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.

Objective: To explore the composition of metabolites in Kombucha fermented by a mixed culture of Acetobacter xylinus, Lactobacillus fermentum and Zygosaccharomyces bailii and to predict its potential active compounds with regulatory effects on metabolic syndrome so as to provide a theoretical basis for the development and utilization of functional Kombucha beverages. Methods: Non-targeted metabolomics based on ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS) was employed to identify the metabolites in the Kombucha, and the mechanism by which the key compounds regulate metabolic syndrome was analyzed by molecular docking and network pharmacology and verified by in vitro activity assays. Results: A total of 1148 metabolites were identified from the novel Kombucha. The results of principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) indicated that 68 metabolites were significantly up-regulated. Network pharmacology analysis selected 10 key functional components, 382 compound targets and 155 disease intersection targets. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 1490 biological processes, 96 cellular components, 145 molecular functions and 172 pathways. Network topological analysis identified eight core targets including INS, IL6, LEP, TNF, ALB, STAT3, IL1B, and TP53. The results of molecular docking showed that kaempferol-7-neohesperidin, raffinose, simmondsin, and taxifolin had the best binding effect to eight receptor proteins. The results of in vitro activity assays showed that the Kombucha had good scavenging activity against 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical and 2,2’-azinobis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical, as well as inhibitory activity against α-amylase, α-glucosidase and pancreatic lipase. Conclusion: The novel Kombucha contained a variety of active metabolites, which exertedvregulatory effects on metabolic syndrome through a multi-component, multi-target and multi-pathway mechanism. The Kombucha may be a functional beverage for regulating metabolic syndrome.