Abstract: 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 1 148 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 1 490 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.

Key words: kombucha; metabolic syndrome; metabolomics; network pharmacology; molecular docking