Abstract: This study aimed to analyze the metabolite composition of water kefir fermented by a mixed bacterial culture and to predict its anti-aging potential, thereby providing theoretical support for the development of functional kefir beverages. Ultra-high performance liquid chromatography-Q Exactive HF-X was employed for untargeted metabolomic analysis of fermentation products to systematically identify metabolite components. The potential anti-aging mechanisms of key metabolites were explored by combined network pharmacology and molecular docking. The results showed that a total of 748 differential metabolites were identified, and 288 significantly upregulated metabolites were selected via principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Network pharmacology identified 10 core bioactive components, 348 metabolite targets, and 159 disease-related intersection targets. Gene Ontology (GO) enrichment analysis revealed 1 691 biological processes, 134 cellular components, and 259 molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis obtained 186 related pathways, and 6 core targets: RAC-alpha serine/threonine-protein kinase, estrogen receptor 1 (AKT1), estrogen receptor 1 (ESR1), heat shock protein 90 alpha family class A member 1 (HSP90AA1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), and proto-oncogene tyrosine-protein kinase Src (SRC) were determined based on network topology. Molecular docking indicated that catechin, kaempferol, epigallocatechin, and 2,7-dihydroxy-4’-methoxyisoflavone had strong binding energies with core target proteins. In vitro assays demonstrated that the novel water kefir exhibited good scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), radical cation, showing significant antioxidant capacity. In conclusion, the novel water kefir exerts potential anti-aging effects through the synergistic action of multiple components, multiple targets, and multiple pathways, which provides a scientific basis for its development as a functional beverage with anti-aging activity.

Key words: water kefir; anti-aging; metabolomics; network pharmacology; molecular docking