to obtain the probiotic suspension for gavage.

[0108] Example 3: Effect of Bifidobacterium bifidum CCFM1424 on the exploratory behavior of aging mice

[0109] Eight-week-old SPF-grade C57BL / 6J male mice were divided into 5 groups, namely the blank group, the model group, the experimental group (CCFM1424 group), the positive substance control group (arginine), and the positive strain control group (FSHHK13M1 group), with 6 mice in each group. They were raised in the Experimental Animal Center of Jiangnan University, fed with ordinary feed, at a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60 dB, and animal illuminance of 15-20 LX (all animal experiment procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0110] The animal experiment was divided into 8 weeks, with 1 week for the adaptation period and 7 weeks for the modeling and intervention period. Starting from the second week, mice in the model group, experimental group, and positive control groups (arginine group and FSHHK13M1 group) were subcutaneously injected with D-galactose (dose 1000 mg / kg BW / d) daily for modeling, while mice in the blank group were subcutaneously injected with an equal volume of sterile normal saline daily. The body weights of all mice were recorded weekly, and the dose of D-galactose was adjusted accordingly. At the same time, intervention was carried out in the second week. The experimental group and the positive strain control group were gavaged with 0.2 mL of the corresponding bacterial suspension with a viable bacteria count of 1×10 9 CFU / mL, the positive substance control group was gavaged with 0.2 mL of arginine at a concentration of 0.4 mg / g BW / d, and the model group and the blank group were gavaged with 0.2 mL of sterile normal saline. The gavage was carried out for 7 weeks in total. During this period, all groups had free access to water and food. The specific process of the animal experiment is shown in Figure 1 as follows.

[0111] Behavioral experiments were carried out in the eighth week. Open field test: The experiment was carried out in a square open field with dimensions of 40*40*35 cm in length, width, and height. The mice were placed in the center of the open field and allowed to move freely for 6 minutes. The proportion of the time the mice spent in the central area in the total duration was calculated. After each mouse completed the experiment, it was disinfected with 75% ethanol to avoid interference.

[0112] The results of the open field test are shown in Figure 2 as follows. It can be seen from Figure 2 (A) that compared with the mice in the model group, the activity trajectory lines of the mice in the blank group, experimental group, and positive control groups were more and denser, indicating that the mice in the blank group, experimental group, and positive control groups were more active in the open field. The percentages of the time the five groups of mice (blank group, model group, CCFM1424 group, arginine group, and FSHHK13M1 group) stayed in the central area were 25.44±5.57%, 6.43±0.43%, 15.05±0.4%, 14.08±1.92%, and 14.43±2.2% respectively. It can be seen from Figure 2 (B) that supplementing CCFM1424 can increase the time the aging mice stayed in the central area. The increase in the time staying in the central area is closely related to the exploratory behavior of the mice, indicating that supplementing CCFM1424 can improve the exploratory behavior of aging mice.

[0113] Example 4: Effect of Bifidobacterium bifidum CCFM1424 on the spatial learning and cognitive ability of aging mice

[0114] The animal experiment design was the same as that in Example 3.

[0115] Behavioral experiments were conducted in the 8th week. Morris water maze experiment: The water maze experiment was carried out in a circular bucket with a diameter of 120 cm and a height of 35 cm. A platform was placed at the midpoint of the third quadrant. The bucket was filled with water at 22 °C, just covering the platform by 1 cm, and powdered milk was added to the water to make it opaque. The water maze experiment included a 4-day consecutive place navigation experiment and a 1-day spatial exploration experiment. In the place navigation experiment, each mouse was trained 4 times a day, entering from the entry points in the four quadrants respectively, and the interval between each trial was 2 hours. The time (escape latency) taken by each mouse to find the hidden platform within 60 s was recorded. If the mouse could not find the platform within 60 s, the escape latency of the mouse was recorded as 60 s, and at the same time, the mouse was guided to the platform and observed standing on the platform for 10 s. On the fifth day, the hidden platform was removed for the spatial exploration experiment. Each mouse was placed into the water from the third quadrant, and information such as the number of times the mouse crossed the center of the target quadrant (where the platform was previously located) and the swimming trajectory within 60 s was recorded. During the experiment, information such as the number of times the experimental mice crossed the platform and the swimming trajectory was recorded using the overhead camera equipped with the device.

[0116] The Morris water maze is widely used to study the spatial learning and memory abilities of mice. Each group of mice underwent 4 consecutive days of place navigation training and their escape latencies were recorded. The results are shown in Figure 3 (A). From the data in the figure, it can be seen that as the training time extended, the escape latencies of each group of mice decreased. Compared with the model control group, the escape latencies of the CCFM1424 group on the 2nd and 3rd days were significantly reduced (p < 0.05), indicating that CCFM1424 intervention could significantly improve the decline in spatial learning ability caused by aging in mice. The spatial exploration experiment was carried out on the 5th day. The hidden platform was removed, and data on the escape latency, the number of times crossing the platform, and the total distance in the target quadrant of the mice within 60 s were recorded. The results are shown in Figure 3 (B - E). As can be seen from the figure, compared with the model group, the escape latency of the CCFM1424 group was significantly reduced (p < 0.01, a decrease of 82.9%), and the number of times the mice crossed the platform (p < 0.001, an increase of 300%), the total distance in the target quadrant (p < 0.01, an increase of 45.75%), and the cumulative duration in the target quadrant (p < 0.01, an increase of 118%) were significantly increased, indicating that CCFM1424 intervention could improve the cognitive function decline caused by aging.

[0117] Example 5: Effects of Bifidobacterium bifidum CCFM1424 on astrocytes in the brain tissue of aging mice

[0118] The animal experiment design was the same as that in Example 3.

[0119] After the experiment, the mice were dissected according to ethical requirements. The brain tissues were taken and fixed in 4% paraformaldehyde solution for 36 h. After gradient dehydration with ethanol, clearing with xylene, paraffin embedding, sectioning, and GFAP staining, the brain tissue sections were scanned under a microscope to observe the sectional conditions of the brain tissues.

[0120] Brain aging in mice is related to astrocytes. The aging of astrocytes can mediate neuroinflammation and induce learning and memory deficits. The immunohistochemical results of brain tissue sections are as Figure 4 shown. Compared with the blank control group, D-galactose induced the activation of astrocytes, showing a highly branched state and a large number. After treatment with CCFM1424, the number and activity of astrocytes decreased. The results indicate that CCFM1424 intervention can significantly improve the appearance of senescent astrocytes.

[0121] Example 6: Effects of Bifidobacterium bifidum CCFM1424 on Oxidative Stress in the Brain and Liver Tissues of Aging Mice

[0122] The animal experiment design was the same as that in Example 3.

[0123] After the experiment, the mice were dissected according to ethical requirements. The brain and liver tissues of the mice were weighed by amount, mixed with physiological saline at a ratio of 1:9, and broken using a tissue disruptor under the conditions of 65 Hz and 30 s (circulating 5 - 8 times). Finally, centrifugation was performed at 3000×g for 10 min, and the supernatant was taken to detect the activities of SOD (product number: A001 - 3 - 2, purchased from Nanjing Jiancheng), CAT (product number: A007 - 1 - 1, purchased from Nanjing Jiancheng), and GSH - Px (product number: A005 - 1 - 2, purchased from Nanjing Jiancheng) and the concentration of MDA (product number: A003 - 1 - 2, purchased from Nanjing Jiancheng) in the brain tissues through a kit. The protein concentration of the supernatant was measured using a BCA protein concentration assay kit. The results are shown in Figure 5-6 .

[0124] The content of MDA in the liver and brain tissues can indirectly reflect the degree of tissue oxidative damage, and the content of MDA is also a marker for testing the success of the aging model induced by D - galactose. Figure 5 (A) and Figure 6(A) showed that the MDA content in the brain and liver tissues of the model control group increased significantly (p<0.05), indicating the success of the D-galactose-induced aging model. It can be seen from the figure that compared with the model group, the MDA content in the CCFM1424 group decreased significantly (p<0.05, 44.36% decrease in brain tissue and 39.86% decrease in liver tissue), suggesting that CCFM1424 can reduce the MDA content in vivo, alleviate the degree of oxidative stress damage to cells, thereby playing an antioxidant role and delaying the aging process. The activities of CAT, SOD, and GSH-Px can reflect the ability of the body to scavenge free radicals, and the increase in the activities of these antioxidant enzymes shows an anti-aging effect. From Figure 5 (B)- Figure 5 (D) and Figure 6 (B)- Figure 6 (D), it can be seen that the activities of CAT, SOD, and GSH-Px in the brain and liver tissues of the senescent mice in the model group were significantly lower than those in the blank control group, indicating that the ability of the model group mice to scavenge free radicals was significantly reduced. However, CCFM1424 treatment can significantly increase the activities of CAT, SOD, and GSH-Px in the brain and liver tissues (the activities of antioxidant enzymes in liver tissue increased by 52.54%, 40.78%, and 49.23% respectively, and the activities of antioxidant enzymes in brain tissue increased by 131.56%, 51.79%, and 37.86% respectively), suggesting that CCFM1424 has a significant antioxidant function.

[0125] Example 7: Effects of Bifidobacterium bifidum CCFM1424 on the muscle tissue morphology and forelimb grip strength of senescent mice

[0126] The animal experiment design was the same as in Example 3.

[0127] At the 8th week, the grip strength test of mice was carried out: the mice were placed flat on the grid plate of the grip strength meter, the tail of the mice was grasped and slowly pulled backward, the data was read, and the experiment was repeated five times and the maximum value was recorded. At the end of the experiment, the equipment was wiped with 75% (v / v) ethanol to eliminate the influence of odor. The results are as Figure 7 shown.

[0128] After the experiment, the mice were dissected according to ethical requirements, and the tibialis anterior muscle of the mice was taken and fixed in an environmentally friendly GD fixing solution, embedded in paraffin, sectioned at a thickness of 5 mm, and stained with hematoxylin and eosin. The stained sections were observed using a pathological section scanner, and images were randomly collected. The results are as Figure 8 shown.

[0129] The grip strength results are as Figure 7As shown, the blank group, model group, CCFM1424 group, positive strain group, and positive substance group were 130.13 ± 9.08 N, 103.03 ± 7.82 N, 131.03 ± 5.35 N, 119.97 ± 8.08 N, and 122.07 ± 11.32 N, respectively. Compared with the blank group, the forelimb grip strength of the mice in the model group was significantly decreased (p < 0.05), and the forelimb grip strength of the mice in the CCFM1424 group was significantly increased compared with the model group (p < 0.05).

[0130] Figure 8 The H&E staining results of the tibialis anterior muscle of mice were shown. The muscle fiber structure of the mice in the blank group was arranged tightly and regularly in shape, with a polygonal cross-section and small gaps between muscle fibers; the muscle fibers of the mice in the model group were arranged disorderly, with irregular fiber shapes, and the gaps between fibers increased in some areas; after intervention with CCFM1424, positive strain, and positive substance, the pathological symptoms of the muscle tissues of senile mice were improved to varying degrees. The CCFM1424 group showed that the arrangement of muscle fibers was relatively restored to normal, with a relatively regular shape, the gaps between muscle fibers decreased, and the muscle fibers in some areas were restored to a tight arrangement, improving the muscle lesions in the model group to a certain extent.

[0131] Example 8: Effects of Bifidobacterium bifidum CCFM1424 on Serum Cytokines of Senile Mice

[0132] The animal experiment design was the same as that in Example 3.

[0133] After the experiment, in accordance with ethical requirements, the mice were sacrificed by exsanguination from the eyeballs and dissected. The mouse blood was allowed to stand for 2 h and then centrifuged at 3000 r / min for 15 min. The mouse serum was taken, and the levels of cytokines TNF-α (product number: SBJ-M0030-96T, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.), IL-1β (product number: SBJ-M0027-96T, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.), and IL-6 (product number: SBJ-M0657-96T, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.) in the mouse serum were measured using a kit (product number 439807, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.). The results are shown in Figure 9 as shown.

[0134] The concentration of serum TNF-α is shown in Figure 9 A, which is Figure 9From the quantitative results of A, the TNF-α concentration in the blank group was 200.29 ± 10.26 ng / L, while the TNF-α concentration in the model group was 268.98 ± 10.77 ng / L. After CCFM1424 intervention, the TNF-α concentration was 211.41 ± 9.47 ng / L, which was significantly lower than that in the model group (p < 0.05), a decrease of 21.4%. After mice were gavaged with the positive substance (TNF-α concentration was 212.62 ± 17.28 ng / L), it was also significantly lower than that in the model group (p < 0.05), a decrease of 20.95%. After gavage with the positive strain (TNF-α concentration was 229.59 ± 7.37 ng / L), it was also significantly lower than that in the model group, a decrease of 14.64%.

[0135] The serum IL-1β concentration is shown in Figure 9 B. The IL-1β concentration in the blank group was 47.94 ± 0.67 ng / L, while the IL-1β concentration in the model group was 56.4 ± 1.33 ng / L. After CCFM1424 intervention, the IL-1β concentration was significantly decreased (p < 0.05) to 45.73 ± 1.27 ng / L, a decrease of 18.92% compared with the model group. After mice were gavaged with the positive substance (IL-1β concentration was 46.73 ± 1.12 ng / L), it was also significantly lower than that in the model group (p < 0.05), a decrease of 10.99%. After gavage with the positive strain (IL-1β concentration was 52.5 ± 0.83 ng / L), it was also decreased compared with the model group, but there was no significant difference.

[0136] The serum IL-6 concentration is shown in Figure 9 C. After CCFM1424 intervention, the IL-6 concentration (concentration was 20.59 ± 1.85 pg / mL) was significantly lower than that in the model group (IL-6 concentration was 25.92 ± 1.12 pg / mL) (p < 0.01), a decrease of 20.56%. After mice were gavaged with the positive substance (IL-6 concentration was 21.27 ± 2.66 pg / mL) and the positive strain (IL-6 concentration was 22.48 ± 1.23 pg / mL), they were also decreased compared with the model group, but there was no significant difference.

[0137] Example 9: Effect of Bifidobacterium bifidum CCFM1424 on colonic inflammation in senile mice

[0138] The animal experiment design was the same as that in Example 3.

[0139] After the experiment, the mice were dissected according to ethical requirements, and 1 cm of the distal cecum of the colon was placed in 4% paraformaldehyde solution for fixation for 36 h. After gradient dehydration with ethanol, transparency with xylene, paraffin embedding, and section H&E staining, the colon section was observed by scanning.

[0140] As shown in Figure 10 Figure [Figure number not provided], there was focal aggregation of lymphocytes in the model group, while this phenomenon was significantly improved after CCFM1424 intervention. Therefore, CCFM1424 can effectively alleviate the severity of colon tissue pathology caused by aging.

[0141] Example 10: Effect of Bifidobacterium bifidum CCFM1424 on goblet cells in the colon of aging mice

[0142] The animal experiment design was the same as in Example 3.

[0143] After the experiment, the mice were dissected according to ethical requirements. A 1-cm segment of the distal cecum of the colon was removed and fixed in 4% paraformaldehyde solution for 36 h. After dehydration through a gradient of ethanol, clearing in xylene, embedding in paraffin, and sectioning for AB-PAS staining, the colon sections were scanned to observe the colon profile. The results are shown in Figure 11 .

[0144] As shown in Figure 11-12 Figure [Figure number not provided], the crypts of the blank mice were normal, and goblet cells were evenly distributed and abundant in number. In the model group, there was partial loss of goblet cells and partial depletion of mucin. After CCFM1424 intervention, the number of goblet cells in the colon tissue was significantly increased compared with the model group (p < 0.05). Therefore, CCFM1424 can more effectively alleviate the loss of goblet cells in aging mice.

[0145] Example 11: Effect of Bifidobacterium bifidum CCFM1424 on cytokines in the colon of aging mice

[0146] The animal experiment design was the same as in Example 3.

[0147] After the experiment, the mice were dissected according to ethical requirements. The colon tissue of the mice was weighed and mixed with PBS at a ratio of 1:9. The mixture was disrupted using a tissue homogenizer at 65 Hz for 30 s (with 5 - 8 cycles), and finally centrifuged at 3000×g for 10 min. The supernatant was used to detect the levels of cytokines TNF-α (product number: SBJ-M0030-96T, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.), IL-1β (product number: SBJ-M0027-96T, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.), IL-6 (product number: SBJ-M0657-96T, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.), and IL-10 (product number: SBJ-M0073-96T, purchased from Nanjing Senbeijia Biotechnology Co., Ltd.) in the colon tissue using an ELISA kit. The protein concentration of the supernatant was measured using a BCA protein concentration assay kit. The results are shown in Figure 13 .

[0148] The TNF-α concentration in colon tissue is shown in Figure 13 A. The TNF-α concentration in the blank group was 12.62 ± 1.7 ng / mg protein, while that in the model group was 23.68 ± 5.76 ng / mg protein. After CCFM1424 intervention, the TNF-α concentration was 12.83 ± 1.78 ng / mg protein, which was significantly lower than that in the model group (p < 0.05), a decrease of 45.82%. After mice were gavaged with the positive substance (TNF-α concentration was 13.27 ± 3.66 ng / mg protein), it was also significantly lower than that in the model group (p < 0.05), a decrease of 31.42%. After gavage with the positive strain (TNF-α concentration was 19.35 ± 3.66 ng / mg protein), there was also a decrease compared with the model group, but the difference was not significant.

[0149] The IL-1β concentration in colon tissue is shown in Figure 13 B. The IL-1β concentration in the blank group was 3.04 ± 0.5 ng / mg protein, while that in the model group was 6.11 ± 0.14 ng / mg protein. After CCFM1424 intervention, the IL-1β concentration decreased significantly (p < 0.05) to 3.84 ± 0.23 ng / mg protein, a decrease of 37.15% compared with the model group. After mice were gavaged with the positive substance (IL-1β concentration was 3.47 ± 0.54 ng / mg protein), it was also significantly lower than that in the model group (p < 0.05), a decrease of 31.15%. After intervention with the positive strain (IL-1β concentration was 5.04 ± 0.49 ng / mg protein), there was also a decrease compared with the model group, but the difference was not significant.

[0150] The IL-6 concentration in colon is shown in Figure 13 C. After CCFM1424 intervention, the IL-6 concentration (concentration was 2.09 ± 0.14 pg / mg protein) was significantly lower than that in the model group (IL-6 concentration was 3.42 ± 0.64 pg / mg protein) (p < 0.01), a decrease of 30.89%. After mice were gavaged with the positive substance (IL-6 concentration was 1.75 ± 0.57 pg / mg protein), it was also significantly lower than that in the model group (p < 0.05), a decrease of 39.02%. After intervention with the positive strain (IL-6 concentration was 2.87 ± 0.32 pg / mg protein), there was a decrease compared with the model group, but the difference was not significant.

[0151] The IL-10 concentration in colon tissue is shown in Figure 13For D, the IL-10 concentration in the blank group was 35.29 ± 5.23 pg / mg protein, while that in the model group was 18.68 ± 1.27 pg / mg protein. After CCFM1424 intervention, the IL-10 concentration increased significantly (p < 0.05) to 39.78 ± 5.42 pg / mg protein. After mice were intragastrically administered with a positive substance (IL-10 concentration was 34.03 ± 5.24 pg / mg protein), it also increased significantly compared with the model group (p < 0.05); while after intervention with a positive strain (IL-10 concentration was 24.83 ± 1.27 pg / mg protein), there was also an increase compared with the model group, but the difference was not significant.

[0152] Example 12: Effects of Bifidobacterium bifidum CCFM1424 on the fecal microbiota of aging mice

[0153] The animal experiment design was the same as that in Example 3.

[0154] Before the end of the experiment, mouse feces were collected for determination of fecal microbiota 16S rRNA. According to the operation instructions of the DNA extraction kit, DNA in the feces of the blank group, model group and CCFM1424 group was extracted. V3-V4 region PCR amplification was performed with primers (341F: 5’-CCTAYGGGRBGCASCAG-3’, 806R: 5’-GGACTACNNGGGTATCTAAT-3’). After detection by gel electrophoresis, the target band was recovered according to the QIAquick Gel Extraction Kit instruction manual, and then a sequencing library was constructed according to the sample DNA concentration. DNA sequencing was performed on the Illumina HiSeq platform and analyzed using QIIME2. Principal Component Analysis (PCA) was performed using the Liangchuan Cloud website (https: / / www.omicstudio.cn / tool), and Linear Discriminant Analysis (LDA) was performed on the website (https: / / www.bic.ac.cn / BIC / # / analysis). The selection of differential strains was based on the significance of LDA and t-test (p < 0.05 and LDA > 3). The results are as Figure 14-16 shown

[0155] α and β diversities were used to evaluate the diversity changes of fecal microbiota after CCFM1424 intervention ( Figure 14)。The α-diversity was characterized by the Shannon index and the Pielou index. The results showed that the α-diversity of the gut microbiota in senescent mice decreased significantly, while after administration of CCFM1424, the α-diversity of the gut microbiota in mice returned to normal. The β-diversity was analyzed by PCA, and the results indicated that there were significant differences among the blank group, the model group, and the CCFM1424 group (p = 0.001).

[0156] Figure 15 (A) shows the changes in the gut microbiota composition at the phylum level among the blank group, the model group, and the CCFM1424 group. The results indicated that significant changes occurred at the phylum level in D-galactose-induced senescent mice, mainly manifested as an increase in the relative abundances of Firmicutes and Verrucomicrobia, while the relative abundances of Bacteroidetes and Proteobacteria decreased. The intervention with CCFM1424 increased the relative abundances of Bacteroidetes and Proteobacteria and decreased the relative abundances of Firmicutes and Verrucomicrobia.

[0157] Figure 15 (B) demonstrates the changes in the gut microbiota composition at the genus level among the blank group, the model group, and the CCFM1424 group. It can be seen from the figure that the relative abundances of most genera changed. D-galactose-induced senescent mice decreased the relative abundances of beneficial bacteria such as Anaerotruncus and Prevotellaceae UCG-001, and increased the relative abundances of harmful bacteria such as Defluviitaleaceae UCG-011. Defluviitaleaceae UCG-011 has been reported to be related to colitis and is enriched in a mouse model of acute ulcerative colitis. After the intervention with CCFM1424, the relative abundances of beneficial bacteria such as Anaerotruncus and Prevotellaceae UCG-001 increased, while the harmful bacterium Defluviitaleaceae UCG-011 decreased after the CCFM1424 intervention. Anaerotruncus has been reported to be related to gut health and is positively correlated with the ability of microorganisms to synthesize spermidine (spermidine has anti-inflammatory, anti-aging and other effects). Prevotellaceae UCG-001 has been reported to participate in regulating the healthy metabolism of the host by affecting the production of short-chain fatty acids and activating related signaling pathways.

[0158] The differential genera of intestinal microbiota between the blank group and the model group, and between the model group and the CCFM1424 group were analyzed using LDA (p < 0.05 and LDA value > 3). It can be seen from the LDA score plot of the blank group and the model group ( Figure 16 (A)) that bacteria such as Prevotellaceae UCG-001, Clostridiaceae1_unclassified, and Clostridium sensustricto 1 were enriched in the blank group, while Faecalibaculum was enriched in the model group. It can be seen from the LDA score plot of the CCFM1424 group and the model group ( Figure 16 (B)) that beneficial bacteria such as Ruminococcus 1, Eubacterium ruminantium group, and Dubosiella were enriched in the CCFM1424 group, while opportunistic pathogens such as Enterococcus were enriched in the model group. Bacteria such as Eubacterium ruminantium group and Dubosiella have been reported to be one of the main producers of short-chain fatty acids, and short-chain fatty acids play an important role in reducing inflammation and improving cognition.

[0159] Example 13: Effects of Bifidobacterium bifidum CCFM1424 on fecal metabolites of aging mice

[0160] The animal experiment design was the same as that in Example 3.

[0161] Mouse feces were collected before the end of the experiment for untargeted metabolomics determination. Weighed 20 mg of fecal samples into 1.5 mL centrifuge tubes, added 200 μL of H2O for homogenization (68 Hz, 15 s, 5 times), added 800 μL of an equal volume of methanol-acetonitrile mixed solution (pre-cooled at -20 °C in advance) to precipitate proteins, vortexed for 30 s, then sonicated in an ice bath (4 °C) for 30 min, placed the samples in a -20 °C refrigerator for incubation for 1 h for secondary precipitation, centrifuged at 15000 rpm at 4 °C for 15 min, took the supernatant into a 2 mL centrifuge tube and evaporated to dryness using a rotary evaporator, re-dissolved with an acetonitrile and aqueous solution with a volume ratio of 1:1, centrifuged at 15000 rpm at high speed for 15 min at 4 °C, transferred an appropriate volume and loaded it into an injection vial for on-machine detection. Untargeted metabolomics determination was performed using liquid chromatography-mass spectrometry. The chromatographic column used was an ACQUITY UPLC BEH Amide column, and the column temperature was 40 °C. The mobile phases were: phase A was an aqueous solution of 25 mM ammonium hydroxide and 25 mM ammonium acetate, and phase B was acetonitrile. The flow rate was set at 0.3 mL / min, and the injection volume of the extracted sample was 2 μL. Data were processed using Compound Discoverer 3.3 software (Thermo Fisher Scientific, USA). Sparse partial least squares discriminant analysis (sPLS-DA), random forest, volcano plot, clustering, and pathway enrichment analysis were performed using MetaboAnalyst 6.0 (https: / / www.metaboanalyst.ca), as Figure 17-19 shown.

[0162] After data preprocessing by screening metabolites with an mzCloud score greater than 80, removing duplicate metabolites in different databases, and metabolites with unidentified names, a total of 232 relatively clear metabolites were finally obtained in the blank group, model group, and CCFM1424 group. From Figure 17 the sPLS-DA plot, it can be seen that there are obvious differences among the blank group, model group, and CCFM1424 group, indicating that D-galactose-induced aging has a certain effect on the metabolites in feces, and CCFM1424 intervention also significantly affects the composition of metabolites.

[0163] Volcano plot analysis showed that there were a total of 14 metabolites with significant differences between the blank group and the model group (FC > 1.5 or < 0.83 and p < 0.05). D-galactose-induced aging caused a decrease in 9 metabolites such as D-sphingosine and 4-hydroxybenzoic acid, and an increase in 5 metabolites such as succinic acid and α-lactose ( Figure 18 ). And CCFM1424 intervention could increase the level of D-sphingosine ( Figure 19)。D-sphingosine has been reported to be involved in signal transduction and neuroprotection in nerve cells. These results indicate that aging induced by D-galactose leads to changes in metabolites in feces. In addition, an increase in spermidine was also observed after the intervention of CCFM1424 compared with the model group. Studies have shown that spermidine plays a key role in maintaining cell health, delaying aging, improving neurodegenerative diseases, and preventing a variety of aging-related diseases.

[0164] In summary, the probiotic provided by the present invention has good effects in alleviating cognitive impairment, colon inflammation, muscle damage caused by aging, and improving the changes in the gut microbiota and gut metabolites caused by aging.

[0165] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Bifidobacterium bifidum CCFM1424, characterized in that, The Bifidobacterium bifidum CCFM1424 was deposited at the Guangdong Provincial Culture Collection of Microorganisms on September 20, 2024, with the deposit number GDMCC No: 65151.

2. A microbial preparation containing the Bifidobacterium bifidum CCFM1424 described in claim 1.

3. The microbial agent according to claim 2, wherein In the microbial preparation, the concentration of Bifidobacterium bifidum CCFM1424 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A product, characterized in that, The product contains the Bifidobacterium bifidum CCFM1424 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. The product according to claim 4, wherein The product is a food, and the food is a dairy product, soy product or fruit and vegetable product fermented and produced using the Bifidobacterium bifidum CCFM1424 described in claim 1 or the microbial preparation described in claim 2 or 3; Or the food is a beverage or snack containing the Bifidobacterium bifidum CCFM1424 described in claim 1 or the microbial preparation described in claim 2 or 3.

6. Use of the Bifidobacterium bifidum CCFM1424 described in claim 1 or the microbial preparation described in claim 2 or 3 in the preparation of a drug for alleviating aging.

7. The application according to claim 5, characterized in that, The alleviation of aging includes the effects of at least one of the following (a) to (k): (a) Can significantly alleviate the decline in cognitive ability caused by aging; (b) Improve the morphology of astrocytes in the brain tissue of aging individuals; (c) Significantly alleviate oxidative stress in the brains and livers of elderly individuals, and the antioxidant indexes include at least one of superoxide dismutase, catalase, malondialdehyde or glutathione peroxidase; (d) Reduce the levels of pro-inflammatory factors in the serum of elderly individuals, and the pro-inflammatory factors include at least one of TNF-α, IL-1β or IL-6; (e) Improve the morphology of muscle tissue in aging individuals; (f) Increase the grasping force of elderly individuals; (g) Reduce the infiltration of inflammatory cells and the number of goblet cells in the colon tissue of elderly individuals; (h) Reduce the levels of pro-inflammatory factors in the colon tissue of elderly individuals, and the pro-inflammatory factors include at least one of TNF-α, IL-1β or IL-6; (i) Increase the levels of anti-inflammatory factors in the colon tissue of elderly individuals, and the anti-inflammatory factors include IL-10; (j) Improve the disorder of the gut microbiota caused by aging; (k) Improve the disorder of gut metabolites caused by aging.

8. Use of the Bifidobacterium bifidum CCFM1424 described in claim 1 or the microbial preparation described in claim 2 or 3 in the preparation of a drug for improving the gut microbiota and gut metabolite disorders caused by aging.

9. The application according to claim 8, wherein The improvement of gut microbiota disorders caused by aging refers to upregulating the relative abundances of beneficial bacteria such as Anaerotruncus, Prevotellaceae UCG-001, Eubacterium ruminantium group, and Dubosiella, and downregulating the relative abundances of opportunistic pathogens such as Defluviitaleaceae UCG-011 and Enterococcus.

10. The application according to claim 8, characterized in that, The improvement of gut metabolite disorders caused by aging refers to upregulating the contents of spermidine and / or D-sphingosine and downregulating the content of adenine.

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