Bioactivity-Incorporated Metabolomics Approach for Efficient Screening of Bioactive Compounds in Kombucha with Varying Citrus Juice and Sugar Levels

Kombucha is gaining global popularity due to its recognized health benefits, supported by testimonials and confirmed through cellular and clinical studies. Citrus-enriched Kombucha, known for its refreshing qualities, has seen increased demand. This study examined metabolic changes during the production of citrus-enriched Kombucha using Hamlin and LB8–9 Sugar Belle varieties with varying initial sucrose concentrations. Targeted and untargeted metabolomics revealed that higher sucrose levels increased concentrations of sugars, organic acids, and flavonoids. A notable conversion from aromadendrin to quercetin in the flavonoid synthesis pathway was observed in Hamlin Kombucha, possibly due to accelerated malolactic fermentation. The bioactivities of nine compounds, namely, neodiosmin, N-acetyl-dl-tryptophan, limonene, diosmin, indole-3-acetyl-l-aspartic acid, phloretin, (l)-2-aminoadipate, kaempferol 3-rhamninoside, and (R)-mevalonate, were successfully predicted using bioactivity-incorporated metabolomics, avoiding labor-intensive individual bioactivity assays. The model’s predictive power was validated by applying authentic standards in corresponding bioassays. This approach demonstrates that metabolomics combined with bioactivity assays can effectively predict the bioactivity of individual compounds in complex samples, offering a time-saving and less laborious alternative to traditional fractionation methods.