Application of bifidobacterium breve in production of vitamins or preparation of anti-aging products

Abstract

The invention relates to application of bifidobacterium breve in production of vitamins or preparation of anti-aging products, and belongs to the technical field of biological medicines. The invention provides an application of bifidobacterium breve XA-1267 in production of vitamins or preparation of products for supplementing vitamins. Researches show that the bifidobacterium breve XA-1267 can produce vitamin B3 at high yield, which shows that the strain can be used for producing vitamins and preparing products (such as probiotic products) for supplementing vitamins. Animal experiments prove that the bifidobacterium breve XA-1267 can improve the level of an anti-aging marker in an aging model mouse body, improve the muscle function and immune disorder condition of the aging model mouse body, relieve the oxidative stress degree in the aging model mouse body, reduce the uric acid level in the aging model mouse body and reverse metabolic disorder in the aging model mouse body. A product for supplementing vitamins prepared from the strain can improve core symptoms and accompanying symptoms of aging, and has an anti-aging function.

Description

Technical Field

[0001] This invention relates to the application of Bifidobacterium breve in the production of vitamins or the preparation of anti-aging products, and belongs to the field of biomedical technology. Background Technology

[0002] Niacin, the most common form of vitamin B3, is a water-soluble vitamin widely found in various foods and is one of the essential nutrients for maintaining normal metabolism and physiological functions. Niacin participates in various redox reactions in the body, supporting the energy metabolism of carbohydrates, fats, and amino acids. It also plays an important role in maintaining skin health, nervous system function, and the integrity of the digestive tract epithelium. When dietary intake of niacin or its metabolic precursors (such as tryptophan) is insufficient, the body cannot synthesize enough coenzyme molecules to maintain normal metabolism, leading to niacin deficiency (also known as vitamin B3 deficiency). Niacin deficiency is clinically characterized by dermatitis, glossitis, enteritis, mental abnormalities, and peripheral neuritis; in severe cases, it can lead to death. Therefore, timely niacin supplementation is necessary to prevent its occurrence and progression.

[0003] Aging refers to the decline of normal physiological functions in an organism. It is a complex physiological process driven by genetic, cellular damage, and environmental factors. The aging process is accompanied by the gradual decline of multiple system functions, including cellular senescence, loss of protein homeostasis, gut microbiota dysbiosis, and chronic inflammation. With the increasing trend of population aging, the economic burden of aging and age-related diseases is rising. Therefore, anti-aging and improving the quality of life for the elderly have become important needs.

[0004] Among the factors contributing to aging, weakened intestinal absorption leads to a gradual decline in the body's utilization of vitamins, resulting in niacin deficiency. Furthermore, numerous studies have confirmed that niacin is not only an essential vitamin for maintaining metabolic homeostasis, but long-term niacin deficiency may also promote the occurrence and development of age-related diseases. Therefore, a dynamic, mutually reinforcing relationship exists between aging and niacin deficiency, making timely niacin supplementation crucial for treating niacin deficiency and combating aging.

[0005] Currently, the main approach to increasing niacin levels is through exogenous supplementation, such as direct oral administration of niacin or nicotinamide supplements. However, studies have shown that direct niacin supplementation has certain limitations. First, the absorption and conversion efficiency of orally administered niacin in the body is significantly affected by individual differences, gut microbiota status, and liver metabolic capacity, leading to significant fluctuations in its bioavailability. Second, high-dose niacin intake can cause a series of adverse reactions, such as skin flushing, itching, and liver damage, limiting its safety in long-term supplementation. Therefore, there is an urgent need to find products that can more safely and effectively increase the body's niacin levels to improve niacin deficiency and achieve anti-aging effects. Summary of the Invention

[0006] To address the above problems, this invention provides a strain of Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve The application of XA-1267 in the production of vitamins or the preparation of vitamin supplements, wherein the Bifidobacterium breve is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 26614.

[0007] In one embodiment of the present invention, the vitamin supplement product has any of the following functions: (a) Prevention and / or treatment of vitamin deficiencies; (b) Anti-aging; (c) and / or, prevention and / or treatment of age-related diseases.

[0008] In one embodiment of the present invention, the vitamin includes vitamin B3 (i.e., niacin). The vitamin deficiency mentioned includes vitamin B3 deficiency (i.e., niacin deficiency). The age-related diseases include age-related immune dysregulation and / or age-related uric acid elevation. The age-related immune dysregulation diseases include inflammation; the age-related uric acid elevation diseases include hyperuricemia, gout, and / or nephritis.

[0009] In one embodiment of the present invention, the anti-aging includes increasing the levels of anti-aging biomarkers, improving age-related muscle function decline, improving age-related immune dysregulation, alleviating age-related oxidative stress, reducing age-related uric acid elevation, and / or reversing age-related metabolic disorders; the aging biomarkers include nicotinamide adenine dinucleotide (NAD). + ).

[0010] In one embodiment of the present invention, the improvement of anti-aging biomarker levels includes improving anti-aging biomarker levels by increasing nicotinamide adenine dinucleotide levels, increasing nicotinamide adenine dinucleotide precursor levels, and / or improving tryptophan metabolism. The improvement in age-related muscle function decline includes improving grip strength; The improvement of age-related immune dysregulation includes improving age-related immune dysregulation by increasing the levels of anti-inflammatory factors and / or decreasing the levels of pro-inflammatory factors. The mitigation of aging-induced oxidative stress includes mitigating aging-induced oxidative stress by reducing the levels of oxidative stress products and / or increasing the levels of antioxidants. The reversal of aging-induced metabolic disorders includes reversing aging-induced metabolic disorders by improving aging-related metabolic pathways.

[0011] In one embodiment of the present invention, increasing the level of nicotinamide adenine dinucleotide precursor includes increasing the level of vitamin B3 (i.e., niacin) and / or increasing the level of trigonelline.

[0012] In one embodiment of the present invention, the improvement of tryptophan metabolism includes increasing the level of anthranilic acid and / or increasing the level of indole-3-propionic acid (IPA).

[0013] In one embodiment of the present invention, the anti-inflammatory factor includes β-sitosterol; the pro-inflammatory factor includes 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and / or palmitic acid.

[0014] In one embodiment of the present invention, the oxidative stress product includes malondialdehyde; the antioxidant includes glutathione.

[0015] In one embodiment of the present invention, the aging-related metabolic pathways include the metabolic pathways of nicotinic acid and nicotinamide metabolism, the metabolic pathway of methylhistidine metabolism, the metabolic pathway of β-alanine metabolism, and / or the metabolic pathway of the malate-aspartate shuttle system.

[0016] In one embodiment of the present invention, the product is a probiotic product; the probiotic product includes probiotic preparations and freeze-dried powder; the probiotic preparations include liquid preparations, powders, drops, capsule preparations, granules, tablets and / or powders.

[0017] In one embodiment of the present invention, the product comprises live, inactivated and / or metabolites of Bifidobacterium breve.

[0018] In one embodiment of the present invention, the viable count of Bifidobacterium breve in the product is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0019] In one embodiment of the present invention, the product further comprises excipients.

[0020] In one embodiment of the present invention, the excipients include a carrier, solvent, propellant, solubilizer, co-solvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesion agent, binding agent, penetration enhancer, pH adjuster, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, encapsulating agent, humectant, absorbent, diluent, flocculant and anti-flocculation agent, filter aid and / or release inhibitor.

[0021] In one embodiment of the present invention, the carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0022] The technical solution of this invention has the following advantages: This invention provides the application of *Bifidobacterium breve* XA-1267 in the production of vitamins or the preparation of vitamin supplements. Studies have shown that *Bifidobacterium breve* XA-1267 can produce high levels of vitamin B3 (niacin), indicating that this strain can be used for vitamin production and the preparation of vitamin supplements (such as probiotic products). Based on this, animal experiments have confirmed that *Bifidobacterium breve* XA-1267 can significantly increase the levels of anti-aging markers in aging model mice (including by increasing the levels of nicotinamide adenine dinucleotide, trigonelline (a precursor to nicotinamide adenine dinucleotide), and improving tryptophan metabolism, thereby increasing the levels of anti-aging markers in aging model mice), significantly improve muscle function in aging model mice (including improving grip strength in the forelimbs and hindlimbs), and significantly improve immune dysregulation in aging model mice (including by increasing the levels of anti-inflammatory factors such as β-sitosterol and reducing the levels of other anti-inflammatory factors). The strain significantly reduced the levels of pro-inflammatory factors such as 9,10-dihydroxy-12Z-octadecenoic acid and palmitic acid in mice, thereby improving immune dysregulation in aging model mice. It also significantly alleviated oxidative stress in aging model mice (including by reducing the levels of oxidative stress products such as malondialdehyde and increasing the levels of antioxidants such as glutathione, thereby alleviating aging-induced oxidative stress responses), significantly reduced uric acid levels in aging model mice, and effectively reversed aging-induced metabolic disorders. Therefore, the vitamin supplements prepared from this strain can significantly improve the core and accompanying symptoms of aging, possess anti-aging functions, and have great application potential. Attached Figure Description

[0023] Figure 1 : The concentration of nicotinic acid, a metabolite produced in vitro by different strains.

[0024] Figure 2Animal experiment flowchart.

[0025] Figure 3 : Nicotinamide adenine dinucleotide levels in mice from different groups.

[0026] Figure 4 Trigonelline levels in mice from different groups.

[0027] Figure 5 Forelimb tension in mice from different groups.

[0028] Figure 6 : Hind limb tension in mice of different groups.

[0029] Figure 7 The levels of anthranilic acid in mice from different groups.

[0030] Figure 8 : Levels of IL-10 cytokine secreted by PBMC cells in different groups.

[0031] Figure 9 : Levels of IL-13 cytokine secreted by PBMC cells in different groups.

[0032] Figure 10 : Levels of IL-4 cytokine secreted by PBMC cells in different groups.

[0033] Figure 11 Indole-3-propionic acid levels in mice from different groups.

[0034] Figure 12 β-sitosterol levels in mice from different groups.

[0035] Figure 13 : Levels of 9,10-dihydroxy-12Z-octadecenoic acid in mice from different groups.

[0036] Figure 14 Palmitic acid levels in mice from different groups.

[0037] Figure 15 : Malondialdehyde levels in mice from different groups.

[0038] Figure 16 : Glutathione levels in mice from different groups.

[0039] Figure 17 Uric acid levels in mice from different groups.

[0040] Figure 18 PCA analysis results of metabolomics in mice from different groups (cations).

[0041] Figure 19PCA analysis results of metabolomics in mice from different groups (anions).

[0042] Figure 20 Pathway analysis results (cations) of metabolomics in different groups of mice.

[0043] Figure 21 Pathway analysis results of metabolomics in mice from different groups (anions).

[0044] Figures 3-19 In the mean squares, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001. Detailed Implementation

[0045] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0046] For experiments in the following examples where specific experimental steps or conditions are not specified, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. The *Bifidobacterium breve* XA-1267 involved in the following examples is described in the patent authorization text with publication number CN117431190B.

[0047] Experiment Example 1: Gene screening of probiotics with high niacin production capacity In recent years, the close relationship between gut microbiota and aging has received widespread attention (see "Wilmanski, Diener et al. 2021"). A team from the University of Pennsylvania discovered that microbes carry... pcnA Gene-expressed nicotinamide / pyrazinamidinase can promote the conversion of nicotinamide to nicotinic acid by microorganisms through deamidation (see "Chellappa, McReynolds et al. 2022" and "Feng, Guo et al. 2023"). Therefore, the genome carries... pncA Strains possessing genes and high expression activity of nicotinamide / pyrazinamidinase have a strong ability to synthesize nicotinic acid, which can be achieved through microbial... pncA Sequence feature analysis of genes can be used to predict the activity of their encoded proteins, enabling the screening and discovery of probiotics that produce high levels of niacin.

[0048] This experimental example involves sequencing the genomes of a series of bacteria to obtain their genomic sequences, and then targeting the genomic sequences... pncAThe genes were analyzed, and multi-dimensional data prediction and integration based on protein sequence similarity and three-dimensional structural similarity were performed to infer the activity of the encoded proteins, thereby screening for optimal strains. The specific experimental procedure is as follows: Strain DNA was extracted using the SDS method, and the quality of the extracted DNA was determined by agarose gel electrophoresis. The DNA was then analyzed using Qubit. ® 2.0 Quantification of extracted DNA concentration was performed. Bacterial genome sequencing was conducted at Novogene Co., Ltd., using the Illumina platform to construct 350bp small fragment libraries for next-generation sequencing of the bacterial genome. Next-generation sequencing data were assembled using SPAdes 3.15.3 to facilitate subsequent gene prediction. A database was constructed using pncA protein sequences downloaded from the UniProt database.

[0049] In prediction pncA When determining the activity of gene-encoded proteins, gene prediction is first performed using protein sequence alignment. Protein sequence similarity is predicted using the software Kofamscan (v1.3.0), with Kofamscan scores used as the predictive criterion for similarity. Kofamscan is a tool for protein sequence analysis that can predict protein function and classification by comparing protein sequences with entries in the KEGG Orthology (KO) database (see "Aramaki, Takuya et al. 2020").

[0050] Furthermore, with the improved accuracy of AI algorithm models in predicting protein structures, using protein three-dimensional structural similarity comparison has become a new method for predicting the existence of specific proteins. AI-driven structure prediction not only improves the efficiency of structure comparison but also enables the analysis of large-scale genomic data. The PLMSearch method (https: / / dmiip.sjtu.edu.cn / PLMSearch#, see the literature "Liu, Wei et al. 2024", a software that utilizes protein language model representation for ultrafast and sensitive homology search, widely used in genome research and functional screening of new proteins) was employed. Using structural similarity >90% and a comparison score >100 as criteria, the target protein structure of candidate strains was predicted and scored.

[0051] Based on the above method, this experimental example analyzed all Bifidobacterium breve strains in The XBIOME GUT BIOBANK (XGB), a strain library independently isolated and constructed by Shenzhen Unknown Biotechnology Co., Ltd. Bifidobacterium breve The analysis and screening were conducted, and all candidate strains were analyzed based on their genome sequences. pncAThe activity of encoded proteins was predicted and scored (only the scores of the top fifteen strains were extracted, as shown in Table 1), and the results are as follows: Bioinformatics predictive analysis results indicate that the candidate strains screened in the list exhibit high activity in nicotinic acid synthesis-related pathways, suggesting potential application value in high-yield nicotinic acid production and anti-aging. However, considering that the above conclusions are based on computational models and database comparisons, and that bioinformatics screening only provides a certain reference for predicting strain function, the scoring results may not necessarily be completely consistent with the actual efficacy rankings in experiments. Further in vitro and animal experiments are needed to verify the specific biological functions and efficacy of these candidate strains.

[0052] Table 1. Probiotic genome screening based on pncA protein sequence and structural similarity.

[0053] Experiment Example 2: Detection of the ability of Bifidobacterium breve to produce nicotinic acid in vitro In Example 1, by predicting and integrating multi-dimensional data on protein sequence similarity and three-dimensional structural similarity, a method was developed targeting microorganisms. pncA The activity of the encoded proteins was predicted and screened using genes. The selected candidate strains possessed the potential to express highly active nicotinamide / pyrazinamidinase, thus potentially exhibiting anti-aging functions through high nicotinic acid production. This provided a list of candidate strains for further verification of the anti-aging function of probiotics. However, since the results of bioinformatics analysis require multi-dimensional verification, Example 1 was based on microorganisms... pncA The scoring and ranking of gene-encoded protein activity is only one indicator of a strain's ability to produce nicotinic acid. Therefore, the top fifteen candidate strains selected in Example 1 were further validated through in vitro nicotinic acid production assays. The specific experimental procedure is as follows: 1. Preparation of bacterial cell supernatant test sample One mL of the candidate strain's culture was inoculated into 25 mL of MRS medium (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number M8540) and anaerobically cultured at 37°C for 18 h to obtain the activated solution. The activated solution was then inoculated into modified GAM medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) at an inoculation rate of 4% (v / v) and anaerobically cultured at 37°C for 18 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000g for 10 min at room temperature (25°C). In a biosafety cabinet, the supernatant of each sample was transferred to a new centrifuge tube. The supernatant sample was aspirated with a syringe and filtered through a 0.22 μm filter membrane to obtain the supernatant sample.

[0054] 2. Detection of nicotinic acid concentration in supernatant samples Remove the supernatant sample from the -80℃ freezer, freeze-thaw at room temperature, resuspend the pipette tip, and perform serial dilutions to 400-fold: add 50 μL of supernatant sample to 950 μL of sample diluent to obtain a 20-fold dilution; add 50 μL of the 20-fold diluted supernatant sample to 200 μL of sample diluent to obtain a 100-fold dilution; add 200 μL of the 100-fold diluted supernatant sample to 600 μL of sample diluent to obtain a 400-fold dilution; use a Vitamin B3 (niacin and nicotinamide) detection kit (purchased from Shenzhen Ruisai Biotechnology Co., Ltd.). Limited Company (product number RS-VA-005), add 150 μL of test medium, 150 μL of positive control (nicotinamide), and 150 μL of sample diluted 400 times to the microwells respectively. Cover the strip or microwell with sealing film to ensure complete sealing. Incubate at 37°C in the dark for 24 h. After incubation, carefully tear off the sealing film and use a pipette tip to break up all foam on the liquid surface. Read the turbidity OD value at 610 nm using an ELISA reader. Subtract the test medium data (baseline) from the sample test data, and then calculate the nicotinic acid content according to the standard curve.

[0055] The test results for nicotinic acid content are as follows: Figure 1 As shown, compared with the control culture medium, some candidate strains demonstrated the ability to produce niacin, with candidate strain XA-1267 exhibiting the most outstanding performance, producing a large amount of niacin. Other *Bifidobacterium breve* strains, such as strains 8-15, showed very poor niacin production, or even produced almost no niacin at all. Therefore, *Bifidobacterium breve* XA-1267 possesses a strong ability to produce niacin, which can more effectively increase the level of niacin in the body, thereby supplementing niacin and further achieving the goal of anti-aging.

[0056] Experimental Example 3: Effects of Bifidobacterium breve XA-1267 on aging mouse models To further clarify the anti-aging effects and mechanisms of action of Bifidobacterium breve XA-1267, this experiment used aging model mice as experimental animals to investigate the effects of Bifidobacterium breve XA-1267 on the levels of anti-aging markers, muscle function, immune regulation, oxidative stress, uric acid levels, and metabolomics in vivo. The specific experimental procedure is as follows: 1. Preparation of bacterial suspension test samples 1 mL of the preserved culture of Bifidobacterium breve XA-1267 was inoculated into 25 mL of modified GAM medium and anaerobically cultured at 37°C for 18 h to obtain the activated solution. The activated solution was inoculated into modified GAM medium at an inoculation rate of 4% (v / v) and anaerobically cultured at 37°C for 18 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000g for 10 min at room temperature (25°C) and the bacterial cells were collected. The bacterial cells were washed with D-phosphate buffer (product number G4200-500M, purchased from Wuhan Saiweier Biotechnology Co., Ltd.) at 1 / 10 of the fermentation broth volume and centrifuged again at 8000g for 10 min at room temperature (25°C) and the washed bacterial cells were collected. 10% (v / v) glycerol aqueous solution at 1 / 10 of the fermentation broth volume was added to the washed bacterial cells to obtain the bacterial suspension. The bacterial suspension was dispensed into 2 mL centrifuge tubes, 1 mL per tube, and stored at -80°C.

[0057] Before administration via gavage, remove the bacterial suspension from the -80°C freezer, thaw it by incubating at 37°C for 5 min, and centrifuge at 8000g for 5 min at room temperature (25°C) to collect the revived bacterial cells. Add an equal volume of phosphate buffer containing 1% (w / v, g / 100mL) L-cysteine ​​hydrochloride (catalog number G4202-500mL, purchased from Wuhan Saiweier Biotechnology Co., Ltd.) to the revived bacterial cells, wash the cells, and centrifuge again at 8000g for 10 min at room temperature (25°C) to collect the washed revived bacterial cells. Resuspend the washed revived bacterial cells in phosphate buffer containing 1% (w / v, g / 100mL) L-cysteine ​​hydrochloride to a bacterial concentration of 2×10⁻⁶. 10 CFU / mL was used to obtain the bacterial solution for gavage.

[0058] 2. Laboratory animals Mice aged 18-24 months correspond to elderly individuals aged 56-69 years, at which point various biomarkers begin to change, making them suitable for aging-related studies. With increasing age and entering old age, older mice exhibit hair loss; multiple physiological functions begin to decline, muscle strength gradually decreases, muscle atrophy leads to slow movement and incoordination, and cognitive decline are also observed.

[0059] Thirty male 20-month-old SPF-grade C57BL / 6J mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used. The SPF-grade mice were housed in individually ventilated cages, with a stocking density of 5 mice per cage. All mice were ear-tagged and numbered. The ambient temperature and humidity were maintained at 23±1°C and 50±10%, respectively, with an automatic light-dark cycle of 12 hours. Unless otherwise specified in the experimental methods below, the mice had free access to food and water. The entire animal experiment lasted 28 days, and the specific experimental schedule is as follows: Figure 2 .

[0060] 3. Bowel preparation and drug administration in laboratory animals 3.1 Antibiotic bowel cleansing in mice Antibiotic bowel cleansing was performed on mice. The specific steps for antibiotic bowel cleansing were as follows: Vancomycin (Sigma Aldrich, CAS No. 1404-93-9), neomycin (Sigma Aldrich, CAS No. 1405-10-3), and metronidazole (Sigma Aldrich, CAS No. 443-48-1) were dissolved in sterile water and administered orally to the mice. The oral dose of vancomycin was 50 mg / kg, neomycin was 100 mg / kg, and metronidazole was 100 mg / kg. The total solution volume administered was 0.1 mL per 10 g of body weight. The solutions were freshly prepared daily and administered orally every 12 hours for 7 consecutive days. The dosage was determined based on the mouse's body weight on the day of administration.

[0061] During the antibiotic bowel cleansing period in mice (7 days), a 1 mg / mL ampicillin solution was prepared using sterile drinking water and changed every three days. During the antibiotic bowel cleansing period in mice (7 days), all procedures must be performed in a biosafety cabinet, including bedding changes, drinking water changes, food addition, and gavage. It is forbidden to open the cage lid outside the biosafety cabinet or expose the mice to the space outside the biosafety cabinet.

[0062] 3.2 Grouping of mice and administration of drugs by gavage Thirty mice were randomly divided into a negative control group (PBS), a positive control group (NMN), and a probiotic XA-1267 intervention group (XA-1267). In the probiotic XA-1267 intervention group, mice were administered Bifidobacterium breve XA-1267 bacterial suspension by gavage at a fixed time each day at a volume of 0.1 mL / 10 g body weight. The dose of Bifidobacterium breve XA-1267 administered by gavage to each mouse was approximately 4 × 10⁻⁶. 8 CFU. The positive control group was administered a solution of NMN (Manton Biotechnology Co., Ltd., CAS No. 1094-61-7) (soluble in PBS buffer) at a fixed time each day, at a dose of 0.1 mL / 10 g body weight. Each mouse received 200 mg / kg of NMN via gavage. The control group received an equal volume of PBS buffer via gavage. Both groups were administered the solution continuously for 21 days. Mouse bedding was changed twice weekly in a sterile operating room.

[0063] 4. Effects of Bifidobacterium breve XA-1267 on aged mice 4.1. Effects of Bifidobacterium breve XA-1267 on nicotinamide adenine dinucleotide (NAD) in aged mice. + The influence of level The aging process of the body is closely related to the dynamic changes in nicotinamide adenine dinucleotide (NADP) levels in the body. Therefore, NADP can serve as an anti-aging biomarker, and the detection of NADP levels in the body has become a key evaluation indicator for the development of anti-aging products. Although detecting NADP levels based on in vitro cell models can provide preliminary screening for anti-aging effects, the results are limited by the fact that cell culture cannot truly simulate the complex microenvironment and metabolic homeostasis changes in the body, making it difficult to accurately reflect the true kinetic changes of NADP. Therefore, in order to accurately evaluate the anti-aging effect of Bifidobacterium breve XA-1267, this experiment directly detected the changes in NADP levels in aged mice after intervention, providing more valuable experimental evidence for evaluating the anti-aging effect of XA-1267.

[0064] 4.1.1 Detection Method Upon reaching the experimental endpoint, blood samples were collected from mice via the iris and placed in plastic blood collection tubes containing EDTA anticoagulant. The blood samples were processed by the analyzer: 5 μL of sample was thoroughly mixed with 4 times its volume of 0.5N (equivalent concentration) perchloric acid to lyse the sample. The lysate was then diluted 10-fold with a buffer solution containing 500 mM HEPES and 500 mM NaCl (pH 7.2). 10 μL of the diluted solution was added to 90 μL of measurement buffer containing 0.5 nM sensor, 30 μM furazine, 50 mM HEPES, and 50 mM NaCl (pH 7.2). The sensor's bioluminescence signal was detected using the analyzer's built-in photon detector (Hamamatsu Photonics): the NLuc luminescence signal was detected at a wavelength of 460 nm (bandwidth 30 nm), and the RFP luminescence signal was detected at a wavelength of 580 nm (bandwidth 20 nm). The concentration of nicotinamide adenine dinucleotide (NADNU) was calculated based on the titration curve and the emission ratio (R) of the sensor at 580 nm to 460 nm. Detection was performed using a standard NADNU solution of known concentration as described above. Referring to the molecular tool developed by Professor Kai Johnsson for the analysis and measurement of metabolites in blood (“see Yu, Qiuliyang et al. 2018”), the sensor emission ratio (R) was fitted to the NADNU concentration, and the maximum sensor ratio (Rmax) was obtained based on the Hill-Langmuir equation. max ), minimum ratio (R) min The half-effective concentration (c50) and Hill coefficient (h) were also considered. The concentration of nicotinamide adenine dinucleotide in unknown samples was calculated using the following equation: .

[0065] 4.1.2 Experimental Results The detection results of nicotinamide adenine dinucleotide concentration are as follows: Figure 3 As shown, the mean change in nicotinamide adenine dinucleotide (NDR) levels in mice before and after intervention in the negative control group was around 1, indicating no significant change in NDR levels (FC=1.02). In contrast, the mean change in NDR levels in mice before and after NMN intervention in the positive control group was greater than 1 (FC=1.33), and significantly higher than that in the negative control group (P < 0.001, t.test). Similarly, the mean change in NDR levels in mice before and after XA-1267 intervention in the probiotic XA-1267 group was also greater than 1 (FC = 1.22), and significantly higher than that in the negative control group (P = 0.046, t.test). There was no significant difference in NDR levels between the NMN group and the XA-1267 group (P = 0.18, t.test). These results indicate that Bifidobacterium breve XA-1267 can target the nicotinamide adenine dinucleotide synthesis pathway in the body, significantly increasing the concentration of nicotinamide adenine dinucleotide, an anti-aging marker, in aging model mice, and has great application potential in anti-aging treatment.

[0066] 4.2 Effects of Bifidobacterium breve XA-1267 on trigonelline levels in aged mice 4.2.1 Detection Method Upon reaching the experimental endpoint, blood samples were collected from mice via the iris and placed in plastic blood collection tubes containing EDTA anticoagulant. After thawing at 4°C, 100 μL of the blood sample was added to 300 μL of a methanol / acetonitrile / water solution (2:2:1, v / v) pre-cooled to 4°C, vortexed, sonicated at 4°C for 30 min, allowed to stand at -20°C for 10 min, and centrifuged at 14000g at 4°C for 20 min. The supernatant was then vacuum-dried to obtain the sample. For mass spectrometry analysis, 300 μL of acetonitrile aqueous solution (acetonitrile:water = 1:1, v / v) was added to the sample to reconstitute it, vortexed, and centrifuged at 14000g at 4°C for 15 min. The supernatant was then injected into a HILIC column of an ultra-high performance liquid chromatography (UHPLC) system for separation and analysis.

[0067] 4.2.2 Experimental Results Trigonelline is a plant alkaloid with multiple biological activities (see Membrez, Migliavacca et al. 2024). Supplementation with trigonelline can rapidly increase the level of nicotinamide adenine dinucleotide in the body. Furthermore, trigonelline levels are positively correlated with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle; increased levels can enhance muscle capacity in mice and prevent fatigue during aging, demonstrating the potential of increased levels of this metabolite to treat age-related muscle decline.

[0068] The test results of trigonelline concentration are as follows: Figure 4 As shown, the trigonelline content in mice before and after NMN intervention was significantly higher in the positive control group than in the negative control group (FC positive control group / negative control group = 2.2, P negative control group VS positive control group = 0.04, t.test). The effect of XA-1267 intervention was even more pronounced, with trigonelline content in mice significantly higher than in the negative control group and significantly better than in the positive control group (FC XA-1267 experimental group / negative control group = 4.0, P negative control group VS XA-1267 experimental group = 0.02, t.test). These results indicate that *Bifidobacterium breve* XA-1267 has a regulatory effect on trigonelline concentration and can significantly promote the endogenous secretion of trigonelline in the body, showing great promise for anti-aging treatment.

[0069] 4.3 Effects of Bifidobacterium breve XA-1267 on muscle function in aged mice 4.3.1 Detection Method Forelimb maximum pulling force test: First, calibrate and zero-point adjust the rat and mouse grip dynamometer. Place the mouse horizontally above the grid, keeping the trunk horizontal, with the forelimbs fully placed on the grip dynamometer test rod. Hold the mouse by the tail and apply maximum force to the mouse tail with the test rod parallel to it, causing the mouse to completely detach from the test rod. Record the maximum pulling force of the mouse. Test 3 times a day, with an interval of more than 30 minutes between each test.

[0070] Hind limb grip strength test: First, calibrate and zero-point adjust the grip strength meter for both mice and rats. Place the mouse horizontally above the grid while keeping its trunk horizontal, allowing its hind limbs to grip the test bar. Apply maximum force to the mouse's tail with the test bar parallel to the test bar until the mouse completely detaches from the test bar. Record the maximum pulling force of the mouse. Test 3 times a day, with an interval of more than 30 minutes between each test.

[0071] 4.3.2 Experimental Results Muscle function decline is a prominent feature of aging, characterized by progressive reduction in muscle mass, strength, and function. It has become a significant factor threatening the health of the elderly, potentially leading to disability or even death. Therefore, evaluating the level of aging from the perspective of improving muscle function is of great importance. Currently, measuring the grip strength of aged mice provides a direct way to assess changes in their muscle ability.

[0072] The results of the maximum tensile force test of the forelimb are as follows Figure 5 As shown, compared with the negative control group, no significant change was observed in the forelimb pulling strength of mice before and after NMN intervention in the positive control group (FC positive control group / negative control group = 1.08, P negative control group VS positive control group = 0.18, t.test). However, the forelimb pulling strength of mice in the XA-1267 intervention group was significantly higher than that of the negative control group (FC XA-1267 experimental group / negative control group = 1.31, P negative control group VS XA-1267 experimental group = 4 × 10⁻⁶). -4 The experimental group (t.test) was significantly better than the positive control group (FC XA-1267 experimental group / positive control group = 1.22, P XA-1267 experimental group VS positive control group = 8.3×10). -3 ,t.test).

[0073] The results of the hind limb grip strength test are as follows: Figure 6 As shown, compared with the negative control group, the forelimb pulling strength of mice in the positive control NMN intervention group was significantly better than that of the control group (FC positive control group / negative control group = 1.11, P negative control group VS positive control group = 0.027, t.test). The forelimb pulling strength of mice in the XA-1267 intervention group was also significantly higher than that of the negative control group (FC XA-1267 experimental group / negative control group = 1.19, P negative control group VS XA-1267 experimental group = 0.005, t.test), and there was no significant difference with the NMN group (FC XA-1267 experimental group / positive control group = 1.08, P XA-1267 experimental group VS positive control group = 0.12, t.test).

[0074] The above results confirm that intervention with Bifidobacterium breve XA-1267 can significantly improve the forelimb and hindlimb muscle strength in aging model mice. From the perspective of improving muscle function, Bifidobacterium breve XA-1267 has great application potential in anti-aging and muscle capacity improvement treatment.

[0075] 4.4 Effects of Bifidobacterium breve XA-1267 on tryptophan metabolism in aged mice 4.4.1 Detection Method The concentration of anthranilic acid in blood samples was determined according to the method described in section 4.2.1.

[0076] 4.4.2 Experimental Results Tryptophan is an essential amino acid whose metabolic pathway plays a crucial role in various physiological environments. With further research, it has been discovered that tryptophan can be metabolized through metabolic pathways to synthesize nicotinamide adenine dinucleotide, an anti-aging marker that plays an important role in aging (see "Covarrubias, Perrone et al. 2021"). Anthranilic acid is a key intermediate metabolite in the tryptophan metabolic pathway, and it interacts with NAD+. + Synthesis, immune regulation, and metabolism of neuroactive substances are closely related (see "Mondanelli, Iacono et al. 2019").

[0077] The detection results of anthranilic acid concentration are as follows: Figure 7 As shown, the anthranilic acid (ANCA) content in mice in the positive control NMN intervention group was significantly higher than that in the negative control group (FC positive control group / negative control group = 2.2, P negative control group VS positive control group = 0.03, t.test). Similarly, the ANCA content in mice in the XA-1267 intervention group was also significantly higher than that in the negative control group (FCXA-1267 experimental group / negative control group = 3.4, P negative control group VS XA-1267 experimental group = 0.02, t.test). These results indicate that *Bifidobacterium breve* XA-1267 can regulate the concentration of ANCA, a metabolite related to nicotinamide adenine dinucleotide (NADP), indirectly supplementing the body's NADP by significantly increasing key substances in the tryptophan metabolic pathway, thereby improving metabolism in aging animal models and demonstrating great application potential in anti-aging treatment.

[0078] 4.5 Effects of Bifidobacterium breve XA-1267 on immune regulation 4.5.1 Effects of Bifidobacterium breve XA-1267 on immune regulation of PBMC cells PBMCs are a mixed population of immune cells isolated from human peripheral blood, mainly consisting of lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. It is one of the most commonly used and classic in vitro immune research models because it is readily available, rich in key immune cells, functionally active, and highly correlated with the human immune status, thus realistically simulating the body's immune response. By detecting changes in PBMC status, secretion of related immune factors, and cell differentiation trends, the effects of relevant drug stimulation on immune regulation can be analyzed.

[0079] 4.5.1.1 Detection Method After removing PBMC cells (purchased from Shanghai Aoneng Company) from the liquid nitrogen tank, they were thawed by shaking in a 37°C water bath for 3 minutes to obtain thawed PBMC cells. The thawed PBMC cells were resuspended in 5 mL of PBS buffer preheated to 37°C, centrifuged at 400xg for 10 minutes, and the supernatant was discarded to obtain precipitate A. Precipitate A was then resuspended in 5 mL of cell culture medium (90% RPMI 1640 medium + 10% fetal bovine serum + 50 μg / mL penicillin-dextrin antibiotics, where % refers to volume ratio, RPMI...). Cells were resuspended in 1640 medium (purchased from Gibco), fetal bovine serum (purchased from Solarbio), and penicillin antibody (purchased from Gibco), and cell counts and viability were performed to obtain resuspension A. 8 mL of cell culture medium was added to resuspension A, and the mixture was incubated at 5% (v / v) CO2 and 37°C for 6 h to obtain culture medium. The culture medium was centrifuged at 400 x g for 10 min, the supernatant was discarded, and cell counts were performed to obtain precipitate B. Precipitate B was resuspended in cell culture medium to a concentration of 1 × 10⁻⁶. 6 The cells / mL yielded resuspension B.

[0080] First, 100 μL of resuspension solution B was added to each well of a 96-well plate. Cells were then divided into three groups: an LPS group (LPS added, purchased from Sigma-Aldrich, final LPS concentration 1 μg / mL), a PHA group (PHA added, final PHA concentration 2.5 μg / mL), and an untreated group. The LPS and PHA groups were activated by LPS / PHA stimulation of PBMCs from a resting state to an activated state, allowing for subsequent observation of the cells' immune response to *Bifidobacterium breve* XA-1267. After LPS / PHA addition, the 96-well plates were incubated at 5% (v / v) CO2 at 37°C for 2 hours. After incubation, cells containing 1 × 10⁻⁶ cells were added to the plates. 7 A 10 μL / well solution of *Bifidobacterium breve* XA-1267 (in physiological saline) was added to each well of a 96-well plate. A positive control and a blank control (CON) were also included. The positive control group received an equal volume of the positive control drug mesylate (20 mg / mL), while the blank control group received an equal volume of physiological saline. After the addition of *Bifidobacterium breve* XA-1267 and mesylate, the 96-well plates were incubated anaerobically at 37°C for 2 hours. After incubation, the viable *Bifidobacterium breve* XA-2103 cells were washed away from the 96-well plates with PBS buffer containing 10% (m / v) antibiotics. Fresh RPMI 1640 medium was added to each well at 125 μL, and the plates were incubated for another 22 hours at 37°C with 5% (v / v) CO2. After incubation, the supernatant was collected by centrifugation, and the cytokine levels in the supernatant were detected using a flow cytometry kit (from Biolegend).

[0081] 4.5.1.2 Experimental Results IL-10 is a key anti-inflammatory factor regulating innate immunity and inflammatory responses. It plays a crucial role in immune regulation primarily by inhibiting the expression of NF-κB pathway-dependent pro-inflammatory factors (such as TNF, IL-6, IL-1β, and IL-12β) in monocytes, macrophages, and dendritic cells (see "Mishra, Bikash et al. 2025"). The effect of Bifidobacterium breve XA-1267 on the immune response can be analyzed by detecting the IL-10 secreted by PBMC cells. Results are as follows... Figure 8 As shown, XA-1267 intervention can stimulate PBMC secretion of IL-10. Compared with the blank culture medium control, XA-1267 stimulation significantly increased IL-10 secretion (FC XA-1267 experimental group / blank control group = 4.1, P XA-1267 experimental group / blank control group = 0.0474, one-way ANOVA). Under LPS stimulation, compared with the blank culture medium control, XA-1267 showed the same trend as the positive control, significantly increasing IL-10 secretion (FC XA-1267 experimental group / blank control group = 15.7, P XA-1267 experimental group / blank control group < 0.001, one-way ANOVA). These results indicate that Bifidobacterium breve XA-1267 can have an anti-inflammatory effect on immune cells PBMCs. Under LPS induction, it can significantly promote the expression and secretion of the anti-inflammatory factor IL-10, thereby improving the inflammatory response and showing great application potential in immunomodulatory therapy.

[0082] IL-13 possesses significant anti-inflammatory and immunomodulatory effects, particularly in acute inflammatory environments, tissue repair, and metabolic homeostasis. Primarily produced by Th2 cells, IL-13 participates in the entire inflammatory response, influencing its development and outcome (see "de Vries, J E. 1998"), and plays a crucial role in immune regulation. By detecting the IL-13 levels secreted by PBMC cells, the effect of Bifidobacterium breve XA-1267 on the immune response can be analyzed. Results are as follows... Figure 9As shown, XA-1267 intervention can stimulate PBMC secretion of IL-13. Compared with the blank culture medium control, XA-1267 stimulation significantly increased the secretion of IL-10 (P: XA-1267 experimental group / blank control group < 0.001, one-way ANOVA). Under PHA stimulation, compared with the blank culture medium control, XA-1267 stimulation significantly increased the secretion of IL-13 (FC: XA-1267 experimental group / blank control group = 2.5, P: XA-1267 experimental group / blank control group = 0.0117, one-way ANOVA). These results indicate that Bifidobacterium breve XA-1267 can have an anti-inflammatory effect on immune cells PBMCs. Under PHA induction, it can significantly promote the expression and secretion of the anti-inflammatory factor IL-13, thereby improving the inflammatory response and showing great application potential in immunomodulatory therapy.

[0083] IL-4 is a pleiotropic cytokine mainly produced by activated T lymphocytes, mast cells, and basophils. It plays a crucial role in immune regulation by modulating immune cells to alleviate chronic inflammation and promoting the polarization of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages (see "Zhou, Guangyin et al. 2025"). By detecting the IL-4 secreted by PBMCs, the effect of Bifidobacterium breve XA-1267 on the immune response can be analyzed. Results are as follows... Figure 10 As shown, XA-1267 intervention can stimulate PBMC secretion of IL-4. Compared with the blank culture medium control, XA-1267 showed the same trend as the positive control, with XA-1267 stimulation increasing IL-4 secretion (FC XA-1267 experimental group / blank control group = 1.5). Under PHA stimulation, XA-1267 stimulation also increased IL-4 secretion compared with the blank culture medium control (FC XA-1267 experimental group / blank control group = 1.3). These results indicate that Bifidobacterium breve XA-1267 can have an anti-inflammatory effect on immune cells PBMCs. Under PHA induction, it can significantly promote the expression and secretion of the anti-inflammatory factor IL-4, thereby improving the inflammatory response and showing great application potential in immunomodulatory therapy.

[0084] 4.5.2 Effects of Bifidobacterium breve XA-1267 on immune regulation in aging mouse models 4.5.2.1 Detection Method The concentrations of indole-3-propionic acid, β-sitosterol, 9,10-dihydroxy-12Z-octadecenoic acid and palmitic acid in blood samples were determined according to the method described in section 4.2.1.

[0085] 4.5.2.2 Experimental Results Aging is characterized by systemic chronic inflammation, accompanied by cellular senescence, immune senescence, and organ dysfunction. Inflammation is now widely recognized as one of the core drivers of aging, and the accumulation of intracellular inflammation is closely related to the occurrence and development of aging. Targeting the inflammatory network is a potential target for anti-aging; therefore, intervention against pro-inflammatory / anti-inflammatory factors related to chronic inflammation holds promise for delaying aging.

[0086] Indole-3-propionic acid (IPA) is a tryptophan metabolite produced by gut microbes. It can regulate immune responses by binding to aryl receptors or heat shock proteins, thereby reducing the occurrence of inflammatory responses, and is an anti-inflammatory factor (see "Gao, Sun et al. 2025").

[0087] The detection results of indole-3-propionic acid concentration are as follows: Figure 11 As shown, the indole-3-propionic acid content in mice in the positive control NMN intervention group was higher than that in the negative control group (FC positive control group / negative control group = 1.1), while the indole-3-propionic acid content in mice in the XA-1267 intervention group was significantly higher than that in the negative control group (FC XA-1267 experimental group / negative control group = 3.5, P negative control group VS XA-1267 experimental group = 2.77 × 10⁻⁶). -3 The experimental group (t.test) and the positive control NMN intervention group (FC XA-1267 experimental group VS positive control group = 3.1, P XA-1267 experimental group VS positive control group = 4.33×10) were compared with the positive control NMN intervention group (FC XA-1267 experimental group VS positive control group = 4.33×10). -3 (t.test). This result indicates that Bifidobacterium breve XA-1267 has a significant regulatory effect on the concentration of the anti-inflammatory factor indole-3-propionic acid.

[0088] β-sitosterol is a naturally occurring plant sterol that inhibits the activity of metalloproteinases and has significant anti-inflammatory and antioxidant effects. It is an anti-inflammatory factor (see "Khan, Nath et al. 2022").

[0089] The results of the β-sitosterol concentration detection are as follows: Figure 12As shown, the β-sitosterol content in mice in the positive control NMN intervention group was higher than that in the negative control group (FC positive control group / negative control group = 1.2), while the β-sitosterol content in mice in the XA-1267 intervention group was significantly higher than that in the negative control group (FC XA-1267 experimental group / negative control group = 2.2, P negative control group vs XA-1267 experimental group = 0.01, t.test) and the positive control NMN intervention group (FC XA-1267 experimental group vs positive control group = 1.8, P XA-1267 experimental group vs positive control group = 0.01, t.test). These results indicate that *Bifidobacterium breve* XA-1267 has a regulatory effect on the concentration of the pro-inflammatory factor β-sitosterol.

[0090] 9,10-Dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) is an endogenous lipid mediator produced from linoleic acid (LA) via the cytochrome P450 (CYP) cyclooxygenase pathway, playing a complex role in inflammation, metabolism, and damage repair (see "Hildreth, Kodani et al. 2020"). High concentrations of 9,10-DiHOME may induce immune stress, thereby mediating inflammatory responses. 9,10-DiHOME may also cause mitochondrial swelling, affecting cellular function, and is a pro-inflammatory factor.

[0091] The detection results of the concentration of 9,10-dihydroxy-12Z-octadecenoic acid are as follows: Figure 13 As shown, the level of 9,10-dihydroxy-12Z-octadecenoic acid in mice in the positive control NMN intervention group was lower than that in the negative control group (FC positive control group / negative control group = 0.6, P negative control group VS positive control group = 0.01, t.test), while the level of 9,10-dihydroxy-12Z-octadecenoic acid in mice in the XA-1267 intervention group was also significantly lower than that in the negative control group (FCXA-1267 experimental group / negative control group = 0.5, P negative control group VS XA-1267 experimental group = 0.01, t.test). These results indicate that *Bifidobacterium breve* XA-1267 has a regulatory effect on the concentration of the pro-inflammatory factor 9,10-dihydroxy-12Z-octadecenoic acid.

[0092] Palmitic acid is a saturated fatty acid that acts as an intracellular signaling molecule in the occurrence and development of various diseases, leading to increased inflammatory responses. It may also promote the accumulation of intracellular ROS, causing apoptosis, and is a pro-inflammatory factor (see the literature "Korbecki and Bajdak-Rusinek 2019").

[0093] The results of palmitic acid concentration detection are as follows: Figure 14As shown, the palmitic acid content in mice in the positive control NMN intervention group was lower than that in the negative control group (FC positive control group / negative control group = 0.6, P negative control group VS positive control group = 1.03 × 10⁻⁶). -3 (t.test), and the palmitic acid content in the mice in the XA-1267 intervention group was also significantly lower than that in the negative control group (FC XA-1267 experimental group / negative control group = 0.5, P negative control group VS XA-1267 experimental group = 6.84×10). -4 (t.test). This result indicates that Bifidobacterium breve XA-1267 has a regulatory effect on the concentration of the pro-inflammatory factor palmitic acid.

[0094] The above results confirm that intervention with Bifidobacterium breve XA-1267 can alleviate inflammation, target the inflammatory network of aging organisms, and thus improve immune dysregulation in aging animal models, showing great application potential in anti-aging treatment.

[0095] 4.6 Effects of Bifidobacterium breve XA-1267 on oxidative stress levels in aging mouse models 4.6.1 Detection Method Upon reaching the experimental endpoint, mice were anesthetized by intraperitoneal injection of sodium pentobarbital and subsequently euthanized by cervical dislocation. The skin of the hind legs was removed to fully expose the gastrocnemius muscle, which was then completely isolated. Physiological saline was added to the gastrocnemius muscle at a ratio of 100 mg of gastrocnemius muscle to 400 μL of physiological saline. The tissue was ground at 4°C and centrifuged to collect the supernatant suspension. The protein concentration in the tissue sample was determined using a BCA assay kit (Shanghai Beyotime Biotechnology Co., Ltd., Cat No. P0010), the glutathione concentration using a total glutathione (T-GSH) / oxidized glutathione (GSSG) colorimetric assay kit (Elabscience, Cat No. E-BC-K097-M), and the malondialdehyde (MDA) concentration using a malondialdehyde (MDA) colorimetric assay kit (Elabscience, Cat No. E-BC-K025-M).

[0096] 4.6.2 Experimental Results Numerous studies have shown that oxidative stress is one of the core drivers of aging. Long-term oxidative stress not only accelerates the activation of cellular senescence signaling pathways but also forms a mutually reinforcing positive feedback loop with chronic inflammation, jointly propelling the aging process. Therefore, alleviating oxidative stress and restoring the homeostasis of the antioxidant system is considered one of the important strategies for delaying aging.

[0097] Malondialdehyde (MDA) is the end product of cell membrane lipid peroxidation, and an increase in its content indicates a greater degree of oxidative damage in the body (see reference "Muluye, Rekik A et al., 2016").

[0098] The test results for malondialdehyde concentration are as follows: Figure 15 As shown, the in vivo malondialdehyde (MDA) content in the positive control NMN intervention group was lower than that in the negative control group (FC positive control group / negative control group = 0.88), while the in vivo MDA content in the XA-1267 intervention group was significantly lower than that in the negative control group (FC XA-1267 experimental group / negative control group = 0.54, P negative control group vs XA-1267 experimental group = 0.004, one-way ANOVA) and the positive control NMN intervention group (FC XA-1267 experimental group vs positive control group = 0.61, P XA-1267 experimental group vs positive control group = 0.0287, one-way ANOVA). These results indicate that *Bifidobacterium breve* XA-1267 has a significant regulatory effect on the oxidative stress product MDA and can alleviate oxidative stress responses in the body.

[0099] Glutathione (GSH) is the main non-enzymatic antioxidant in cells, which can scavenge free radicals and maintain redox homeostasis. A decrease in its content reflects a weakening of antioxidant capacity (Reference "Chen, Mingming et al., 2022").

[0100] The results of glutathione concentration detection are as follows: Figure 16 As shown, the glutathione level in mice in the positive control NMN intervention group was significantly higher than that in the negative control group (FC positive control group / negative control group = 1.32, P positive control group VS negative control group = 0.025, one-way ANOVA), while the glutathione level in mice in the XA-1267 intervention group was significantly higher than that in the negative control group (FC XA-1267 experimental group / negative control group = 1.49, P negative control group VS XA-1267 experimental group = 0.0011, one-way ANOVA). These results indicate that *Bifidobacterium breve* XA-1267 has a significant regulatory effect on the non-enzymatic antioxidant glutathione and can enhance the body's antioxidant stress response.

[0101] 4.7 Effects of Bifidobacterium breve XA-1267 on uric acid levels in aged mice 4.7.1 Detection Method The concentration of uric acid in the blood sample was determined by the method described in section 4.2.1.

[0102] 4.7.2 Experimental Results Uric acid is the final product of purine metabolism in the human body and is mainly excreted through the kidneys. With increasing age, the body's metabolic capacity declines, especially kidney function, leading to a gradual accumulation of uric acid in the body (see "Han, Yao et al. 2025"). Hyperuricemia is closely related to gout; high concentrations of uric acid can induce crystal deposition in joints and kidneys, causing kidney damage and leading to a series of complications such as nephritis. Simultaneously, high uric acid is also considered a key influencing factor for cardiovascular and metabolic diseases. According to the "Multidisciplinary Expert Consensus on the Diagnosis and Treatment of Hyperuricemia-Related Diseases in China (2023 Edition)," the incidence of hyperuricemia in the elderly population continues to rise with age, and high uric acid levels in the body can also continuously trigger inflammatory responses.

[0103] The test results for uric acid concentration are as follows: Figure 17 As shown, the uric acid content in mice in the positive control NMN intervention group was lower than that in the negative control group (FC positive control group / negative control group = 0.2, P negative control group VS positive control group = 0.01, t.test), while the uric acid content in mice in the XA-1267 intervention group was also significantly lower than that in the negative control group (FC XA-1267 experimental group / negative control group = 0.2, P negative control group VS XA-1267 experimental group = 0.01, t.test). These results indicate that *Bifidobacterium breve* XA-1267 can regulate the concentration of the metabolite uric acid, significantly reduce uric acid levels in the body, alleviate metabolic disorders in aged mice, and show great promise for anti-aging treatment.

[0104] 4.8 Effects of Bifidobacterium breve XA-1267 on metabolomics in aged mice 4.8.1 Detection Method Supernatant was extracted from mouse blood samples and metabolomics analysis was performed according to the method described in section 4.2.1. The analysis method is as follows: Metabolomics PCoA analysis: PCoA (principal coordinates analysis) is a visualization method for studying the similarity or difference of data. It directly performs eigenvalue decomposition on the distance matrix between samples and is an unconstrained data dimensionality reduction analysis method that can be used to study the similarity or dissimilarity of sample community composition. Based on the processed data, beta diversity analysis is performed, and PCoA plots are drawn to observe the distribution between groups.

[0105] Metabolic pathway analysis: To further focus on specific metabolic pathways and construct biomarker metabolic pathways, the specific analysis method was as follows: The collected blood biomarker metabolic profiles were input into the MetPA dialog box. The Iuput (Input) tab was selected, followed by either the HMDB or KEGG label (metabolite name number). Submit was clicked, and the species was selected: Mus musculus (mouse). Hypergeometric Test and Pathway Topology Analysis were performed. Relative-betweeness Centrality was used, followed by registration for pathway model analysis. Signal pathway analysis was performed using the KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.kegg.jp / ) database. The HMDB (The Human Metabolome Database, http: / / www.hmdb.ca / ) database was used to annotate metabolite molecules, analyze related enzymes or transport proteins and their properties. The MetPA network software (MetaboAnalyst 6.0) was used to visualize the metabolite pathways.

[0106] 4.8.2 Detection Method The results of the metabolomics PCoA analysis are as follows: Figure 18 and Figure 19 As shown, there were significant differences in species composition among the aging model group, the positive drug group, and the XA-1267 group under anion mode (PERMANOVA P = 0.045). The aging model group and the positive drug control group showed clear spatial separation on the PCoA plot, while the metabolic profile of the XA-1267 probiotic intervention group significantly converged with that of the positive drug control group. Its data points formed a clear cluster boundary with the aging model, indicating that intervention with Bifidobacterium breve XA-1267 can significantly reverse aging-induced metabolic disorders.

[0107] The results of the KEGG pathway enrichment analysis are as follows: Figure 20 and Figure 21 As shown, XA-1267 probiotic intervention significantly reversed age-related metabolic disorders. In the cationic mode, niacin and nicotinamide metabolism, along with methylhistidine metabolism, were the core targets. Niacin and nicotinamide are coenzymes NAD+. + and NADP + A key precursor to NAD+, it plays a crucial role in cellular energy metabolism and redox balance. Upregulating this pathway can improve the body's ability to maintain NAD+ levels. +Concentration and improvement of age-related indicators. Methylhistidine is a specific biomarker for skeletal muscle protein breakdown and is closely related to muscle metabolism. Improving this pathway can enhance age-related exercise indicators. In anionic mode, β-alanine metabolism and the malate-aspartate shuttle system are key targets. β-alanine is a key precursor for carnosine synthesis, playing a dominant role in maintaining muscle pH homeostasis and resistance to exercise fatigue. Improving this metabolic pathway is expected to improve exercise endurance and performance indicators. The malate-aspartate shuttle system is a central hub for cytoplasmic NADH reoxidation, dominating transmembrane electron transport in eukaryotic cell energy metabolism. Upregulation of this metabolic pathway can improve the body's maintenance of NAD. + Concentration, thereby improving aging-related indicators.

[0108] The above results confirm that Bifidobacterium breve XA-1267 can improve metabolic disorders in aging model mice, upregulate key aging-related metabolic pathways, and promote the restoration of metabolic homeostasis in aging organisms.

[0109] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Bifidobacterium breve ( Bifidobacterium breve Its application in the production of vitamins or the preparation of vitamin supplements is characterized by, The aforementioned Bifidobacterium breve is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26614.

2. The application as described in claim 1, characterized in that, The vitamin supplement product has any of the following functions: (a) Prevention and / or treatment of vitamin deficiencies; (b) Anti-aging; (c) and / or, prevention and / or treatment of age-related diseases.

3. The application as described in claim 2, characterized in that, The vitamins mentioned include vitamin B3; The vitamin deficiency mentioned includes vitamin B3 deficiency; The age-related diseases include age-related immune dysregulation and / or age-related uric acid elevation. The age-related immune dysregulation diseases include inflammation; the age-related uric acid elevation diseases include hyperuricemia, gout, and / or nephritis.

4. The application as described in claim 2, characterized in that, The anti-aging measures include increasing the levels of anti-aging biomarkers, improving age-related muscle function decline, improving age-related immune dysregulation, alleviating age-related oxidative stress, reducing age-related uric acid elevation, and / or reversing age-related metabolic disorders; the age biomarkers include nicotinamide adenine dinucleotide.

5. The application as described in claim 4, characterized in that, The improvement of anti-aging biomarker levels includes improving anti-aging biomarker levels by increasing nicotinamide adenine dinucleotide levels, increasing nicotinamide adenine dinucleotide precursor levels, and / or improving tryptophan metabolism. The improvement in age-related muscle function decline includes improving grip strength; The improvement of age-related immune dysregulation includes improving age-related immune dysregulation by increasing the levels of anti-inflammatory factors and / or decreasing the levels of pro-inflammatory factors. The mitigation of aging-induced oxidative stress includes mitigating aging-induced oxidative stress by reducing the levels of oxidative stress products and / or increasing the levels of antioxidants. The reversal of aging-induced metabolic disorders includes reversing aging-induced metabolic disorders by improving aging-related metabolic pathways.

6. The application as described in claim 5, characterized in that, The increase in nicotinamide adenine dinucleotide precursor levels includes increasing vitamin B3 levels and / or increasing trigonelline levels.

7. The application as described in claim 5, characterized in that, The improvement in tryptophan metabolism includes increasing levels of anthranilic acid and / or increasing levels of indole-3-propionic acid.

8. The application as described in claim 5, characterized in that, The anti-inflammatory factor includes β-sitosterol; the pro-inflammatory factor includes 9,10-dihydroxy-12Z-octadecenoic acid and / or palmitic acid.

9. The application as described in claim 5, characterized in that, The oxidative stress products include malondialdehyde; the antioxidants include glutathione.

10. The application as described in claim 5, characterized in that, The aging-related metabolic pathways include the metabolic pathways of nicotinic acid and nicotinamide metabolism, the metabolic pathway of methylhistidine metabolism, the metabolic pathway of β-alanine metabolism, and / or the metabolic pathway of the malate-aspartate shuttle system.

11. The application as described in claims 1 to 10, characterized in that, The product is a probiotic product; the probiotic product includes probiotic preparations and freeze-dried powder; the probiotic preparation includes liquid preparations, powders, drops, capsules, granules, tablets and / or powders.

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