Identification of volatile compounds and metabolic pathway during ultrasound-assisted kombucha fermentation by HS-SPME-GC/MS combined with metabolomic analysis

•The fermentation conditions were optimized by single factor experiments and response surface methodology.•Metabolomics analysis platform was employed to identify the characteristic metabolism during the fermentation of kombucha.•25 potentially characterized metabolites with VIP > 1 were selected by metabolomic analysis.•Metabolic pathways of volatile components reveal underlying mechanisms of kombucha metabolite changes. The current work combines headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC/MS) with multivariate analysis fusion metabonomics for examining metabolite profile changes. The correlation with metabolic pathways during the fermentation of kombucha tea were comprehensively explored. For optimizing the fermentation process, ultrasound-assisted factors were explored. A total of 132 metabolites released by fermented kombucha were detected by HS-SPME-GC/MS. We employed the principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to present the relationship between aroma components and fermentation time, of which the first two principal components respectively accounted for 60.3% and 6.5% of the total variance. Multivariate statistical analysis showed that during the fermentation of kombucha tea, there were significant differences in the phenotypes of metabolites in the samples, and 25 characteristic metabolites were selected as biomarkers. Leaf alcohol was first proposed as the characteristic volatile in the fermentation process of kombucha. Furthermore, we addressed the generation pathways of characteristic volatiles, their formation mechanisms, and the transformational correlation among them. Our findings provide a roadmap for future kombucha fermentation processing to enhance kombucha flavor and aroma.