Predicting the Anti-aging Activity of Novel Water Kefir by Combining Network Pharmacology and Molecular Docking

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 1691 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.